SaSSOH

September 4-5, 2024

Abstracts

Contents

Schedule (Day 1–2)

2–3

General Information & Guidelines

4–8

Student Session

9

Map

10

Profile and Abstract of Invited Speakers

11-20

Abstract (Oral Presentation)

21-33

Abstract (Poster Presentation)

34-94

1

Program Day 1
September 4th (Wed), 2024
8:15-9:00

Registration

9:00-9:05

Opening Remarks
Prof. Motohiro Horiuchi (Dean, Graduate School of Infectious Diseases, Hokkaido University)

9:05-9:10

Announcement from the Committee

Session A

Vererinary Science 1
Chair: Dr. Nozomu Yokoyama, Kanittha Darawiroj

9:10-9:25

O-1

Yuki Kawakami (Laboratory of Veterinary Internal Medicine, HU)

9:25-9:40

O-2

Hayato Nakamura (Laboratory of Infectious Diseases, HU)

Lecture 1

L-1

Comparative Oncology ~Lessons from canine and human histiocytic sarcoma~
Dr. Hirotaka Tomiyasu

9:40-10:30

Laboratory of Veterinary Internal Medicine, Department of Veterinary Medical Sciences, Graduate Schoolof Agricultural and Life Sciences, The University of Tokyo, Japan
10:30-10:45

Break

Poster session 1
Flash talk (Chair: Dr. Naganori Nao)

10:45-11:30
11:30-12:00

P-1

Poster core session 1

12:00-12:30

P-2

Poster core session 2

12:30-13:00

Extended poster session

Session B

Microbiology 1
Chair: Dr. Tomohiro Okagawa, Mari Ikehata

13:00-13:15

O-3

Mebuki Ito (Division of Risk Analysis and Management, HU)

13:15-13:30

O-4

Paula Andrea Jimenez Jimenez (Laboratory of Parasitology, HU)

13:30-13:45

O-5

Didik Pramono (Laboratory of Molecular Immunology and Infectious Disease, Yamaguchi Univ.)

Lecture 2

L-2

Understanding oncogenesis induced by BLV/HTLV retroviruses opens opportunities for therapeuticintervention
Dr. Luc Willems

13:45-14:35

Laboratory of Molecular and Cellular Epigenetics, Grappe Interdisciplinaire de Génoprotéomique Appliquée (GIGA), University of Liège, Belgium
14:35-15:00

Break
Microbiology 2

Session C

Chair: Dr. Tomokazu Tamura, Daiki Kobayashi
15:00-15:15

O-6

Kento Mori (Department of Microbiology and Immunology, Faculty of Medicine, HU)

15:15-15:30

O-7

Keisuke Maezono (Laboratory of Public Health, HU)

15:30-15:45

O-8

Takuma Ariizumi (Division of Molecular Pathobiology, HU)

Lecture 3

L-3

The initiation of dengue virus replication and its inhibitor from marine organisms

15:45-16:35

Dr. Sarin Chimnaronk
Institute of Molecular Biosciences, Mahidol University, Thailand

2

Program Day 2
September 5th (Thur), 2024
8:30-9:00

Registration

Session D

Microbiology 3
Chair: Dr. Jirachaya Toyting, Temuulen Erdenebat
Maryam Sani Lawal (Division of Collaboration and Education, HU)

9:00-9:15

O-9

9:15-9:30

O-10 Haruto Eguchi (Laboratory of Public Health, HU)

9:30-9:45

O-11 Pawarut Narongpun (Division of Bioresources, HU)

Lecture 4

L-4

Insights into prion neurodegeneration and transmission from human brain organoids
Dr. Cathryn L. Haigh

9:45-10:35

Rocky Mountain Laboratories, NIH, United States
10:35-10:45

Break

Poster session 2
Flash talk (Chair: Dr. Yuki Otani)

10:45-11:30
11:30-12:00

P-3

Poster core session 3

12:00-12:30

P-4

Poster core session 4

12:30-13:00

Extended poster session

Session E

Veterinary Science 2
Chair: Dr. Shouta Nakayama, Anastasiia Kovba

13:00-13:15

O-12 Nelly Banda (Laboratory of Toxicology, HU)

13:15-13:30

O-13 Anastasiia Kovba (Laboratory of Wildlife Biology and Medicine, HU)

Lecture 5

L-5

13:30-14:20

One Health implications for the conservation of large felids in Asia
Dr. Martin Gilbert (Cornell University, United States)
Cornell K. Lisa Yang Center for Wildlife Health, College of Veterinary Medicine Cornell University, UnitedStates

14:20-14:40

Break

Student Session
Organizers: Dela Ria Nesti, Anastasiia Kovba, Nelly Banda, Loc Tan Huynh, Paula Jiménez
14:40-17:10

Fostering complementary teamwork towards One Health!

17:10-17:30

Break

17:30-18:30

Closing CeremonyAwardsClosing Remarks
Prof. Motohiro Horiuchi (Coordinator of the WISE Program; Dean, Graduate School of Infectious
Diseases, Hokkaido University)

3

GENERAL INFORMATION & GUIDELINES
General Information
Oral presentation: face-to-face (Lecture Hall)
Poster presentation: face-to-face (Meeting rooms)l

SaSSOH 2024 is closed to the public. Please refrain from recording or distributing presentations
without the consent of the presenter.

l

We cannot protect your data perfectly. Please be aware that your presentation may be watched by anybody.
Please discuss with your PI whether you can show the data or not.

l

Use English. No Japanese.

Information of SaSSOH 2024
https://sassoh.vetmed.hokudai.ac.jp/upcoming-sassoh/
Survey for Next Year (by Sep 13th, 2024)
https://forms.gle/CmCwGWBam71Gzy1H8

4

General Guideline for Oral PresentationlVenue: Lecture Hall, Lecture buildinglPresentation Data: Please bring your PowerPoint file to the lecture hall from 8:15 to 9:00 on September 4th(from 13:30 to 14:30 on September 3rd for Hokkaido University students), transfer the data to PC and preview
it.

l

We have only windows PC in the lecture hall. No mac.

l

Widescreen (16:9) is recommended for slides.

lPresentation time
* 1 bell ring: 1 min before the end of talk, 2 bells: end of talk, 3 bells: end of discussionl-Invited: 50 min talk (including discussion)-

Student oral: 12 min talk + 3 min discussion

Please keep the time for the presentation to ensure smooth proceedings.

5General Guideline for Poster PresentationlVenue: Meeting rooms, Lecture BuildinglPoster Display/Removall-Display: September 4th, 8:15–9:00-

Removal: September 5th, 14:20–15:00

Poster Size
Each board size is 900 mm W x 2,000 mm H. A0 size: 841 mm x 1189 mm may be appropriate if you prepare
your poster as a single sheet. Each poster panel is tagged with a poster number.

lPoster Core Time
The poster number determines which session you will present at:-

Poster core session 1, September 4th, 11:30-12:00: P1-1~P1-16-

Poster core session 2, September 4th, 12:00-12:30: P2-1~P2-16-

Poster core session 3, September 5th, 11:30-12:00: P3-1~P3-15-

Poster core session 4, September 5th, 12:00-12:30: P4-1~P4-16

They are luncheon sessions. You can have small lunch exchanging a ticket provided at registration in themorning.
Question sheet is available for asking questions and make contact at the extended poster session.

6General Guideline for Flash Talk
Give a short talk (1 min) to provide a brief summary of your research. Future-plan is OK if you don’t have enoughdatalVenue: Lecture Hall, Lecture BuildinglPresentation file: 1 slide of power point (wide screen 16:9)

lSchedulel-Poster Session A, September 4th, 11:00–11:30: P1-1~P1-16 and P2-1~P2-16-

Poster Session B, September 5th, 11:00–11:30: P3-1~P3-15 and P4-1~P4-16

Wait for your presentation in line.

Closing Ceremony
This year's SaSSOH will provide an opportunity for speakers and participants to gather and get to know each other.
Light meal will be served. We hope you will take this opportunity to deepen your socializing.
l

Venue: Meeting rooms, Lecture BuildinglTime: September 5th, 17:30–18:30

7

Organizing Committee
Shintaro Kobayashi

Yuki Otani

Laboratory of Public Health, Faculty of Veterinary

Social Cooperation Unit, One Health Research Center,

Medicine, Hokkaido University

Hokkaido University

Toyotaka Sato

Tatsuki Sugi

Laboratory of Veterinary Hygiene, Faculty ofDivision of Collaboration and Education, International

Veterinary Medicine, Hokkaido University

Institute for Zoonosis Control, Hokkaido University

Shiro Murata

Tomokazu Tamura

Laboratory of Infectious Diseases, Faculty of

Department of Microbiology and Immunology, Faculty

Veterinary Medicine, Hokkaido Universityof Medicine, Hokkaido University

Keisuke Aoshima

Nozomu Yokoyama

Laboratory of Comparative Pathology, Faculty ofLaboratory of Veterinary Internal Medicine, Faculty of

Veterinary Medicine, Hokkaido University

Veterinary Medicine, Hokkaido University

Michael Henshaw

Junko Hayashi

International Affairs Office, Faculty of Veterinary

International Affairs Office, Faculty of Veterinary

Medicine, Hokkaido University

Medicine, Hokkaido University

Naganori Nao

Masaki Sato

Integrated Database Unit, One Health Research Center,

International Affairs Office, Faculty of Veterinary

Hokkaido University

Medicine, Hokkaido University

Collins Nimako

Maiko Nakano

One Health Research Center, Hokkaido University

International Affairs Office, Faculty of Veterinary
Medicine, Hokkaido University

Yuma Ohari
Division of Risk Analysis and Management,International Institute for Zoonosis Control, Hokkaido
University
Organized by

HOKKAIDO UNIVERSITY WISE Program for One Health Frontier Graduate School of Excellence
Website: https://onehealth.vetmed.hokudai.ac.jp
Facebook: https://www.facebook.com/vetmed.wiseprogram/
WISE Office
Faculty of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Sapporo, Japan 060-0818
ohf@vetmed.hokudai.ac.jp
8

STUDENT SESSION
General information
The Student Session is an original creation by students that provides additional opportunities for attendees to meetand collaborate, regardless of personal research interest.
Student Session 2024: Fostering complementary teamwork towards One Health!
The 2024 student session endeavors to achieve complementary teamwork that is important on the road to achieving
One Health, that includes human, animal and environmental health. It aims to improve the understanding of the
One Health foundation components: communication, collaboration and coordination.
In this age of emerging and re-emerging diseases increase, absolute cooperation of the various sectors of One
Health is required, to safeguard animal and human health. Decisions made in each sector are important and how
resources are shared is cardinal in today’s economy. As experts in One Health, comprehension of how to navigate
such situations is crucial.
To reach a healthy society and reduce health problems, we all must understand the scarcity of resources and how
they affect decision making. Each component of One Health is important just as each body organ is important fororganism functionality. This session will allow the participants to work together to cure a body by first solving the
problem of its organs. It will help them understand how making decisions within each organ will overall affect thehealth of the body, thereby embodying the concept of how the three components of One Health work together.
Website: https://sassoh.vetmed.hokudai.ac.jp/upcoming-sassoh/student-session/
Venue
Practice Rooms 1, 2, and 4
Registration
Period: August 12nd, 2024 – August 24th
To better understand the game before you play, the Student Committee will provide an event guide after yourregistration.
Student Committee
Dela Ria Nesti

Loc Tan Huynh

Division of Collaboration and Education, Graduate

Laboratory of Microbiology, Graduate School of

School of Infectious Disease

Infectious Diseases

Anastasiia Kovba
Paula Jimenez

Wildlife Biology and Medicine Laboratory, Graduate

Laboratory of Parasitology, Graduate School of

School of Veterinary Medicine

Infectious Disease
Nelly Banda
Laboratory of Toxicology, Graduate School of
Veterinary Medicine

9MAP
n Faculty of Veterinary Medicine, Hokkaido University

Main Building

LectureBuilding

Lecture building (for oral and poster presentation)

10

PhotoProfile of Invited Speakers

Hirotaka Tomiyasu
Associate Professor
Graduate School of Agricultural and Life Sciences,
Veterinary Medical Sciences,
The University of Tokyo

Education
BVSc, DVM

2010

Faculty of Agriculture, the University of Tokyo

PhD in Veterinary Medicine

2014

Graduate School of Agricultural and Life Sciences, theUniversity of Tokyo

Professional Appointments
2014-2015

Postdoctoral Associate, Veterinary Clinical Sciences, College of Veterinary
Medicine, University of Minnesota, USA

2015-2016

Project Researcher, Veterinary Medical Center, Graduate School of Agriculturaland Life Sciences, the University of Tokyo, Japan

2016-2018

Project Assistant Professor, Veterinary Medical Center, Graduate School of
Agricultural and Life Sciences, the University of Tokyo, Japan

2018-2021

Assistant Professor, Department of Veterinary Internal Medicine, Graduate Schoolof Agricultural and Life Sciences, the University of Tokyo, Japan2021-present

Associate Professor, Department of Veterinary Internal Medicine, Graduate
School of Agricultural and Life Sciences, The University of Tokyo, Japan

Major Research Interests
Translational research and clinical trials based on the investigations of molecular aberrations incanine and feline diseases including tumors.

1111

September 4th, 9:40-10:30

L-1

Comparative Oncology ~Lessons from canine and human histiocytic sarcoma~
Hirotaka Tomiyasu
Laboratory of Veterinary Internal Medicine, Department of Veterinary Medical Sciences, Graduate School of Agricultural and Life
Sciences, The University of Tokyo
atomi@g.ecc.u-tokyo.ac.jp

One Health approach includes comparative oncology as one of the key focuses, as it plays an important role inelucidating the mechanisms of initiation and progression of both human and animal tumors. Translational
research is an important approach to achieve meaningful health outcomes in medical and veterinary sciences.
In this lecture, I will discuss about the findings of our recent study that utilized these approaches to investigate
the clinical and molecular pathology of histiocytic sarcoma. Histiocytic sarcoma is a neoplastic disease derived
from dendritic cells or monocytes/macrophages that is often diagnosed in dogs. However, it is extremely rare in
humans, resulting in a lack of intensive studies to develop effective treatments and improve the patient prognosis.
We have investigated the molecular pathology of canine histiocytic sarcoma through a comprehensive analysis
of genetic aberrations using next-generation sequencing, and we revealed the dysregulation of several
intracellular signaling pathways, such as ERK and Akt pathways, in the tumor cells. It was also shown thathuman and canine histiocytic sarcoma share common molecular abnormalities, including the dysregulation ofthese pathways and the mutations in the specific genes, such as PTPN11 and TP53. Furthermore, this diseasehas been shown to be highly heterogeneous in terms of molecular aberrations among canine cases, and thesensitivities to molecular-targeted drugs that target the dysregulated pathways varied among cell lines. The
results obtained in our study indicated the need for “precision medicine” based on the molecular pathology ofeach canine histiocytic sarcoma case. Since this disease is extremely rare in humans, as described above, I
believe that elucidations of molecular pathology of canine histiocytic sarcoma would also make a significant
contribution to human medicine from the perspective of comparative oncology.

12Profile of Invited Speakers

Photo

Luc Willems
Research Director of the National Fund for Scientific Research
Professor at University of Liege
Laboratory of Molecular and Cellular Epigenetics,
GIGA, University of Liège

Education
Master in Bioengineering

1984

University of Gembloux (Liege), Belgium

PhD in Bioengineering

1988

University of Gembloux (Liege), Belgium

Habilitation for Higher Education

1995

University of Gembloux (Liege), Belgium

Professional Appointments
1984-1988

Research Assistant of the National Fund for Scientific Research, Belgium

1988-1991

Senior Research Assistant of the National Fund for Scientific Research, Belgium

1991-1995

Research Associate of the National Fund for Scientific Research, Belgium

1995-1999

Senior Research Associate of the National Fund for Scientific Research, Belgium

1999-present

Research Director of the National Fund for Scientific Research, Belgium

2010-present

Professor of Molecular Biology at Gembloux Agro Bio-Tech, University of Liège,
Belgium

2011

Permanent Member of the Royal Academy of Medicine of Belgium

Major Research Interests
The ongoing research focuses on the epigenetic mechanisms of cancer induced by environmental
factors (asbestos fibers and viral infections). The current research topics include mesothelioma andretrovirus-induced leukemia (HTLV/BLV). In a translational perspective, the goal is to understandthe basic mechanisms of cancer in order to develop novel therapeutic and preventive approaches.

13

September 4th, 13:45-14:35

L-2

Understanding oncogenesis induced by BLV/HTLV retroviruses opens opportunities fortherapeutic intervention
Luc Willems (1, 2, 3)(1) Laboratory of Molecular and Cellular Epigenetics, Grappe Interdisciplinaire de Génoprotéomique Appliquée (GIGA), University of
Liège(2) Gembloux Agro-Bio Tech (GxABT), University of Liège(3) National Fund for Scientific Research
Luc.Willems@uliege.be

My research team investigates the mechanisms associated with infection by two evolutionarily related
retroviruses: bovine leukemia virus (BLV) and human T-lymphotropic virus (HTLV-1). Approximately 25
million people worldwide are infected with HTLV-1. Transmission occurs primarily from mother to child,between sexual partners, through contaminated blood products, and by needle sharing. Infected individuals risk
developing a rapidly progressive leukemia (ATL) and a debilitating, sometimes fatal neurologic condition(HAM/TSP). BLV naturally infects cattle, yak, zebu, and water buffalo. In bovines, major symptoms of BLV
infection include lymphoma (enzootic bovine leukemia or EBL) and persistent lymphocytosis (PL).
Approximately one-third of BLV-infected bovines develop PL, while tumors affect 5–10% of animals after longlatency periods (4–10 years). At the asymptomatic stage, BLV infection is associated with reduced milk
production, shortened longevity, and immune suppression. Because most animals do not show obvious
symptoms, BLV has been neglected in many regions worldwide. Nevertheless, BLV prevalence has a significant
economic impact, according to recent prediction models.
A comparative virology approach to BLV and HTLV-1 has revealed that viral persistence is a highly dynamic
process, characterized by continuous viral replication efforts and active host immune response control. Evidenceof a robust immune response is demonstrated by the presence of virus-specific cytotoxic T cells and high titersof neutralizing antibodies. Silencing viral expression is crucial for the virus to evade immune detection. HTLV1 and BLV replicate via an infectious cycle upon expression of progeny virions, as well as by mitotic divisionof provirus-carrying cells (clonal expansion). According to the currently most accepted model, two viral proteins(Tax and HBZ) are hypothesized to have the greatest impact on viral replication and cell transformation. The
modes of action of Tax and HBZ are remarkably pleiotropic, involving various cell signaling pathways that
stimulate proliferation.
Knowledge of the basic mechanisms of viral replication has opened prospects for preventive and therapeuticinterventions. In particular, reverse genetics has provided useful insights into viral persistence, genetic
determinants of pathogenesis, and potential novel therapies.

14

PhotoProfile of Invited Speakers

Sarin Chimnaronk
Associate Professor
Institute of Molecular Biosciences, Mahidol University, Thailand

Education
Master of Science (M.Sc.).

2001

Department of Integrated Biosciences, Graduate School of
Frontier Sciences, The University of Tokyo, Japan

Ph.D. in Biosciences

2004

Department of Integrated Biosciences, Graduate School of
Frontier Sciences, The University of Tokyo, Japan

Professional Appointments
2005–2007

Postdoctoral fellowship, X-ray crystallography group (Prof. Isao Tanaka) at
Faculty of Sciences, Hokkaido University, Sapporo, Japan

2007–present

PI of the Laboratory of RNA Biology at the Institute of Molecular Biosciences,
Mahidol University, Nakhon Pathom, Thailand

Major Research Interests
My particular interest is in the function and structure of the RNA-protein interface. About 80% of
the human genome is transcribed to RNA and those transcripts usually require protein partners
to be functional. The functions of cellular RNAs are therefore regulated via the sequence andstructure of RNA elements and the interaction mode with proteins. However, the knowledge as to
how the interactions of RNA structures with their protein counterparts contribute to function islimited. I have been exploring the interface between viral RNA genome and cellular proteins at
Mahidol University in Thailand. In particular, we identified two crucial viral RNA interfaces in the
dengue virus genome responsible for viral RNA replication and pathogenesis. With support fromthe National Research Council of Thailand (NRCT), we are exploring more on the dengue RNA
genome and working on the SARS-CoV-2 RNA interface. We propose that the RNA-protein
interface can be a new target for drug development for infectious diseases.

15

September 4th, 15:45-16:35

L-3

The initiation of dengue virus replication and its inhibitor from marine organisms
Sarin Chimnaronk
Institute of Molecular Biosciences, Mahidol University, Thailand.
sarin.chi@mahidol.ac.th

Dengue virus (DENV) belongs to the genus Flavivirus of the family Flaviviridae, and it is primarily transmitted to humans
via Aedes aegypti and Aedes albopictus mosquitoes. WHO reported a historic high of over 6.5 million cases and more than
7,300 dengue-related deaths in 2023. There is currently no drug for specific treatment of dengue infection. DENV consistsof four antigenically related but distinct DENV serotypes (DENV-1, DENV-2, DENV-2, DENV-3, and DENV-4). These
DENVs can cause a spectrum of symptoms ranging from asymptomatic dengue infection to dengue fever (DF), andoccasionally to dengue hemorrhagic fever (DHF) and life-threatening dengue shock syndrome (DSS). DENV is a positivesense RNA virus possessing a ~10.7-kb single-stranded RNA genome, which is replicated in infected host cells by theviral non-structural protein 5 (NS5). NS5 contains the center of the RNA-dependent RNA polymerase (RdRp) activity in
its C terminal domain, indispensable for de novo synthesis of viral RNA. We probed the entire DENV RNA genome forinteractions with the NS5 using the yeast three-hybrid scan. I will describe the specific interaction between the C-terminal
thumb subdomain of NS5 RdRp and an apical loop in the 3´ stem-loop (SL) element in the 3´-untranslated region (UTR)of the viral genome for initiation of the negative strand synthesis. In addition, we performed a large screening campaign
and identified a novel chemotype from marine species that inhibited DENV NS5’s activities and viral replication in humanhepatic cells for all DENV serotypes with IC50 values of 2 - 4 μM. We used X-ray crystallography analysis to unveil the
new mechanism of action of the marine inhibitor against DENV NS5. Our results provided a new model of viral replicationand showed that marine species are unique and important sources for discovering novel lead types against infectious
diseases.

16

Profile of Invited Speakers

Photo

Cathryn Louise Haigh
Chief
Prion Cell Biology Unit
Laboratory of Neurological Infections and Immunity (LNII),
DIR, NIAID, NIH, Rocky Mountain Laboratories, USA

Education
Bachelor of Biomedical Sciences (B.Sc.)

2002 University of Wales Institute Cardiff (UWIC), UK

Ph.D. in Biology and Biochemistry

2006

University of Bath, UK

Professional Appointments
2000-2001
2006
2006-2014
2008-20112015-20162016

2017-present

Trainee Biomedical Scientist. Haematology, Immunology and Blood/Tissue
Banking. Swansea NHS Authority, Wales UK.
Post-doctoral researcher. Department of Biology and Biochemistry, University ofBath, Claverton Down, Bath, BA2 7AY, UK.
Senior Research Officer. Department of Pathology, The University of Melbourne.
Parkville VIC 3010, Australia.
Honorary Research Fellow. Mental Health Research Institute of Victoria. 155 Oak
St, Parkville VIC 3052.
Senior Research Officer. Department of Medicine (Royal Melbourne Hospital),
The University of Melbourne. Parkville VIC 3010, Australia.
Senior Diagnostic Scientist. Australian National CJD Registry. Department ofMedicine (Royal Melbourne Hospital)/Florey Institute of Neuroscience and Mental
Health, The University of Melbourne. Parkville VIC 3010, Australia.
Tenure-track Investigator. Laboratory of Neurological Infections and Immunity
DIR, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA.

Major Research Interests
The Prion Cell Biology Unit investigates the cellular pathways causing neuronal death anddysfunction in prion diseases and explores the basic functions of prion proteins. Key research areas
include prion protein's role in cellular redox homeostasis, its impact on disease progression andtoxicity, its influence on neural stem cell behavior, and the effects of its post-translational
modifications. The Unit also advances tools for prion disease research, including a human cerebral
organoid model that mimics features of human prion infection. This model helps study cellularresponses to different Creutzfeldt-Jakob Disease sub-types and the role of hereditary PRNP
mutations in prion diseases.

17

September 5th, 9:45-10:35

L-4

Insights into prion neurodegeneration and transmission from human brain organoids
Cathryn Haigh
National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH), USA
cathryn.haigh@nih.gov

Prion diseases are rare transmissible diseases affecting humans and animals. Following onset of symptoms they are always
fatal but the mechanisms by which the brain degenerates are still poorly understood. To aid discoveries that would inform
us about the pathogenic mechanisms of human disease, we developed a human cerebral organoid model of human prion
infection. Human cerebral organoids are small spheres of human brain tissue that self-structure as they grow. They canbe differentiated from embryonic or induced pluripotent stem cells and are viable for long culture periods (>1 year), thus
they are suitable for studying the evolution of infection over long periods. Our investigations have found that prion
infection causes neuroelectrophysiological dysfunction with a shift toward excitotoxicity and altered post-synaptic
composition. This is accompanied by changes indicating impaired mitochondrial fission and fusion dynamics, with some
infections additionally showing changes within the extracellular matrix, neurite structure and signal transduction pathways.
Improving our understanding of the pathogenic features of prion diseases allows us to consider ways to intervene to reducethe damage associated with the infection. The organoid model permits examination of anti-prion activity and neurorestorative actions of putative therapeutics. A further application of the organoid models is examining cross-species
transmission of prion diseases. Since bovine spongiform encephalopathy transmitted to humans causing the emergenceof a new variant of Creutzfeldt-Jakob Disease, cross-species transmission has been an ongoing concern. This concern
recently has been most focused on chronic wasting disease, which affects cervids and has been found in a number ofcountries. We have been using the human cerebral organoid model to test whether CWD prions can trigger human prion
infection.

18

PhotoProfile of Invited Speakers

Martin Gilbert
Associate Professor of Practice - Wildlife HealthCornell K. Lisa Yang Center for Wildlife HealthCornell University

Education
Bachelor of Science (Hons) in Zoology

1996

University of Glasgow, Scotland

Bachelor of Veterinary Medicine & Surgery

1999

University of Glasgow, Scotland

Master of Research

2012

University of Glasgow, Scotland

PhD in Comparative Medicine

2016

University of Glasgow, Scotland

Professional Appointments
Octʼ 21 – Present
Decʼ 16 – Sepʼ21
Aprʼ 14 – Octʼ16
Jul ʼ11 – Mar ʻ14
Jan ʻ08 – Jun ʻ11
Aug ʼ04 – Dec ʼ07
Dec ʼ00 – Aug ʼ04
Sep ʼ00 – Dec ʼ00
Jul ʼ00 – Sep ʼ00
Aug ʼ99 – Mar ʼ00

Associate Professor of Practice, Cornell University
Senior Research Associate, Cornell University
Carnivore Health Specialist, Wildlife Conservation Society
MCEIRS Project (WCS-PI), Wildlife Conservation Society
Assoc Director – Asia, Global Health Program, Wildlife Conservation Society
Field Veterinarian, Field Veterinary Program, Wildlife Conservation Society
Field Veterinarian, Asian Vulture Crisis Project, The Peregrine Fund, Pakistan
Veterinarian, Kanyana Wildlife Rehabilitation Centre, Lesmurdie, Australia
Assistant Bird Manager, SANCCOB, Treasure Oil Spill, South Africa
Field Veterinarian/Biologist, The Peregrine Fund, Madagascar

Major Research Interests
As the Cornell Wildlife Health Center's Wild Carnivore Health Specialist, Dr. Martin Gilbert leads
projects focused on understanding how disease affects threatened carnivore populations anddeveloping practical measures to mitigate conservation impacts on key species. With over two
decades working in international settings, Dr. Gilbert has achieved significant milestones,
including the identification of diclofenac as the agent responsible for the regional collapse ofvulture populations across South Asia, and research that identified canine distemper virus as athreat to the survival of Amur tigers in the Russian Far East, and potentially in other range
countries.

19

September 5th, 13:30-14:20

L-5

One Health implications for the conservation of large felids in Asia
Martin Girbet
National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH), USA Cornell K. Lisa Yang Center for Wildlife
Health, College of Veterinary Medicine Cornell University, Ithaca, NY 14853, United States.
m.gilbert@cornell.edu

Asia is home to five species of large felids, more than any other continent, which are among the region’s most enigmaticand highly threatened wildlife. Their populations are now heavily depleted due to habitat conversion, dwindling preynumbers, persecution for their body parts, and conflicts with humans. Health professionals can contribute to theconservation of Asia’s large felids in a variety of ways. Research to understand the epidemiology of canine distemper
virus and its impact on Amur tigers (Panthera tigris altaica) in the Russian Far East has enabled us to design management
strategies to control this direct threat to their population viability. Pathogens such as African swine fever virus, or Sarcoptes
scabiei can also apply indirect pressures on large felids by reducing numbers of key prey species such as wild boar (Sus
scrofa) or mountain ungulates on which large felids rely. Embracing the One Health concept presents opportunities toaddress competition and conflicts that simultaneously threaten large felids, humans, and their domestic animals.
Interventions that improve the health and productivity of ruminant livestock maintain or increase income for pastoralist
communities from smaller herds with a lighter ecological footprint, while reducing disease in wild ungulate prey.
Additionally, innovative approaches using chemical signals from large felid urine have shown promise as deterrents in'bio-barriers' to manage problematic predatory behaviors. Recognizing that competition for resources and health areintegral drivers of conflict between large felids, domestic animals, and humans allows us to implement mitigation measuresthat foster coexistence, promote conservation, and ensure sustainable livelihoods across these wild Asian ecosystems.

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Relationship between Airway Pressure and Shear Wave Speed at the Lung Surface
Yuki Kawakami (1), Kensuke Nakamura (1), Noboru Sasaki (2), Nozomu Yokoyama (1), Mitsuyoshi Takiguchi(1)(1)Laboratory of veterinary internal medicine, Hokkaido Univerisity, Japan(2)Hokkaido University Veterinay Teaching Hospital, Japan

Introduction:
Ultrasonography of the lungs is widely used in both medical and veterinary practice due to its non-invasivenature. Ultrasound shear wave elastography (USWE) provides an objective measure of tissue elasticity and has
been clinically applied to the evaluation of lung surfaces. However, it remains unclear whether USWE canaccurately assess the lung surface. In this study, we investigated whether changes in the lung surface can bedetected by USWE and evaluated its reproducibility. To verify whether USWE can detect lung surface changes,
how changes in airway pressure affect USWE, and the reproducibility of lung surface USWE.
Methods:
Oxygen was delivered into excised porcine lungs (n=3) to maintain airway pressures at 10 cmH2O, 20 cmH2O,
and 30 cmH2O. Shear Wave Speed (SWS) was measured at each airway pressure. SWS measurements were
performed five times for one lung at each airway pressure, and each set of measurements was repeated threetimes. Intraclass correlation coefficients (ICC) were calculated for one, three, and five measurements todetermine reliability. Pearson's correlation coefficient between airway pressure and lung surface SWS was also
calculated.
Results:
ICCs at each airway pressure were as follows: at 20 cmH2O, ICC(1,1) = -0.013, ICC(1,3) = -0.323, ICC(1,5) =-0.066; at 30 cmH2O, ICC(1,1) = 0.371, ICC(1,3) = 0.605, ICC(1,5) = 0.747. Pearson's correlation coefficient
was r = 0.97.
Discussion:
The high correlation coefficient suggests that USWE is capable of capturing changes in the lung surface. ICC
tended to be highest at an airway pressure of 20 cmH2O and increased with the number of measurements. When
USWE is performed on lungs inflated to an airway pressure of 20 cmH2O, a minimum of five measurements islikely to provide clinically acceptable reliability. In the present study, SWS changes in lung surface dynamics
induced by elevated internal airway pressure may encompass several factors: (1) an augmentation in elastic
modulus attributed to tension generated on the lung surface, (2) alterations in lung surface morphology, and (3)modifications in tissue density at the measurement site.
Conclusion
USWE can effectively detect changes in the lung surface induced by airway pressure. To achieve clinically
acceptable reliability, USWE should be performed at least five times on lungs inflated to an airway pressure of
20 cmH2O.

21

O-2

Comprehensive expression analysis of immune checkpoint molecules in BLV-infected cattle
Hayato Nakamura (1), Tomohiro Okagawa (2), Mari Ikehata (1), Naoya Maekawa (2), Yasuhiko Suzuki (2) (3)(4) (5), Shiro Murata (1) (2), Kazuhiko Ohashi (1) (2) (6), Satoru Konnai (1) (2) (3)(1)Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Japan(3)Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Japan(4)Division of Bioresources, International Institute for Zoonosis Control, Hokkaido University, Japan(5)Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University,
Japan(6)International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Japan

Bovine leukemia virus (BLV) is a retrovirus that causes B-cell lymphoma called enzootic bovine leukosis (EBL)in cattle and is widespread in many countries, including Japan. Our recent studies have revealed that T cells in
BLV-infected cattle exhibit elevated expression of immune checkpoint molecules, such as programmed death1 (PD-1), lymphocyte-activation gene 3 (LAG-3), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4), andT cell immunoglobulin domain and mucin domain-3 (TIM-3), contributing to functional exhaustion of T cells,
the maintenance of persistent infection, and disease progression. These findings suggest that the upregulationof multiple immune checkpoint molecules is involved in the mechanism of T cell exhaustion in BLV infection.
However, it remains unclear which immune checkpoint molecules play a central role in the induction of immune
exhaustion, warranting comprehensive immunological analysis.
In this study, we aimed to further elucidate the mechanisms of immune exhaustion via immune checkpoint
molecules in BLV-infected cattle. Utilizing an eight-color flow cytometric analysis for the expression analysis
of four immune checkpoint molecules, we found a significant increase in the proportion of CD4+ and CD8+ T
cells co-expressing TIM-3 and PD-1 in the tumorized lymph nodes of EBL cattle compared to the lymph nodesof healthy cattle. The proportion of PD-1+TIM-3+ T cells was markedly higher compared to those of T cells
expressing CTLA-4 or LAG-3. Additionally, a similar increase in PD-1 and TIM-3 co-expression was observedin tumor-infiltrating T cells in lymphosarcomas formed in non-lymphoid organs of EBL cattle. Interestingly, we
also found that the PD-1+TIM-3+ T cells in EBL cattle exhibited increased expressions of CTLA-4 and LAG-3.
Similar increased expression was observed in peripheral blood, indicating a significant increase in the proportionof T cells co-expressing TIM-3 and PD-1, particularly in EBL cattle.
This study revealed the detailed expression profiles of immune checkpoint molecules in BLV-infected cattle anda marked increase in the proportion of PD-1+TIM-3+ T cells in EBL cattle compared to that of T cells expressing
other immune checkpoint molecules. Furthermore, the subset of PD-1+TIM-3+ T cells exhibited a phenotype of
more pronounced exhaustion. Future investigations focusing on the gene expression and functional changes in
the PD-1+TIM-3+ exhausted T cell subset will be crucial in elucidating the mechanisms of exhaustion induced
by BLV infection.

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O-3

Ecological factors interacting with the tick-borne virus infection: Raccoons and Yezo virus in
Hokkaido, Japan
Mebuki Ito (1) (2), Miku Minamikawa (1) (3), Anastasiia Kovba (4), Hideka Numata (5), Tetsuji Itoh (6),Nariaki Nonaka (5), Ryo Nakao (5), Ryosuke Omori (7), Yuma Ohari (2), Michito Shimozuru (1) (8), Toshio
Tsubota (1), Keita Matsuno (2) (8) (9) (10), Mariko Sashika (1)(1)Laboratory of Wildlife Biology and Medicine, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Division of Risk Analysis and Management, International Institute for Zoonosis Control, Hokkaido University, Japan(3)Center for Ecological Research, Kyoto University, Japan(4)Laboratory of Wildlife Biology and Medicine, Graduate School of Veterinary Medicine, Hokkaido University, Japan(5)Laboratory of Parasitology, Faculty of Veterinary Medicine, Hokkaido University, Japan(6)Laboratory of Wildlife Management, Department of Environmental and Symbiotic Sciences, College of Agriculture, Food andEnvironment Sciences, Rakuno Gakuen University, Japan(7)Division of Bioinformatics, International Institute for Zoonosis Control, Hokkaido University, Japan(8)One Health Research Center, Hokkaido University, Japan(9)Institute for Vaccine Research and Development, HU-IVReD, Hokkaido University, Japan(10)International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Japan[Background] Yezo virus (YEZV) is a novel tick-borne virus isolated from febrile patients in Hokkaido, Japan.
While YEZV infection is prevalent in ticks and wild animals such as raccoons (Procyon lotor), the ecology ofthe virus remains largely unknown. The aim of this study is to reveal the environmental and host factors
interacted with YEZV infection and quantify the relationship between tick infestation intensity and YEZV
infection in wild animals. [Materials and Methods] Sera from 514 raccoons captured in the central part of
Hokkaido in 2018-2023 were used to investigate the history of YEZV infection by detecting anti-YEZV
antibodies with ELISA. Among these raccoons, the number of infesting ticks was quantified in 244 raccoonscaptured in 2022-2023. Generalised linear models (GLMs) were used to analyse the effects of environmental
and host factors on the number of infesting ticks per species/developmental stage and the possession of antiYEZV antibodies in raccoons. The GLM was also used to quantify the effect of tick infestation intensity on thepossession of anti-YEZV antibodies. [Results and discussion] Adult Ixodes ovatus was the predominant tickinfesting raccoons in Hokkaido, followed by adult Ixodes tanukis and Ixodes persulcatus. The size of forest area
and the length of forest edges around the capture site of raccoons had significant positive effects on the numberof these ticks infesting raccoons. Additionally, male raccoons were significantly infested by more ticks thanfemales. The total prevalence of anti-YEZV antibodies in raccoons captured in 2018-2023 was 6.2%. GLM
analysis showed that possession of anti-YEZV antibodies in raccoons was significantly positively influenced
by the intensity of adult I. ovatus infestation. This result emphasizes the importance of I. ovatus on the YEZV
circulation by demonstrating that the higher exposure of ticks enhances the likelihood of tick-borne virustransmission from ticks to wild animals. On the other hand, the environmental and host factors that significantly
affected the possession of anti-YEZV antibodies were the size of forest area and the length of water edgesaround the capture site and the age of raccoon, which did not fully match the factors that affected tick infestation
intensities. This study demonstrates the close relationship and uniqueness of tick infestation on wild animals
and the subsequent transmission of tick-borne viruses, as well as providing a better understanding of the YEZV
maintenance mechanisms from ecological perspectives.

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O-4

One Health approach to identify and characterize tick-borne pathogens using tick cell cultures
Paula Andrea Jimenez Jimenez (1), Yongjin Qiu (1), Mackenzie Kwak (1), Nariaki Nonaka (1), Ryo Nakao (1)(1)Laboratory of Parasitology, Department of Disease Control, Faculty ofVeterinary Medicine, Hokkaido University, Sapporo, Japan

Background: Emerging tick-borne pathogens have become a growing concern as they pose a significant threat
to public health in Japan and worldwide. Pathogens, such as intracellular bacteria, viruses, and protozoa, havebeen increasingly reported and are associated with a range of diseases affecting both animals and humans. As
the geographic distribution of ticks and their pathogens expands in Japan, a detailed investigation is required.
This study aims to isolate and characterize tick-borne pathogens from ticks collected across Japan, with special
focus on Amami and Tokunoshima Islands. Target pathogens include known disease sources such as Rickettsia,
Ehrlichia, Anaplasma, Neoehrlichia, and emerging pathogens yet to be identified. Further analysis to evaluate
their pathogenic potential in animals will be conducted to develop a serological assay which involves IFA(indirect fluorescent antibody) tests targeting companion animals such as dogs and cats. The IFA tests will utilize
blood and serum samples, to assess the presence of antibodies against the isolated pathogens, offering valuable
information on the pathogens' ability to infect companion animals and their zoonotic risk. Methods: Ticks weresystematically collected from diverse regions of Japan using the flagging technique. Collected specimens wereidentified to the species level using morphological keys and molecular methods. Pathogen isolation wasachieved by co-culturing of tick homogenates with tick cell lines such as ISE6, BME/CTVM23, and AAE2 andmammalian cell lines (L929F). These cultures are being monitored for pathogen growth, and confirmation ofisolated pathogens to further analysis is being conducted using molecular assays such as conventional PCR andreal-time PCR. Additionally, blood and serum samples from dogs and cats are being collected in Amami Islandto accomplish the serological tests. Results: A total of 225 ticks were collected in Tokunoshima Island including
adults of Amblyomma testudinarium (n=38), Haemaphysalis hystricis (n=12), Haemaphysalis formosensis(n=52), and Haemaphysalis megaspinosa (n=1), and nymphs of Haemaphysalis spp. (n=122). Tickhomogenates from different tick species were inoculated into each cell line. Cell morphology was observed
daily under an inverted microscope to determine cytopathic effects (CPE), bacterial growth or the occurrenceof intracellular morulae. After 8 days, L929F cells culture exhibited significant alterations, including increased
cell death, noticeable cell aggregation, and CPE. Meanwhile, tick cell lines showed no changes during the firstweek after inoculation. In addition, microscopic examination in all cell line cultures revealed the presence ofbacterial-like microorganisms. Since, this an ongoing experimental process, preliminary results warrant further
analyses before drawing conclusions.

24

O-5

FeLIX is a restriction factor for mammalian retrovirus infection
Didik Pramono (1) (2), Dai Takeuchi (1), Masato Katsuki (1), Loai AbuEed (1) (2), Dimas Abdillah (1) (2),Tohru Kimura (3), Junna Kawasaki (4), Ariko Miyake (1) (2), Kazuo Nishigaki (1) (2)(1)Laboratory of Molecular Immunology and Infectious Disease, The Joint Graduate School of Veterinary Medicine, Yamaguchi
University, Yamaguchi, Japan(2)Research Institute for Cell Design Medical Science, Yamaguchi University, Yamaguchi, Japan.
(3)The Joint Graduate School of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
(4)Graduate school of medicine, Chiba University, Japan.

Retroviruses are unique in using reverse transcriptase to convert RNA genomes into DNA, infecting germ cells,
and transmitting to offspring. Endogenous retroviruses (ERVs) are remnants of ancestral viral infections. Feline
leukemia virus (FeLV) is an exogenous and endogenous retrovirus in domestic cats. It is classified into several
subgroups (A, B, C, D, E, and T) based on viral receptor interference properties or receptor usage. ERV-derived
molecules benefit animals, conferring resistance to infectious diseases. However, the soluble protein encodedby the defective envelope (env) gene of endogenous FeLV (enFeLV) functions as a co-factor in FeLV subgroup
T infections. Therefore, whether the gene emerged to facilitate viral infection is unclear. Based on the properties
of ERV-derived molecules, we hypothesized that the defective env genes possess antiviral activity that would
be advantageous to the host because FeLV subgroup B (FeLV-B), a recombinant virus derived from enFeLV env,
is restricted to viral transmission among domestic cats. When soluble truncated Env proteins from enFeLV were
tested for their inhibitory effects against enFeLV and FeLV-B, they inhibited viral infection. Notably, thisantiviral machinery was extended to infection with the Gibbon ape leukemia virus (GALV), Koala retrovirus A(KoRV-A), and Hervey pteropid gammaretrovirus (HPG). Although these viruses used feline phosphatetransporter 1 (fePit1) and phosphate transporter 2 as receptors, the inhibitory mechanism involved competitive
receptor binding in a fePit1-dependent manner. The shift in receptor usage might have occurred to avoid theinhibitory effect. In particular, this finding may explain why FeLV-B transmission is not observed among
domestic cats. ERV-derived molecules can benefit animals in an evolutionary arms race, highlighting the
double-edged sword nature of ERVs. Overall, these findings highlight the possible emergence of soluble
truncated Env proteins from enFeLV as a restriction factor against retroviral infection and will help indeveloping host immunity and antiviral defense by controlling retroviral spread. In particular, these findings
may involve mechanisms of intra- and interspecies transmission among animals, including in humans.
Keywords: endogenous retrovirus, feline leukemia virus, restriction factor, mammalian retrovirus

25

O-6

Development of an AI model for predicting antigenic properties from amino acid sequence of
dengue virus envelope protein
Kento Mori (1), Tomokazu Tamura (1) (2) (3), Rigel Suzuki (1) (2), Saori Suzuki (1) (2), Takasuke Fukuhara(1) (2) (3) (4)(1)Department of Microbiology and Immunology, Faculty of Medicine, Hokkaido University, Japan(2)Institute for Vaccine Research and Development (IVReD), Hokkaido University, Japan(3)One Health Research Center, Hokkaido University, Japan(4)Laboratory of Virus Control, Research Institute for Microbial Diseases, Osaka University, Japan

Dengue virus (DENV), transmitted by mosquitoes, represents a significant public health concern. Dengue fever
can escalate into severe conditions such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).
A major factor in the severity of DENV infections is antibody-dependent enhancement (ADE), which occurs
when antibodies generated during a primary infection or through vaccination with one DENV fail to neutralize
a subsequent infection by another DENV with distinct antigenic properties. Hence, a comprehensive
understanding of antigenic variations among DENV is crucial. Katzelnick, L. C. et al. (Science, 2021)
characterized 412 DENV strains using plaque reduction neutralization test with a 50% neutralization cutoff(PRNT50) assays on mosquito cells (Aedes albopictus C6/36). They used a panel of antisera against of a diverse
DENV strains that were obtained from 20 African green monkeys (Chlorocebus sabaeus). Although these assays
help understand antigenic diversity, the outcomes are not always practical for all virus variants due to theongoing emergence of diverse DENV strains.
To address these challenges, we developed an AI model combining deep learning and machine learningcomponents. The deep learning component uses Meta's ESM-2 model to transform amino acid sequences of the
DENV envelope protein, which including all serotypes, into mathematical representations (tensors). We
enhanced ESM-2's performance for DENV related tasks by performing domain adaptation with clustered DENV
sequences from NCBI, using a 95% identity threshold. These tensors were input into a machine learningcomponent to predict neutralization titers. We trained an ensemble learning model, combining XGBoost andRandomForest algorithms, using the data from their previous report. XGBoost is suitable for structured data,
while RandomForest builds multiple decision trees for accurate predictions. Optuna was used for
hyperparameter tuning, and StratifiedKFold cross-validation ensured robust model evaluation. In comparison
with epidemiological surveys conducted in Thailand, our AI model demonstrated the capacity to assess temporal
alterations in sequences consistent with the actual progression of epidemics. Consequently, our AI model
achieved a root mean square error of 0.08 and an R2 correlation coefficient of approximately 0.87, predicting
PRNT50 titer scores from amino acid sequences.
In conclusion, our study demonstrates that the model is potential to streamline the assessment of antigenic
properties and enhance vaccine development processes. Compared with the neutralization test, this approachallows prioritization of strains for phenotypic testing. Furthermore, the model's predictions could guide thedevelopment of vaccines that induce broadly neutralizing antibodies, offering protection against multiple DENV
serotypes.

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O-7

Association between deformation of nuclear membrane and innate immune response in West
Nile virus infection
Keisuke Maezono (1), Passawat Thammahakin (1), Kentaro Yoshii (2), Michiyo Kataoka (3), Tadaki Suzuki (3),Thuy Thi Ngoc Duong (1), Haruto Eguchi (1), Hiroaki Kariwa (1), Shintaro Kobayashi (1) (4)(1)Laboratory of Public Health, Faculty of Veterinary medicine, Hokkaido University(2)National research Center for the Control and Prevention of Infectious Diseases (CCPID), Nagasaki University(3)Department of Pathology, National Institute of Infectious Diseases(4)Institute of Vaccine Research and Development (HU-IVReD), Hokkaido University

Background and aims
In eukaryotic cells, structure of a nucleus is maintained by nuclear lamina that consists of polymerized two typesof lamin proteins. The polymerized lamin proteins are dissociated by phosphorylation, leading to nuclear
membrane deformation. The nuclear membrane plays a crucial role in host defense against viral infections byregulating nuclear-cytoplasmic localization of innate immune response factors, such as IRF-3 and NF-κB.
Capsid (C) protein, a structural protein of West Nile virus (WNV), is known to be localized in a nucleus, however,
the implications of the intranuclear localization for viral replication and cell homeostasis remain unclear. In thisstudy, we analyzed the effect of WNV infection on nuclear membrane structure and its association with innate
immune response.
Materials and methods
The structure of nuclear membrane in human SH-SY5Y neuroblastoma cells infected with WNV was analyzed
by transmission electron microscope. Localization of Lamin A/C, a component of nuclear lamina, was analyzed
in cells infected with WNV or expressing C protein by immunocytochemistry. Moreover, phosphorylation andpolymerization of Lamin A/C were analyzed by Phos-tag SDS-PAGE, which can separate phosphorylated
proteins, and by coimmunoprecipitation with Lamin B1, respectively. Then, subcellular localization of innate
immune response factors and IFN-β promoter activity were analyzed in cells expressing C protein byimmunoblotting and luciferase reporter assay.
Results
Nuclear membrane was deformed in cells infected with WNV. WNV infection and C protein expression induced
loss of continuity of Lamin A/C in the nucleus. C protein expression induced phosphorylation of Lamin A/C
and inhibited the polymerization of nuclear lamina. Additionally, C protein expression perturbed nuclearcytoplasmic localization of IRF-3 and NF-κB, and suppressed IFN-β promoter activity.
Conclusion
These results suggested that C protein induced nuclear membrane deformation by phosphorylation of laminproteins, resulting in mislocalization and loss of function of innate immune response factors.

27

O-8

Establishment of lethal mouse model of emerging tick-borne orthonairovirus infection forpathological analysis and validation of antiviral agents
Takuma Ariizumi (1), Keita Matsuno (2) (3), Koshiro Tabata (4), Yukari Itakura (4), Hiroko Kobayashi (1),Michihito Sasaki (1), Hirofumi Sawa (3) (4), Yasuko Orba (1) (3)(1)Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University, Japan(2)Division of Risk Analysis and Management, International Institute for Zoonosis Control, Hokkaido University(3)One Health Research Center, Hokkaido University(4)Institute for Vaccine Research and Development, Hokkaido University

Yezo virus (YEZV) is a novel orthonairovirus (family Nairoviridae) firstly isolated from a patient showing
febrile illness after tick bite in Hokkaido, Japan, in 2019. Patients with YEZV infection showed leukopenia,
thrombocytopenia and elevated liver enzyme levels after the onset of fever. YEZV has the potential to be aburden on public health, but no pathological analyses have been conducted and no effective treatment iscurrently available. In this study, we aim to establish a mouse model for understanding the pathogenesis of
YEZV and evaluation of therapeutic agents.
[Methods]
AG129 mice (type I/II IFN receptor double knockout mice) were inoculated intraperitoneally with 104 focus
formation units of YEZV, and their body weight changes and survival were monitored for 14 days. Blood andtissue samples were collected at 2, 4, and 6 days post-infection (dpi) for virus titration and pathological analysis.
To evaluate the anti-YEZV effect of favipiravir, YEZV-infected AG129 mice were orally treated with favipiravir(100 or 300 mg/kg) once a day from 1 hour post-infection to 3 dpi.
[Results]
AG129 mice inoculated with YEZV showed body weight loss with 100% mortality up to 6 dpi. The liver tissue
structure was disrupted with hepatocyte degeneration and infiltration of inflammatory cells at 4 and 6 dpi. In
the spleen, follicular structures were obscured at 4 and 6 dpi. In immunohistochemistry (IHC) analysis, positive
signals of YEZV antigen were diffusely observed in liver and spleen at 2 and 4 dpi and were decreased at 6 dpi.
The viral titer in each organ was highest at 2 dpi and decreased after 4 dpi.
YEZV-infected mice treated with 100 or 300 mg/kg/day of favipiravir showed a survival rate of 57% or 100% ,respectively. No clear tissue damage was observed in the liver and spleen of mice treated with favipiravir at 4
dpi. In IHC analysis, positive signals for viral antigens detected in the liver and spleen of mice treated withfavipiravir were significantly less than those of mice treated with vehicle. Virus titers in the liver and spleen ofmice treated with favipiravir were below the limit of detection.
[Discussion]
YEZV proliferated mainly in the liver and spleen, resulting in lethal hepatitis. In this mouse model,
administration of favipiravir rescued YEZV-infected mice from lethal infection by inhibiting virus replicationin the liver and spleen. This animal model could be applied to the evaluation of therapeutic agents against YEZV
infection.

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O-9

Upscaling Viral Infectious Disease Monitoring and Surveillance with Metagenomic NextGeneration Sequencing Enhanced by a Group Testing Algorithm (mEGA)
Maryam Sani Lawal (1) (2), Kyoko Hayashida (1) (2), Tatsuki Sugi (1) (2), Chikwe Iheakweazu (3), Chinwe
Ochu (4), Adesuyi Omoare (5), Junya Yamagishi (1) (2)(1)Division of Collaboration and Education, International Institute for Zoonosis Control , Hokkaido University, Japan.
(2)Graduate School of Infectious Diseases, Hokkaido University(3)WHO Hub for Pandemic and Epidemic Intelligence, World Health Organization(4)Planning , Research and Statistics, Nigeria Centre for Disease Control and Prevention(5)National Reference Laboratory, Nigeria Centre for Disease Control and Prevention

Metagenomic next-generation sequencing (mNGS) is transforming pathogen surveillance by enabling
comprehensive detection of pathogens. Despite its potential, the high cost presents significant challenges forwidespread application in routine monitoring efforts, especially in low-income countries where zoonotic andreemerging infections are most prevalent.
This study aims to expand the capacity of viral infectious disease monitoring and surveillance by optimizing
mNGS, enhanced by a group testing algorithm (mEGA) to improve the efficiency and cost-effectiveness of
mNGS by significantly reducing the number of sequencing libraries from 4n to 4n.
Using mEGA, 512 serum samples collected from febrile patients in Nigeria negative for Yellow fever, Measles,
Mpox and Lassa virus PCR tests were reduced to just 20 pools. A viral enriched Illumina sequencing library
was prepared by incorporating a comprehensive viral enrichment panel (Twist bioscience). This process
involved RNA purification, production of an indexed double-stranded DNA library, followed by viral probehybridization for enrichment. Bioinformatic analysis was performed for quality control, host decontamination,
and alignment to database.
As a result, mEGA effectively identified four viral pathogens of high public health significance- Lassa, measles,
hepatitis B, and human parvovirus B19 viruses in the pooled samples and successfully traced them back to their
original sample sources. Using viral specific primers, we validated the presence of mEGA-identified viruses by
qPCR.
The previous limitation of mEGA was the small percentage of viral reads, but with modified viral-enriched
mEGA, we achieved a 1850-fold increase in viral reads. Another limitation of mEGA was its inability to traceviruses when two or more samples contained the same virus. We addressed this by tracing hepatitis B virus tomultiple samples using genomic polymorphism. Upon alignment of mEGA-obtained Lassa viral sequence toreference sequences, we observed low similarity (81.022%) to the Refseq Lassa virus genome(GCA_000851705.1). It is known that the genetic diversity of Lassa virus is quite large; however, the Lassa testin Nigeria targets only one genotype. This explains our detection of Lassa virus in a negative sample, provingthat our method can detect unexpected viruses, including emerging variants undetectable by current targeted
PCR techniques. Implementation of mEGA demonstrated a substantial cost-reduction for library preparationand sequencing, from $13,000 for 512 samples to $3,500 for 20 pools, while still maintaining high sensitivity.
Our approach is applicable to a large number of samples across human, animal, and environmental sectors thatcomprise One Health: therefore, promising for pandemic prevention, preparedness, and response.

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O-10

Transport of dsRNase into vesicle packets facilitates the development for the treatment of West
Nile virus infection
Haruto Eguchi (1), Keisuke Maezono (1), Passawat Thammahakin (1), Kazuhiro Ishibashi (2), Kentaro Yoshii(3), Hiroaki Kariwa (1), Shintaro Kobayashi (1) (4)(1) Laboratory of Public Health , Faculty of Veterinary Medicine, Hokkaido University(2) Division of Plant Molecular Regulation Research, Institute of Agrobiological Sciences, NARO
(3)National Research Center for the Control and Prevention of Infectious Diseases, Nagasaki University(4)Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University

West Nile virus (WNV) causes encephalitis, but no specific treatments for WNV infection has been developed
for humans. Viral proteins induce invagination of ER membrane and form vesicle packets (VPs) in WNVinfected cells. The VPs are known to be site of viral genome replication and generate a long double-stranded
RNA (dsRNA). It was reported that fusion protein of dsRNase and plant host factor localized in VPs inhibited
multiplication of plant viruses. In this study, we examined the effect of fusion protein of dsRNase, degradingflaviviral dsRNA, and Heat shock protein 90 (HSP90) which was reported to be localized in VPs on WNV
infection to develop a treatment for WNV infection.
Viral dsRNA extracted from WNV-infected cells was incubated with RNase III-like protein 2 (RTL2) as arecombinant protein and the degradation was evaluated by electrophoresis. Intracellular localization of fusion
protein of RTL2 and HSP90 (RTL2-HSP90) was analyzed by immunocytochemistry. The effect of RTL2-HSP90
on WNV multiplication was investigated by plaque assay and immunoblotting in WNV-infected cells.
Recombinant RTL2 degraded flaviviral dsRNA. RTL2-HSP90 was localized in VPs of WNV-infected cells. In
addition, the expression of RTL2-HSP90 decreased viral titers and expression levels of viral protein in WNVinfected cells.
These results suggested that transport of RTL2 into VPs by fusion with VPs-locating protein, HSP90, results inantiviral effect on WNV infection.

30

O-11

Emergence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal
Complex 398 through Animal Trading in Thai Pig Population
Pawarut Narongpun (1), Chie Nakajima (1), Jeewan Thapa (1), Yasuhiko Suzuki (1), Pattrarat Chanchaithong(2), Junya Yamagishi (3), Narong Nuanmuang (4), Pimlapas Leekitcharoenphon (4), Frank M. Aarestrup (4)(1)Division of Bioresources, International Institute for Zoonosis Control, Hokkaido University(2)Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University(3)Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University(4)Research Group for Genomic Epidemiology, National Food Institute, Technical University of Denmark

The majority of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among pigs in
Asian countries belongs to clonal complex 9 (CC9). However, in 2019, a new emergence of LA-MRSA CC398
was identified, showing a concerning 87.5% incidence rate among pig herds in Thailand. Additionally, coregenome single nucleotide polymorphism (SNP)-based analyses confirmed zoonotic transmission of LA-MRSA
CC398 between pigs and husbandry workers on several farms. So far, the possible pathways contributing to theemergence of LA-MRSA CC398 have not been examined. Therefore, this study aimed to identify the original
source of LA-MRSA CC398 in Thailand and determine how it was introduced into the Thai pig population.
Thirteen genomes of LA-MRSA CC398 isolated from pigs and workers in central Thailand were sequenced
using Illumina MiSeq and PromethION 2 Solo and assembled using a hybrid approach. We also received fivedraft genomes of Thai LA-MRSA CC398 identified in pork and one slaughtered pig. Additionally, a total of
1,197 draft genomes of S. aureus CC398 from 25 countries, previously deposited in the Pathogen Watch
database during 1998-2021, were downloaded. Subsequently, quality control of 1,215 sequencing data wasconducted using the FoodQC pipeline before the phylogenetic reconstruction using maximum-likelihood andBayesian approaches. Based on the maximum-likelihood phylogeny, Thai S. aureus isolates with distinct
genotypes were placed into different sub-clades. All Thai S. aureus isolates carrying SCCmec type V were highly
related to Danish isolates from humans and cattle. Conversely, the strains with a composite SCCmec element
resided together in a discrete sub-clade. Bayesian inference revealed that CC398-SCCmecV isolates were likely
introduced into Thailand during 2000-2003, followed by pig-to-pig and pig-to-human transmission in various
provinces. In addition, CC398 strains with a composite SCCmec island emerged three years later. According to
the Ministry of Commerce of Thailand, Denmark has been one of the main exporters of breeding pigs since2003, with total imports valued at an average of $1 million per year. This study suggests that LA-MRSA CC398
may have been introduced into Thai pig production through animal trading.

31

O-12

Sex Dependent Intergenerational Effects of Lead in Mouse Model.
Nelly Banda (1), Nyein Chan Soe (1), John Yabe (2) (3), Rio Doya (1), Yohannes Yared Beyene (1), Yoshinori
Ikenaka (1) (4) (5) (6), Mayumi Ishizuka (1), Shouta Nakayama (1)(1)Laboratory of toxicology, Department of Graduate studies, faculty of veterinary Medicine, Hokkaido University, Japan(2)School of Veterinary Medicine, University of Zambia.
(3)School of Veterinary Medicine, University of Namibia.
(4)School of Environmental Sciences and Development, North-West University, South Africa(5)Translational Research Unit, Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Hokkaido University.
(6)One Health Research Center, Hokkaido University

Lead contaminated soils remains one of the major sources of lead (Pb) exposure with high impact on fertility inboth males and females, pregnancy outcomes and child brain development. We investigated inhalation andingestion as medium of Pb exposure via soil in male and female mice. Exposure was then withdrawn prior tomating thus we investigated the lingering effects of Pb on fertility in F0 generation and neurologicaldevelopment in F1 generation. Lead exposure reduced conception rates with a cumulative decrement being
recorded when both males and females are exposed. Male testicular gene expression revealed reduction in
fertility was caused by oxidative stress. Cessation of exposure did not alleviate neurological deficits in F1
generation, as offspring of exposed individuals performed poorly in the water Morris test when compared tocontrol. The severity of the effects was also parent-dependent and sex-dependent. Paternal Pb exposure also
caused some neurological and motor deficits, with effects being more pronounced in female offspring. The
female offspring of paternal Pb exposure showed gene expression of changes occurring more in prefrontal cortex
than in hippocampus, a trend recorded in their sires. This trend suggests that Pb neurological effects may be Xchromosome linked.

32

O-13

SARS-CoV-2 infection surveillance in Hokkaido wildlife
Anastasiia Kovba (1), Naganori Nao (2) (3) (4), Michito Shimozuru (4) (5), Mariko Sashika (5), Chihiro
Takahata (5), Kei Sato (6), Keiya Uriu (6), Masami Yamanaka (7), Masanao Nakanishi (7),Genta Ito (7), Mebuki Ito (8), Miku Minamikawa (8), Kotaro Shimizu (1), Kouichi Goka (9),Manabu Onuma (9), Yuma Ohari (10), Keita Matsuno (2) (4) (10), Toshio Tsubota (5)(1)Laboratory of Wildlife Biology and Medicine, Graduate School of Veterinary Medicine, Hokkaido University(2)Institute for Vaccine Research and Development, HU-IVReD, Hokkaido University(3)Division of International Research Promotion, International Institute for Zoonosis Control, Hokkaido University(4)One Health Research Center, Hokkaido University(5)Laboratory of Wildlife Biology and Medicine, Faculty of Veterinary Medicine, Hokkaido University(6)Division of Systems Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University ofTokyo(7)Shiretoko Nature Foundation, Hokkaido(8)Laboratory of Wildlife Biology and Medicine, School of Veterinary Medicine, Hokkaido University(9)National Institute for Environmental Studies, Ibaraki(10)Division of Risk Analysis and Management, International Institute for Zoonosis Control, Hokkaido University

SARS-CoV-2, the causative agent of COVID-19, has transmitted to various domestic and wildlife species acrossthe globe, raising concerns about virus adaptation to new animal hosts. Particularly concerning are thedocumented cases of virus spillover to wild white-tailed deer populations in North America, followed by deerto-human transmission (spillback). This highlights the importance of effective wildlife infection monitoring toprotect public health. Therefore, we assessed the exposure of wild animals to SARS-CoV-2 in Hokkaido, where
the lack of predators, field abandonment, and increased human acclimatization have resulted in frequent wildlife
invasions of urban areas.
Nasal swabs and serum were collected from raccoons, sika deer, brown bears, and raccoon dogs throughout
Hokkaido from 2022 to 2024 for viral genetic and serological testing, respectively. All 547 nasal swabs tested
negative in the SARS-CoV-2 N RT-qPCR test. Among 550 serum samples, 20 raccoons, 1 brown bear, and 4
sika deer tested positive in SARS-CoV-2 N protein ELISA but negative in other serological tests such as thevirus neutralization test, immunofluorescence assay, and lentivirus-based pseudovirus neutralization test,
suggesting the lack of SARS-CoV-2 specific antibodies in these samples.
In conclusion, it is unlikely that SARS-CoV-2 transmission occurred from humans to wild animals in Hokkaido
during the study period. However, considering the worrying reports of SARS-CoV-2 in wild deer in North
America, continuous wildlife monitoring for virus transmission is needed. Identifying the factors contributing
to wild animals' susceptibility to infection and virus transmission would also lead to better public healthprotection strategies and wildlife management efforts in the global pursuit to achieve One Health.

33

P1-1

Expression of adrenoceptor alpha 1 in the obstructed ureters of feline urolithiasis cases
Sao Oe (1), Md. Zahir Uddin Rubel (1), Masaya Hiraishi (1), Kazuhisa Oyamada (2), Hiroshi Aoki (3), Yuki
Otani (4), Teppei Nakamura (5), Osamu Ichii (1) (4)(1)Laboratory of Anatomy, Department of Basic Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University(2)Matsubara Animal Hospital.
(3) Aoki Animal Hospital.
(4)One Health Research Center, Hokkaido University.
(5)Laboratory of Laboratory Animal Science and Medicine, Department of Applied Veterinary Sciences, Graduate School of Veterinary
Medicine, Hokkaido University.

Ureteral obstruction is a common disease among humans and animals. Movement of urinary stones from thekidney to the ureter can cause ureteral obstruction, which can subsequently lead to inflammation and fibrosis.
Adrenoceptor alpha 1 (ADRA1) antagonism, which aims at smooth muscle relaxation, is used as a treatment
strategy for ureteral obstruction. However, the expression of ADRA1 in the feline ureters has not been fully
investigated. The present study investigated ADRA1 expression and its association with pathological conditions
by examining feline ureters dissected during surgical treatments.
Ureters were collected from clinical cases with ureteral obstruction treated surgically in animal hospitals (n =37 for RNA analysis, n = 4 for histological analysis). The ureters from obstructed lesion to the ureterovesicaljunction were equally cut into three parts, and the obstructed part (kidney side) and the distal part (bladder side)
were used in the present study. The mRNA expression of ADRA1 subtypes (ADRA1A, ADRA1B, ADRA1D) was
examined by RT-PCR and electrophoresis. mRNA expression of ADRA1, smooth muscle actin (ACTA2), andrepresentative collagens (type I: COL1A2, type III: COL3A1) was quantified by TaqMan PCR. The mRNA
expression levels of these genes were compared between dissected parts, and their correlations with age or bodyweight were also examined. Histological ureter sections were stained by picrosirius red to quantify the collagenfibers under polarized light.
All mRNAs of the examined molecules were detected in both parts of the ureter. Feline ADRA1A has seventranscript variants (TV1-7); TV3 and TV7 were detected with individual TaqMan probes, while the other TVs
were detected with a common probe. ADRA1A expression levels, except for TV3, were significantly lower inthe obstructed part compared to the distal part. In the distal part, mRNA expression levels of TV3 and TV7 werepositively correlated with COL1A2 and age respectively, whereas the expression levels of other TVs were
negatively correlated with body weight. Collagen staining visualized fibers rich in thick type I collagen as redcolor, and fibers rich in thin type III collagen as green color. The area of the former fibers in the adventitia wassignificantly larger in the obstructed part compared to the distal part, indicating fibrosis progression.
Thus, the feline ureters maintained the ADRA1 expression even under obstructed condition, and our results
suggest that the expression level of each ADRA1A variant is altered with pathological condition, and age, bodyweight, and collagen expression levels would be relevant factors.

34

P1-2

Evaluation of the antimicrobial effect of gepotidacin on wild type and mutant Salmonella
Typhimiruium
Charlotte Poussier (1), Pondpan Suwanthada (1), Jeewan Thapa (1), Chie Nakajima (1) (2) (3),Yasuhiko Suzuki (1) (2) (3)(1)Division of Bioresources, Hokkaido University, International Institute for Zoonosis Control, Japan(2)International Collaboration Unit, Hokkaido University, International Institute for Zoonosis Control, Japan(3)Hokkaido University Institute for Vaccine Research & Development, Hokkaido University, Japan

Introduction Non-typhoidal Salmonella (NTS) is a primary cause of acute gastroenteritis. Although these
infections are typically mild and self-limiting, NTS can also cause bloodstream infections and serious lifethreatening invasive diseases, and improper treatment can cause fatal end. Fluoroquinolones are the first-choicetreatment for Salmonella infections in adults. These broad-spectrum antimicrobials target bacterial type II
topoisomerases: DNA gyrase and topoisomerase IV. In the last decade, increasing reports of fluoroquinolone
resistance have impeded the treatment of Salmonella infections, exacerbating the high fatality rates of invasive
NTS diseases. Fluoroquinolone resistance is characterized by mutations in the quinolone resistance-determining
region (QRDR) in target genes gyrA, gyrB, parC and parD, encoding DNA gyrase subunits (GyrA and GyrB)
and Topoisomerase IV subunits (ParC and D). A single-point mutation in gyrA induces fluoroquinolone reduced
susceptibility and combinations of mutations or coexistence of mutations in target genes with plasmid mediatedquinolone resistance genes cause fluoroquinolone resistance. The continued emergence of resistance has createdan urgent need for new and effective drugs against Salmonella infections. Novel bacterial topoisomerase
inhibitors (NBTIs) is one of the new promising classes of antimicrobial agents. Gepotidacin, the first-in-classof the NBTIs, operates similarly to fluoroquinolones by selectively inhibiting bacterial DNA replication throughinteraction with type II topoisomerases. It inhibits both DNA gyrase and topoisomerase IV in a proportionate
manner. Although this drug is approved for the treatment of uncomplicated urinary tract infections caused byuropathogens, it has yet to be evaluated for other indications.
Material and methods The Minimum Inhibitory Concentrations (MICs) of gepotidacin against wild-type (WT)and mutant Salmonella Typhimurium was evaluated by in vitro micro broth dilution assays and compared withthose of ciprofloxacin (CIP). We used mutant strains carrying amino acid substitutions D87Y, G81C and S83F
in GyrA.
Results The comparative study showed that while gepotidacin exhibits higher MIC values, the increase in MIC
values from WT to mutant strains is strikingly lower compared to CIP. Specifically, the MICs of gepotidacin
against D87Y and S83F mutants just doubled, while those of CIP for these mutant strains increased by 16-foldand 32-fold, respectively. Moreover, the MIC of gepotidacin against G81C mutant was as same as against the
WT strain, while that of CIP increased by 16-fold. These findings suggest that gepotidacin may offer a more
effective treatment option for Salmonellosis resistant to current fluoroquinolones.

35

P1-3

Influence of photoperiod and ambient temperature on adipose tissue in Hamsters
Khaled Mahmud Sujan (1) (2), Anju Tsukada (1), Zibo Yang (1), Bong Soo Seok (1), Xu Shucheng (1),Mohamed Elfeky (1), Mira Kato-Suzuki (1), Yuko Okamatsu-Ogura (1)(1)Laboratory of Biochemistry, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Department of Physiology, Faculty of Veterinary Science, Bangladesh Agricultural University, Bangladesh

Abstract:
Hibernation is an adaptive strategy characterized by drastic suppression of metabolism, activity, and bodytemperature which allows animals to survive during periods with little or no food availability. Syrian hamsters
are facultative hibernators that can be experimentally induced hibernate when they are placed under the shortday and cold condition typical of winter, hibernation initiates following a pre-hibernation period lasting to 2-3
months. During this pre-hibernation period, brown adipose tissue (BAT), which is responsible for heatproduction, and white adipose tissue (WAT), which is involved in energy storage, are assumed to undergofunctional and quantitative changes to prepare for hibernation, yet detailed information remains limited.
This study was conducted to investigate changes in the adipose tissues of Syrian hamsters under the short-day
and cold condition. Hamsters were divided into two groups (n=3): the control group was exposed to light: dark
12:12 hour at 23°C room temperature, and the short-day cold group (SD-Cold) was exposed to light: dark 8:16
hour at 8°C for 10 weeks. Compared to the control group, the SD-Cold group experienced a significant decreasein body weight and a significant increase in food intake. At the end of the 10-week exposure period, hamsters
were euthanized, and adipose tissues were collected.
While BAT weight did not differ significantly between the groups, the SD-Cold group showed higher mRNAand protein expression of UCP1, a key thermogenic molecule in BAT, along with lower lipid droplets in brown
adipocytes compared to the control group, indicating enhanced activity of BAT. The weights of white adipose
tissue in the inguinal (i-WAT), peri-gonadal (g-WAT), and retroperitoneal (retro-WAT) depots were significantly
lower in the SD-Cold group than in the Control group. The size of adipocytes in the i-WAT, g-WAT, and retroWAT was significantly smaller than that in the Control group, suggesting the utilization of stored lipid forthermogenesis.
In conclusion, exposure to short-day and cold induced significant morphological and functional changes in both
BAT and WAT of Syrian hamsters. The effects of photoperiod, and ambient temperature, respectively, will beanalyzed and discussed during the presentation.

36

P1-4

The intervention of immune suppression status in sheep experimentally infected with bovine
leukemia virus
Wisa Tiyamanee (1), Tomohiro Okagawa (2), Mari Ikehata (1), Hayato Nakamura (1), Naoya Maekawa (2),Shiro Murata (1) (2), Kazuhiko Ohashi (1) (2) (3), Satoru Konnai (1) (2) (4)(1)Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Japan(3)International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Japan(4)Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Japan

Bovine leukemia virus (BLV) is a retrovirus that infects B cells of ruminants and can lead to lymphoma in some
infected cattle after a long latent period. Our previous studies have shed light on the status of T-cell exhaustionof BLV-naturally infected cattle, which is mediated by immunoinhibitory molecules such as programmed death1 (PD-1), PD-ligand 1 (PD-L1), and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3).
However, the understanding of immune exhaustion throughout all stages of BLV infection remains a challenge
due to the long latency of the virus in cattle. Sheep is one of the optimal experimental models for BLV infection
since they develop the clinical disease in a shorter period with a higher frequency compared to natural hosts. In
this study, by using sheep as an experimental model, we aimed to elucidate the kinetics of immune suppression
during BLV infection and intervene it with immunoinhibitory receptor blockade.
Expressions of PD-L1 on B cells and TIM-3 on T cells were analyzed in peripheral blood mononuclear cells(PBMCs) isolated from BLV-experimentally infected sheep. PBMCs were then cultured with anti-bovine PDL1 monoclonal antibodies (mAbs) and anti-bovine TIM-3 mAbs under the stimulation with concanavalin A(ConA). The cultivated PBMCs were subsequently analyzed for the expression of activation-induced markers(AIMs) in T cells using flow cytometry, and the cytokine production was analyzed by using the culture
supernatants.
The expression levels of TIM-3 and PD-L1 were upregulated in PBMCs in sheep during BLV experimental
infection. Moreover, the blockade using anti-TIM-3 mAb increased CD25 expression on CD4+ T cells and CD69
expression on CD8+ T cells in cultured PBMC. Interferon-gamma (IFN-γ) production was also increased in
TIM-3 inhibition, and was higher in a combined blockade of TIM-3 with PD-L1.
These findings suggest that the immunoinhibitory molecules, PD-L1 and TIM-3, are involved in the T-cell
exhaustion of BLV infection in sheep. Furthermore, the inhibition of the TIM-3 and PD-L1 pathway showsenhanced immune responses which is essential for antiviral immunity. Further analysis of immune exhaustion
status and antiviral effects should be elucidated by analyzing other cytokines and functions of T cells in BLVinfected sheep. These studies by using sheep as a model will enhance our understanding of immune exhaustionin cattle and contribute to developing the therapeutic intervention for chronic infectious diseases.

37

P1-5

First report of canine leishmaniasis in Malawi: Emerging neglected zoonotic disease
Boniface Onesmo Chikufenji (1) (2), Elisha Chatanga (3), Tatsuki
Yamagishi (1)Sugi (1), Kyoko Hayashida (1), Junya(1)Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Japan(2)Department of Animal Health and Livestock Development, Ministry of Agriculture, Malawi(3)Department of pathobiology, Faculty of Veterinary Medicine, Lilongwe University of Agriculture and Natural Resources, Malawi

Abstract
Leishmaniasis is one of the zoonotic Neglected Tropical Diseases (NTDs) on the World Health Organization(WHO) list and is caused by a protozoan parasite of Leishmania species. It poses serious health risks to bothanimals and humans globally. The dog is the main reservoir of Leishmania infantum, the causative agent ofcanine leishmaniasis and human visceral leishmaniasis. In Africa, more emphasis has been given to studies onruminants, thereby neglecting domestic dogs, which are in large numbers, live in close contact with humans,
and are potential transmitters and reservoirs of NTDs. Zoonotic leishmaniasis caused by L. infantum is foundin a large part of the world, including the Mediterranean basin, Middle East, Central Asia, China, and Central
and South America, while Sub-Saharan Africa has been considered free from the disease for a long time.
However, an autochthonous canine leishmaniasis case was recently reported in Zambia, suggesting that thedisease has emerged in the area or that its presence had been neglected for a long time. There is no data on the
disease in Malawi, which shares boundaries with Zambia. This study, therefore, aimed to investigate thepresence of L. infantum in canine populations in Malawi for the first time. A total of 257 canine blood samples
were collected in two districts of Malawi, Nkhotakota and Mchinji districts. PCR screening of the DNA using
primers targeting the 18S rRNA region was conducted. Among 100 and 157 dog blood DNA samples fromNkhotakota and Mchinji, 5 (5.0%) and 18 (11.5%) respectively, were PCR positive for L. infantum. The finding
highlights the need for extensive surveillance, targeted control measures and further research to ascertain the
zoonotic potential in humans.
As a next step, we will detect IgG antibodies against the crude lysate of the parasite (LdSLA) and therecombinant antigen LinJ14.1160r4 (rK39) specific for L. donovani complex using ELISA. Later, we will also
investigate other NTDs in dogs, such as trypanosomiasis and rickettsiosis. All three zoonotic diseases show
clinical symptoms like those of malaria, making diagnosis difficult in humans. The baseline data of these
diseases will be important for controlling zoonotic diseases and improving public health.
Keywords: Dogs, Leishmania, Malawi, PCR, Surveillance, zoonosis

38

P1-6

Assessment of Echinococcus multilocularis eggs infectivity in the fox feces collected from theroadside in Nakashibetsu, Hokkaido
Kanyatip Sangarun (1), Naoki Hayashi (1), Kouguchi Hirokazu (2), Takaya Hoketsu (1), Tsutsumi Taiga (1),Kinpei Yagi (1), Ryo Nakao (1), Nariaki Nonaka (1)(1)Laboratory of Parasitology, Graduate School of Infectious Disease, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Hokkaido Institute of Public HealthAlveolar echinococcosis caused by Echinococcus multilocularis is a serious zoonosis found in Hokkaido. The
lifecycle of this parasite is maintained by carnivores (definitive hosts) and wild rodents (intermediate hosts).
The parasitic eggs are distributed into environment through feces of definitive hosts. The presence of
Echinococcus eggs in fox feces has been assessed using PCR and fecal egg-flotation method to evaluate
environmental contamination. However, the infectivity of these eggs has not been clarified. This study analyzes
the infectivity of E. multilocularis eggs in fox feces collected in the field.
A total of 84 and 106 fox fecal samples were collected from roadsides at Nakashibetsu in June 2023 (J-23) and
April 2024 (A-24), respectively, and PCR and the fecal egg-flotation method were applied to these samples. The
parasite eggs were purified from 23 and 20 samples of J-23 and A-24, respectively, and then orally inoculated
into DBA/2 mice. The number of cysts developed in the liver was counted at 4 weeks post-inoculation.
The PCR on copro-DNA showed positive in 39% (33/84) and 41% (43/106) of J-23 and A-24, respectively.
Cysts were developed in 61% (14/23) and 70% (14/20) in the egg-inoculated mice with J-23 and A-24 samples,
respectively, with each infected mouse harboring 1 to 56 cysts. The rate of inoculated eggs that could develop
into cysts ranged from 0 to 69%, with a median of 4%. These findings suggest that the infectivity of the parasite
eggs is efficiently maintained in the fox feces even under harsh environment condition such as those found on
roadsides.

39

P1-7

Evaluation of glycosylation of Otopetrin-1
Omi Sasaki (1), Saori Yano-Nashimoto (2), Soichiro Yamaguchi (2)(1)Laboratory of Physiology, Department of Basic Veterinary Sciences, Graduate school of Veterinary Medicine, Hokkaido University,Japan(2)Laboratory of Physiology, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University, Japan

Otopetrin-1 (OTOP1) was recently identified as a proton (H+) channel which is expressed in type III taste cells
and detects acidic stimuli. However, its post-translational modifications remain unknown. Glycosylation is atypical post-translational modification for many ion channels, playing various roles such as cellular localizationand functional regulation. Therefore, we investigated whether OTOP1 was glycosylated and how theglycosylation affected OTOP1 function by biochemical and physiological approaches.
Firstly, western blot analyses revealed that overexpressed mouse OTOP1 was detected as two bands. The
treatment of OTOP1 with PNGase F, which cleaves N-glycan, and the treatment of the cells with tunicamycin,
which inhibits N-glycan biosynthesis, revealed that the higher molecular weight band was N-glycosylated
OTOP1. N-glycans can be classified into three types: high-mannose type, hybrid type, and complex type. To
identify the type of the N-glycans, we utilized Endo H, which cleaves high-mannose type and some hybrid typeof N-glycans, and kifunensin, which inhibits biosynthesis of hybrid type and complex type of N-glycans. As thehigher molecular weight band was affected by Endo H but not by kifunensine, the N-glycans of OTOP1 weresuggested to be high-mannose type.
To identify the glycosylation sites, we made OTOP1 mutants whose candidate asparagine residues were mutatedto glutamine residues so that N-glycosylation did not occur. Using these mutants, it was revealed that the Nglycosylation sites of OTOP1 were the 238th and 251st asparagine residues.
The functional effects of the glycosylation were investigated by recording H+ currents using a whole-cell patchclamp method. The tunicamycin treatment or the double mutation of both 238th and 251st asparagine residues
made the current density of OTOP1 smaller significantly.
Finally, we performed a cell surface biotinylation assay to measure the OTOP1 expression level at the plasma
membrane. The results showed that the ratio of OTOP1 expression at the plasma membrane to that in thecytoplasm became significantly lower by the prevention of the N-glycosylations.
In conclusion, this study revealed that OTOP1 carried high-mannose type N-glycans at the 238th and 251st
asparagine residues. In addition, it was suggested that the glycosylation contributed toward maximizing thechannel activity of OTOP1 at the plasma membrane by increasing the targeting of OTOP1 to the plasma
membrane. These findings will improve the understanding on the regulation of OTOP1 function.

40

P1-8

Treatment with Colony Stimulating Factor-1 Receptor induces activation of microglia and isrelated to the survival of West Nile virus-infected mice
Passawat Thammahakin (1), Keisuke Maezono (1), Thuy Thi Ngoc Duong (1), Haruto Eguchi (1), Hiroaki
Kariwa (1), Shintaro Kobayashi (1) (2)(1)Laboratory of Public Health, Department of Preventive Veterinary Medicine, Faculty of Veterinary Medicine, Hokkaido University,
Japan(2)Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Japan.

West Nile virus (WNV) has emerged as a cause of viral encephalitis in human. However, the pathogenesis ofencephalitis remains unclear. The pathogenic involvement of microglia activated by WNV infection iscontroversial because of their heterogeneous function in different microglia phenotypes. The receptor for
Colony Stimulating Factor-1 (CSF-1R) is found on microglia and is known to be important for the pathogenesis
of neurodegenerative diseases. Colony Stimulating Factor-1 (CSF-1) and Interleukin-34 (IL-34) are the ligandsof CSF-1R, but they induce different microglia phenotypes. However, the effect of CSF-1 and IL-34 on
microglia phenotypes and the pathogenesis of WNV infection are unknown. In this study, to clarify the role ofmicroglia in WNV infection, the effects of these treatments on the pathogenesis were analysed using a mousemodel of WNV infection.
The mice inoculated with 5 pfu of WNV (NY99-strain) were treated with CSF-1, IL-34, or PBS after 1 day postWNV inoculation. All mice were monitored daily for behavior and body weight until 14 days post-WNV
inoculation. The mouse brain was collected and immunohistopathological analysis was performed with
TMEM119 antibody to investigate the activation of microglia.
In uninfected mice, the alternation of morphology and size of TMEM119-positive microglia was observed bytreatment of CSF-1, or IL-34. The survival rate of the WNV-infected mice treated with CSF-1 was lower andthat with IL-34 was higher than that with PBS. These results suggested that CSF-1 and IL-34 are involved in
the activation of different phenotypes of microglia and have different effects on the pathogenesis of WNV
infection. Currently, these effects are analysed in detail.

41

P1-9

Vaginal osteopontin receptive mechanisms in dairy cows to normalize the endometrial epidermal
growth factor concentrations
Jeremiah Buumba Chijikwa (1), Takashi Tanida (1), Kenichiro Sakaguchi (2) (3), Tomochika Sugiura (4),
Yojiro Yanagawa (5), Seiji Katagiri (5)(1)Laboratory of Theriogenology, Department of Clinical Studies, Graduate School of Veterinary Medicine, Hokkaido University, Japan(2)Laboratory of Laboratory of Veterinary Theriogenology, Faculty of Applied Biological Sciences, Gifu University, Japan(3)Division of Animal Medical Science, Center for One Medicine Innovative Translational Research, Gifu University, Japan(4)Laboratory of Theriogenology, School of Veterinary Medicine, Rakuno Gakuen University, Japan(5)Laboratory of Theriogenology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Japan[Introduction and Objectives] We have previously demonstrated that osteopontin (OPN), a seminal protein,restores the normal endometrial epidermal growth factor (EGF) profile in the uterus and fertility in dairy cows
when infused intravaginally in cattle. Although, the mechanism of OPN action to normalize EGF in the uterus
is unclear, it is speculated that its action is initiated through an immune mediated cascade triggered by binding
to its integrin and/or CD44 receptors. OPN contains multiple integrin and CD44 binding motifs and the presenceof its receptors in the vagina at estrus might influence treatment success. The present study seeks to determine
OPN receptor expression in calves, and in heifers and cows on the day of estrus.
[Materials and Methods] As a preliminary study, a total of 15 animals of Holstein breed (calves, heifers, andcows; 5 each) with an average age of 3, 15 and 48 months, respectively, were used. All animals were apparently
normal. Heifers and cows had normal estrous cycles, uterine cytology, and genital organs on ultrasonography.
The heifers and cows were synchronized for ovulation by hormonal treatment with gonadotropin releasing
hormone (GnRH) and prostaglandin F2 alpha (PGF) using the Ovsynch protocol and vaginal swabs werecollected from the dorsal wall of the vagina on the day of the subsequent estrus. Swabs from calves werecollected once without any synchronization. Expression of OPN receptors (Integrin subunits α4, αv, α9, β1, β3,β5, β6, β7 and CD44) was determined by quantitative PCR (qPCR) for the different groups.
[Results and Discussion] Expression of CD44 and the integrin subunits was consistently detected in the 3 groups
examined. There was also an apparent difference in fold expression among the different receptors, but statisticalsignificance of these differences was not determined. It will be necessary to establish fold changes in expressionof the detected genes as well as compare expression by group to determine variation in expression based on age
and reproductive status.
[Conclusion] Our study aims at establishing a baseline of OPN receptor expression during different
physiological stages of development and at estrus to establish candidates for OPN binding and action. This study
will elucidate the vaginal OPN receptive mechanisms in dairy cows to normalize the endometrial epidermal
growth factor (EGF) concentrations following treatment allowing for a wider treatment window.

42

P1-10Phylogenetic analysis of Glutathione S-transferase (GST) and N-acetyltransferase (NAT) in
Hyaenidae
Shiori Miyamoto (1), Mitsuki Kondo (2) (3), Ayuko Morita (1), Kanami Watanabe (1), Yoshinori Ikenaka (4),Shouta MM Nakayama (1), Mayumi Ishizuka (1)(1)Laboratory of toxicology, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)National Institute for Environmental Studies, Japan(3)Joint Faculty of Veterinary Medicine, Kagoshima University, Japan(4)One Health Research Center, Hokkaido University, Japan

Spotted hyenas, striped hyenas, and brown hyenas are scavengers that feed on dead carcasses. On the African
continent where hyenas live, there has been an increase in cases of poisoning and death of wild animals causedby environmental chemicals such as pesticides, and there is concern about chemical exposure and secondary
poisoning deaths caused by scavengers consuming poisoned corpses. Furthermore, conservation of hyenas isurgently needed due to their declining population and low genetic diversity. When implementing conservation
measures, it is important to assess the risk of chemical substances for each animal species, and xenobiotic
metabolizing enzymes are a necessary part of the risk assessment. Information on xenobiotic metabolizing
enzymes is also useful for predicting the pharmacokinetics of drugs administered in captivity, and thus
contributes widely to conservation. We have analyzed foreign metabolizing enzymes in the order Carnivora andcharacterized cytochrome P450 (CYP), glucanosyltransferase (UGT), and sulfotransferase (SULT) in hyaenas,
but we have not started work on other xenobiotic metabolizing enzymes mentioned above.
[Objective] To analyze and compare the species differences in xenobiotic metabolizing enzymes, mainly
Glutathione S-transferase (GST) and N-acetyltransferase (NAT), in hyaenas and their relatives, and to elucidate
the characteristics of xenobiotic metabolizing ability of hyaenas.
[Materials and Methods] Genome information on hyaenas and related species was obtained from a web-based
database, and candidate genes for GST and NAT were determined. Molecular phylogenetic analysis was
performed using the maximum likelihood method to create a phylogenetic tree from the determined sequences,and each enzyme was compared with those of closely related species.
[Future plan] In addition to GST and NAT, genes related to taste and olfactory receptors will be analyzed in the
future to characterize the biological defense mechanisms of hyaenas.

43P1-11

Exploration of novel marker proteins of M-specific cells responsible for antigen uptake inmucosal epithelial cells
Yurie Makino (1), Shinji Saito (1), Yukari Itakura (1), Gabriel Gonzalez (1), Hirofumi Sawa (1) (2) (3), Koshiro
Tabata (1)(1)Institute for Vaccine Research and Development, Hokkaido University, Japan(2)Division of International Research Promotion, International Institute for Zoonosis Control, Hokkaido University(3)One Health Research Center, Hokkaido University

In influenza virus infection, IgA antibodies play an important role in protecting against the viral infection on
upper respiratory mucosal tissue. Intranasal vaccines induce antigen-specific local IgA antibody responses on
upper respiratory mucosal epithelial cells, resulting in effectively preventing from the viral infection.
Additionally, the vaccines induce the antigen-specific systemic IgG responses, and the IgG antibodies work as
main protective agent against pulmonary infection by influenza virus. Therefore, the intranasal vaccines are
expected to be a next-generation vaccine that enables to acquire both protective IgA and IgG antibody responses
to influenza virus infection.
Current subcutaneous injectable split vaccines are subunit vaccines consisting with partial influenza virus
proteins. When protein-based subunit vaccines are administrated via intranasal route, the antigens are easily
digested by enzymes on the nasal mucosa. Consequently, no antibody responses sufficient to protect against theinfection is induced because of digestion of antigens. Previous studies have shown that mucosal adjuvants need
to be administered intranasally together with protein-based subunit antigens to maintain antigenicity andimprove immunogenicity. However, currently there is no approved-mucosal adjuvants in Japan. Thus, todevelop adjuvant-free intranasal vaccine, we focused on M cells, which contribute to the active uptake of foreign
antigens in mucosal tissues and play a central role in IgA antibody production. In this study, we propose thatbispecific IgA antibodies simultaneously recognizing both of protein antigens and M cell-specific protein
marker are employed as a drug delivery system (DDS) carrier, and the intranasal administration of an antigenantibody (bispecific IgA antibody) complex as a vaccine antigen.
Previously, it has been reported that IgA antibodies acquire tolerance of proteases by binding to secretorycomponents (SC). The DDS carrier, we develop in this study, also contains the SC. Therefore, its tolerance
allows the protein antigen to transport the protein antigen to M cells with keeping its intact antigenicity, resulting
in efficient induction of antibody responses on the nasal tissues.
Glycoprotein 2 (GP2) has been used as a M cell-specific marker. However, GP2 has both membrane-anchoredand secreted forms which may result in loss of M cell specificity. Thus, we attempt to find the other M cellmarker proteins, which are anchored on the surface M cells. This presentation will demonstrate the progressionof about research activities

44

P1-12Development of a Semi-comprehensive Detection Method for Paramyxoviruses and its
Validation in Indonesian Bats
Dela Ria Nesti (1) (2), Kyoko Hayashida (1), Tatsuki Sugi (1), Wayan T. Artama (2), Hery Wijayanto(3), Junya Yamagishi (1)(1)Departement of Collaboration and Education, International Institute for Zoonosis Control, Faculty of Veterinary Medicine, Hokkaido
University, Japan(2)Department of Bioresourses Technology and Veterinary, Vocational College, Universitas Gadjah Mada, Indonesia(3)Department of Anatomy, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia

An outbreak of zoonotic diseases is known as one of the worldwide threats. In Indonesia, they face a high riskof bat-borne virus outbreaks including paramyxoviruses due to abundant bat diversity and sustained hunting
and trading of wildlife. To mitigate the risk, understanding bat virome and their distribution is indispensable.
Therefore, universal detection methods that can simultaneously identify multiple viruses are promising. Here,
we developed a semi-comprehensive detection method integrating RT-PCR and multiplex sequencing with NGS.
The RT-PCR consists of three sets of degenerative primers: RMH (Respirovirus, Morbillivirus, Henipavirus,
and unclassified paramyxovirus), ARb (Avulavirus, Rubulavirus), and PNE (Pneumovirus andMetapneumovirus) covering viruses of Paramyxoviridae family. Additionally, multiplex sequencing wasestablished to increase the cost-efficiency by adding 8 selected nucleotides (index) to primers. To achieve thehighest sensitivity, semi-nested PCR was optimized. Target sequences from eight paramyxoviruses were cloned
into plasmids and used as templates for optimization. When the system was established, 270 bat samples fromtracheal and rectal swabs were used for validation. Sequencing was done by nanopore and Illumina platform.
Meanwhile, another pan-paramyxovirus PCR with PAR primer was applied for additional validation.
Collectively, seven novel sequences from 29 bat samples were successfully detected from both ARb and PAR
PCR systems and nucleotide similarity was 73-86%. Among them, ARb and PAR amplicons from the same
sample were similar to Tuhoko virus3-like sequence in one sample. The result was validated by bridged ampliconusing ARb forward-primer and PAR reverse-primer. Interestingly, tracheal and rectal swabs of the same bat wereidentified differently; Parainfluenza virus5-like sequence was detected from ARb amplicon; and Humanorthorubulavitus2-like sequence was detected from the PAR amplicon. Moreover, ARb amplicon with Tioman
virus-like sequence and PAR amplicon with Henipavirus-like sequence were found in the same samples. Thesesuggest presumable co-infection. The distribution of viruses is not unique to any specific bat species, but it
seems like fruit bats are more abundant in viruses. While Tuhoko virus3 and human orthorubulavirus2 cause
acute febrile illnesses in humans, Henipavirus has been reported with a high mortality rate. Even if theseparamyxoviruses-like identity is low, the possibility of causing similar symptoms should be considered. This
comprehensive, cost-efficient, and sensitive detection method for paramyxoviruses in bats is a promising
approach toward pathogen genome surveillance and zoonotic disease control.
Contact: Dela Ria Nesti,delarianesti87@gmail.com

45

P1-13

Relation of polymorphisms in the transactivation domain in Meq oncoprotein of Marek's diseasevirus, an avian oncogenic herpesvirus, to its pathogenicity
Shunsuke Yamagami (1), Shiro Murata (1) (2), Jumpei Sato (1), Yoshinosuke Motai (1), Shwe Yee Win (1),Fumiya Horio (1), Naoya Maekawa (2), Tomohiro Okagawa (2), Satoru Konnai (1) (2) (3), Kazuhiko Ohashi(1) (2) (4)(1)Laboratory of Infectious Diseases, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Japan(3)Institute for Vaccine Research and Development (GU-IVReD), Hokkaido University, Japan(4)International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Japan

Marek's disease virus (MDV) is an oncogenic herpesvirus that causes malignant lymphomas in chickens(Marek's disease; MD). Although MD is currently controlled by vaccination, field isolates of MDV havecontinuously increased in virulence and sporadically cause MD even in vaccinated chickens. Polymorphisms in
Meq, an MDV oncoprotein, is associated with enhanced virulence of MDV, and particularly, Meq of highly
virulent MDV strains has characteristic amino acid sequences in the proline-rich repeats (PRRs) in thetransactivation domain of Meq. Therefore, in this study, to examine the relationship between polymorphisms in
the PRRs and MDV pathogenicity, we analyzed the effects of Meq which the amino acid substitutions are
introduced into its PRRs on the transcriptional regulations by Meq and MDV pathogenicity. We analyzed theeffects of Meq with mutations in the PRRs on the transactivation activities on the promoters of Meq and Bcl-2,
which is an anti-apoptotic protein and a candidate host factor regulated by Meq. The reporter assays revealed
that Meq with mutations in the PRRs showed higher transactivation activities on both of the promoters thanwild type Meq. In addition, to assess whether mutations in the PRRs affect the pathogenicity, we generated arecombinant MDV (rMDV) based on the RB-1B strain which mutations were introduced into the PRRs (rRB1B_Meq-mutant), and analyzed the effects of mutations on the mortality and tumor incidence in experimentallyinfected chickens. Unexpectedly, the mortality and tumor incidence in chickens infected with rRB-1B_Meqmutant were significantly decreased when compared to those in the rRB-1B-infected group. These results
suggest that the mutations in the PRRs attenuate MDV virulence. Therefore, to elucidate the mechanisms ofenhanced virulence of MDV, further investigation is required, including the effects of other viral factors on
MDV pathogenicity.

46

P1-14The NPYNE sequence in the rabies virus L protein is critical for viral replication.
Fumiki Izumi (1), Machiko Makino (2), Michihito Sasaki (3), Kento Nakagawa (4), Tatsunori Masatani (1) (2)(5), Hirofumi Sawa (6), Makoto Sugiyama (2), Naoto Ito (1) (2) (5)(1)Joint Graduate School of Veterinary Sciences, Gifu University, Japan(2)Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, Japan(3)Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University, Japan(4)The United Graduate School of Veterinary Sciences, Gifu University, Japan(5)Center for One Medicine Innovative Translational Research, Institute for Advanced Study, Gifu University, Japan(6)Institute for Vaccine Research and Development (IVReD), Hokkaido University, Japan[Objective]
The rabies virus (RABV) large (L) protein binds to its essential cofactor, phosphoprotein (P protein), to function
as an RNA-dependent RNA polymerase (RdRp). We previously reported that substitution of the NPYNE
sequence at positions 1929-1933 of the L protein with the APYAA sequence attenuated the P protein binding
capacity of the L protein and thus decreased its RdRp function. This indicates that the NPYNE sequence isimportant for the P protein binding. However, the importance of this sequence in viral replication has not been
examined. In this study, we investigated the importance of the NPYNE sequence in RABV replication.
[Methods]
We established a mutant with the APYAA sequence instead of the NPYNE sequence using a reverse genetics
system of the RABV Nishigahara strain (wild-type virus). To examine the replication efficiency of the mutant,
we infected mouse neuroblastoma-derived NA cells with the wild-type virus or the mutant and measured thetiters in the culture supernatants at 5 days post-infection. Following serial passages of the mutant in NA cells 20
times, a revertant was obtained, and its replication efficiency was also examined. To determine the mutations
that occurred in the genome of the revertant, we conducted whole-genome sequence analysis using a nextgeneration sequencer. Furthermore, to assess the interaction between L and P proteins of the wild-type virus,
mutant, or revertant, we performed co-immunoprecipitation (Co-IP) analysis using 293T cells expressing L and
P proteins of each strain in various combinations.
[Results]
The mutant replicated less efficiently than did the wild-type virus, whereas the revertant replicated more
efficiently than did the mutant. The whole genome sequence analysis revealed that Phe-to-Leu/Gly-to-Arg, Histo-Asn, and Ala-to-Ser mutations were introduced at positions 81/138 in the P protein, at position 21 in theglycoprotein, and at position 1929 in the L protein (L1929), respectively. Compared to the mutant L protein, therevertant L protein had a higher capacity to bind with the wild-type virus and revertant P proteins.
[Discussion]
The APYAA substitution decreased the viral replication efficiency of the wild-type virus, indicating theimportance of the NPYNE sequence in viral replication. It was suggested that the mutation at L1929
compensates the deficiency in the P protein binding capacity that was caused by the APYAA substitution,
resulting in an increase in replication efficiency.

47

P1-15

Analysis of mosquito-borne Sindbis virus and Eilat virus isolated from mosquitoes captured inZambiaChadwic De'sean Mears (1), Koshiro Tabata (2), Michihito Sasaki (1) (2), Hirofumi Sawa (2) (3), Yasuko Orba
(1) (2) (3)(1)Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University, Japan(2)Institute for Vaccine Research and Development, Hokkaido University, Japan(3)One Health Research Center, Hokkaido University, Japan

Mosquito transmitted viruses are responsible for significant global morbidity and mortality. Sindbis virus(SINV) is an arbovirus in the family Togaviridae, genus Alphavirus, which also contains Chikungunya virus(CHIKV). The genus Alphavirus also includes some insect specific viruses, such as Eilat virus (EILV) and
Mwinilunga Alphavirus (MWAV).
During an arbovirus survey of mosquitoes captured in Zambia, SINV (isolate zmq17 M115) and EILV (isolate
zmq19 M44) were detected for the first time in the country. We characterized these viruses in terms of growth
kinetics, and molecular phylogenetic relatedness to other alphaviruses. We compared the growth kinetics of theZambian SINV strain with the laboratory reference strain SINV AR339 in mammalian (Vero, BHK) and insect(C6/36) cells by a plaque formation assay and performed phylogenetic analysis based on multiple alignment ofprotein coding regions of related SINV strains. The growth kinetics of EILV was determined by median tissue
culture infectious dose (TCID50) in C6/36 cells, and phylogenetic analysis was performed based on multiplealignment of nonstructural and structural protein coding sequences of related alphaviruses.
Our results show that the Zambian SINV strain was found to be in Clade D of SINV Genotype 1 along with
Kenya isolate BONI584 and several Central African isolates. The growth of the Zambian SINV isolate wascomparable to that of reference strain AR339 in mammalian cells, but there was some statistical difference ininsect cells. Furthermore SINV required longer incubation times in insect cells to saturate viral growth than in
mammalian cells. The phylogenetic analysis of Zambian EILV isolate showed close proximity with other insectspecific alphaviruses within the ungrouped cluster of the genus Alphavirus. The Zambian isolate shares close
nucleotide identity to the reference EILV isolate (90.4%), and MWAV (75.5%), which was previously identified
from mosquitoes in Zambia. Growth kinetics showed that Zambian EILV attained a saturating titer in C6/36
cells at 6-8 days post infection (dpi) and was unable to replicate in mammalian cells.
Our analysis demonstrates that the SINV isolated in Zambia shows expected growth characteristics from in vitro
experiments and is closely related to medically relevant strains from Central Africa. Lastly the Zambian EILV
isolate showed replication capacity in insect cells, while being unable to replicate in mammalian cells, consistent
with the results of other previously reported insect specific alphaviruses.

48

P1-16

Identification of genes critical for ST131 survival in the dog fecal suspension
Aiko Maeda (1) (2), Toyotaka Sato (1) (2) (3), Motohiro Horiuchi (1) (2) (3)(1)Laboratory of Veterinary Hygiene, Faculty of Veterinary Medicine, Hokkaido University(2)Graduate School of Infectious Diseases, Hokkaido University(3)One Health Research Center, Hokkaido University

Objective:
Antimicrobial resistance is a critical global issue. Fluoroquinolone-resistant Escherichia coli sequence type
131 (FQ-R E. coli ST131) is the major high-risk clone in clinical settings. ST131 has been isolated from clinicaland healthy humans, as well as companion animals (dogs and cats), suggesting potential transmission in thecommunities. Therefore, urgent measures are needed to control its spread. However, the actual dissemination
mechanism of ST131 in communities remains unclear. This study aims to explore the transmission andcolonization mechanisms of ST131 by identifying ST131 factors important for survival in the intestinal
environment of companion animals.
Material and Methods:
We utilized transposon-directed insertion-site sequencing (TraDIS) to identify genes important for ST131
survival in the intestinal environment. We inoculated ST131 transposon mutant library (1.6 × 106 cfu/mL) into
canine fecal suspension prepared to 80% using Mueller Hinton II broth (MHBII) as a simulated intestinal
environment and 80% saline with MHBII as the control. After overnight incubation at 37 °C, bacterial DNA
was extracted and processed to DNA libraries using custom primers for Next-generation Sequencing (NGS). By
TraDIS analysis using the NGS sequencing data, we identified genes with less than 2-fold detection(FDRp<0.05) compared to control as potential ST131 colonization factors. Lambda Red recombination wasused to construct gene specific deletion mutants of ST131 and cultured in the canine fecal suspensions toconfirm their survival in the intestinal environment.
Results:
By TraDIS analysis using canine fecal suspension, we identified 8 genes that are putatively important
bacterial factors for survival in the intestinal environment. We constructed gene-deletion ST131 mutants among
7 of the 8 genes. Among the 7 mutants, two types of the gene-deletion mutants (ΔoxyR and ΔlpxL) significantly
decreased the bacterial growth in the canine fecal suspension compared with that of the parent strain, suggestingthese genes influence survival in the intestinal environment in E. coli ST131.
Discussion:
In this study, we identified 2 crucial ST131 factors by using TraDIS that are important for survival in canine
fecal suspension. These 2 factors encoded by oxyR and lpxL genes. OxyR regulates oxidative stress response
genes by activating the production of protective enzymes like catalase. LpxL is an enzyme involved in lipid A
biosynthesis that contributes to bacterial defense against innate immune responses. Therefore, these functions
are essential for ST131 survival in companion animals' intestinal environments and are suggested to contributeto the colonization and dissemination of ST131 in the community.

49

P2-1

Functional differences of glycosyltransferases in glycopeptidolipid biosynthesis of nontuberculous mycobacteria
Hayato Sugiura (1), Nagatoshi Fujiwara (2), Shinji Maeda (3)(1)Division of Global Epidemiology, Graduate School of Infectious diseases, Hokkaido University, Japan(2)Department of Food and Nutrition, Faculty of Contemporary Human Life Science, Tezukayama University, Japan(3)Faculty of Pharmaceutical Science, Hokkaido University of Science, Japan

Mycobacterium avium and Mycobacterium intracellulare are the most causative bacteria of non-tuberculous
mycobacteria (NTM) infections, accounting for over 70% of NTM infections in Japan. These bacteria arecollectively referred to as the Mycobacterium avium complex (MAC) due to their similar phenotype andgenotype. In recent years, the number of patients infected with NTM has increased, and the incidence of NTM
infection is higher than that of smear-positive TB. This study focuses on serotype-specific glycopeptidolipids(GPLs), which are the outermost layer of the bacteria. The GPL may be one of the bacterial components tocontact with host cells. There are also reports that GPL is related to the virulence of the bacteria. The clarificationof the interaction between GPL structure and host immune cells will facilitate an understanding of themechanisms of pathogenesis.
The present study investigated the M. intracellulare ku11 strain, a clinical isolate from Japan, which has a novel
GPL structure. In the first step, the sequence of the GPL synthase cluster region of the ku11 strain was analyzed
and open reading frame (ORF) was examined. The estimated ORFs (orf1 to orf8) were searched homology using
databases at the amino acid level. With regard to enzyme functions, the results demonstrate that ORF1, ORF6,
and ORF8 exhibited high homology to glycosyltransferases. Additionally, ORF4 and ORF5 showed highhomology with GDP-mannose 4,6-dehydratase and GDP-fucose synthetase, respectively.
The serotype 1 strain (M. intracellulare NF113) was transformed by the pVV16 plasmid, which contained thegene from the ku11 strain. The GPL structure of the mutant was then analyzed. It was observed that theoverexpression of orf1 or orf8 in NF113 resulted in a change to the serotype 1 GPL structure, which led to thetransfer of rhamnose. However, no such alteration was observed in the case of orf6, which is also a
glycosyltransferase.
The objective of this study is to elucidate the function of genes by creating mutants in which specific genes are
disrupted. This is achieved through the overexpression of enzymes involved in the synthesis of cell wall
structures and CRISPR (clustered regularly interspaced short palindromic repeat) -Cas9. In particular, by
elucidating the novel GPL structure and its synthesis mechanism in pathogenic NTM, we will be able to clarify
the difference in reactivity of previously reported GPLs and novel GPL with Toll-like receptors.

50

P2-2

Isolation and PCR-based genetic characterization of multi-drug resistant bacteria from animalderived food products in Mongolia
Mungunsar Chuluun (1) (2), Tuvshinzaya Zorigt (1), Harvey Kakoma Kamboyi (1) (2), Enkhtsetseg
Nyamdorj (3), Gantsetseg Tumuruu (4), Bolortsetseg Denebaldan (4), Enkhtuya Jargalsaikhan (4), Tomoe Kitao(1), Hideaki Higashi (1)(1)Division of Infection and Immunity, International Institute for Zoonosis Control, Hokkaido University, Japan(2)Graduate School of Infectious Diseases, Faculty of Veterinary Medicine, Hokkaido University, Japan(3)Laboratory of Infectious Disease and Immunology, Institute of Veterinary Medicine, Mongolian University of Life Science, Mongolia(4)Laboratory of Food Safety and Hygiene, Institute of Veterinary Medicine, Mongolian University of Life Science, Mongolia

Background: The bacterial antimicrobial resistance (AMR) is a significant public health concern, and the World
Health Organization warned that AMR could cause up to ten million deaths annually by 2050. In Mongolia,
AMR monitoring is underdeveloped due to limited diagnostic infrastructure. Recently, bacterial isolates
producing extended-spectrum β-lactamases (ESBLs), which confer resistance to a broad range of β-lactam
antibiotics, have been reported in the human medical setting. Additionally, antimicrobial usage in livestock islargely unregulated, and there is a significant gap in detailed information regarding AMR bacteria originatingfrom animal-derived foods. This study aims to clarify the AMR characteristics of the ESBL-producing Gramnegative bacterial species isolated from food markets and slaughterhouses in Ulaanbaatar, Mongolia, toward
investigating the potential mechanisms for the genetic transmission of AMR genes.
Methods: Using 448 bacterial samples from processed dairy and meat products, and raw milk in food markets
and slaughterhouses, we screened suspected ESBL-producing Gram-negative bacteria on MacConkey agar
plates supplemented with cefotaxime (CTX), a broad-spectrum, third-generation cephalosporin. The
identification of species was confirmed by Sanger sequencing of the 16S rRNA gene. The minimum inhibitory
concentration (MIC) of CTX-resistant isolates was determined against nine antimicrobials, according to Clinicaland Laboratory Standards Institute guidelines. ESBL genes were detected by polymerase chain reaction (PCR)
and visualized using 1.5% agarose gel electrophoresis.
Results: From the screening, we identified 28 isolates of CTX-resistant Gram-negative bacteria: 11 isolates
were Escherichia coli, 14 were Enterobacter spp., and 3 were Klebsiella spp. Analyses of MICs indicated that
8 out of 28 CTX-resistant strains (28.5%) exhibited MDR phenotype. Resistance to ampicillin (71.4%),
aztreonam (42.8%), and imipenem (21.4%) were most frequently observed, followed by other classes ofantibiotics, including gentamicin (35.7%), and colistin (39.2%). Notably, some strains showed high-level MDR
phenotype. The detection of ESBL genes by PCR revealed that the blaTEM gene is predominant (7 isolates;
63.6%), followed by blaOXA gene (6 isolates; 54.5%), and blaSHV gene (4 isolates; 36.3%) for E. coli isolates.
Conclusion: We identified several high-level MDR Gram-negative bacterial strains isolated from animalderived foods and slaughterhouses in Mongolia. Our future research will involve whole-genome sequencing
analysis of MDR Gram-negative isolates to determine their detailed genetic characteristics. This study will
underscore the critical need for surveillance and management strategies to mitigate the AMR issue, particularly
in environments such as food markets and slaughterhouses, where resistant bacteria may thrive and disseminate.

51

P2-3

Clustering SARS-CoV-2 variants using taxonomic distances and relative reproduction numbers
Richard Musonda (1)(1)Laboratory of Bioinformatics, Graduate School of Infectious diseases, Hokkadio University, Japan

Introduction
Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in 2019, the virus
has continuously evolved by accumulating mutations. This evolution has led to the formation of multiple
variants. It has been observed that the variants with the highest reproductive numbers become the dominantstrains over time through natural selection. Over the course of the SARS-CoV-2 pandemic the number ofvariants circulating at any given time point has increased, possibly because different variants have similar
reproduction numbers. It is therefore crucial to develop a method that can find groups of variants that share thesame relative reproduction numbers and variants of which relative reproduction numbers are different.
MethodIn this study, we utilized 172,082 SARS-CoV-2 sequence metadata downloaded from GISAID for France,
Denmark, the United Kingdom and Spain. We developed an effective algorithm that could efficiently findgroups of variants sharing the same relative reproduction numbers. Our algorithm was based on the renewal
equation which calculates instantaneous relative reproduction numbers and the set partitioning enumeration
which uses restricted growth strings. To reduce the number of maximum likelihood calculations, we introduced
a pruning algorithm based on the Akaike Information Criteria (AIC). Additionally, we take into account the
taxonomic distances based on the pangolin lineage nomenclature when the variants are grouped together.
Results and Discussion
We found that our algorithm could find the best clustering of variants which gives the minimum AIC among all
possible clustering patterns for a given temporal change of observed count of variants in a reasonable amountof computational time when the number of variants was smaller than 20. This number increased when aconstraint on the taxonomic distances between variants to be grouped was introduced. Existing popularclustering methods, such as the k-means clustering algorithm and the Expectation Maximisation algorithm weremuch faster than our algorithm, but these algorithms rarely found the best clustering, while our algorithm isguaranteed to find the best. The main application of this algorithm is to identify important mutations that may
be responsible for the observed increased transmissibility of new variants in circulation.

52

P2-4

Automatic 3D Segmentation of Liver in Computed Tomography Images with a Deep Learning
Model in Dogs
Seungyeon Lee (1), Genya Shimbo (2), Nozomu Yokohama (1), Kensuke Nakamura (1), Mitsuyoshi Takiguchi(1)(1)Laboratory of Veterinary Internal Medicine, Department of Veterinary Clinical Science, Graduate School of Veterinary Medicine,
Hokkaido University, Japan(2)Veterinary Teaching Hospital, Graduate school of Veterinary Medicine, Hokkaido University, Japan

Introduction: Medical image segmentation is crucial in diagnostics, enabling detailed examination of
anatomical structures. It plays a vital role in clinical workflows, aiding surgical/radiation planning, research,and education. In both human and veterinary medicine, manual segmentation, though accurate, is timeconsuming and subject to observer variability. Advances in deep learning have facilitated fully automatic
segmentation, which is efficient and reproducible. However, such technologies are underutilized in veterinary
medicine.
Objective: This study aims to develop a deep-learning model for automatic liver and tumor segmentation incanine CT scans, enhancing diagnostic accuracy and efficiency.
Materials & Methods: CT scans from dogs at Hokkaido University Veterinary Teaching Hospital (2022-2023)
are investigated. All images are contrast-enhanced CT scans. The medical records of the dogs, including dataon age, sex, neutering status, body weight, and laboratory examination results, were also collected. The CT
scans included in this study were manually labeled using a 3D image analysis system (Synapse Vincent, Fuji
Film Co., Ltd., Tokyo, Japan). Pre-processing steps include data completion, noise reduction, and normalization.
The study explores various deep learning architectures (CNN, U-Net, U-Net++, FPN) and uses image
augmentation to create a realistic dataset. Performance metrics such as the Dice Coefficient and Hausdorff
Distance are used to evaluate the model.
Results and Discussion: A total of 161 CT scans from 149 dogs free of mass-like lesions in the liver werecollected and manually labeled. An additional 150 scans from dogs with liver tumors are planned. It is expected
to demonstrate the model's ability to enhance surgical/radiation planning, research, and education by providing
accurate and reliable liver and tumor segmentations.

53

P2-5

Evaluation of exposed antigens as vaccine antigen candidates for controlling poultry red mites
Fumiya Horio (1), Shiro Murata (1) (2), Shwe Yee Win (1), Jumpei Sato (1), Yoshinosuke Motai (1),
Shunsuke Yamagami (1), Takumi Sato (3), Eiji Ohishi (3), Naoya Maekawa (2), Tomohiro Okagawa (2),Satoru Konnnai (1) (2) (4), Kazuhiko Ohashi (1) (2) (5)(1)Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japanious(2)Department of Advances Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Japan(3) Vaxxinova Japan K.K, Japan(4)Institute for Vaccine Research and Development (GU-IVReD), Hokkaido University, Japan(5)International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Japan

Poultry red mite (Dermanyssus Gallinae, PRM) is a hematophagous ectoparasite of poultry, causing significant
economic losses in poultry farming worldwide. Although pesticides are commonly used to control PRMs, theemergence of pesticide-resistant PRMs has been reported, and a novel strategy for controlling these mites, suchas vaccination, are demanded. Among the vaccine antigen candidates identified to date, most of them wereconcealed antigens, which are mainly expressed in midguts, whereas some of the effective antigens wereconsidered as exposed antigens expressed in salivary glands. In this study, therefore, to assess the potential of
exposed antigens for the development of the anti-PRM vaccine, we identified three exposed antigens, DgLipocalin (Dg-LCN), Dg-Defensin (Dg-DEF), and Mite allergen (Dg-MA), and evaluated their feasibilities as
vaccine antigens.
The partial fragments of the Dg-LCN, Dg-DEF, and Dg-MA genes were detected based on the data obtained
from RNA sequencing that we previously conducted, and the sequences of open reading frames were determined.
The recombinant proteins were prepared by E. coli expression system, and immune plasmas were isolated fromchickens immunized with the recombinant proteins of each molecule. The anti-PRM efficacy was evaluated bymonitoring the survival rate of PRMs fed with the immune plasmas. Of the three immune plasmas, anti-DgLCN and anti-Dg-DEF immune plasmas significantly decreased PRMs' survival rate, while anti Dg-MA
immune plasma did not affect mites' survival. The efficacy of the immune plasmas at different developmental
stages showed that anti-Dg-DEF immune plasma was effective especially in protonymphs. These data suggestthat the exposed antigens, including Dg-LCN and Dg-DEF, could be applied for vaccine antigens against PRMs.

54

P2-6

Characterization of major facilitator superfamily as a transporter protein in Babesia ovata
Silviane Aoko Miruka (1)(1)Research Unit for Global Infection Control, National Research Center for Protozoan Diseases, Obihiro University of Agriculture andVeterinary Medicine, Japan.

Characterization of major facilitator superfamily as a transporter protein in Babesia ovata
Silviane Miruka, Misato Sato, Masahito Asada
Research Unit for Global Infection Control, National Research Center for Protozoan Diseases, Obihiro
University of Agriculture and Veterinary Medicine, Japan.
Babesia is a tickborne apicomplexan parasite that causes zoonotic diseases in livestock, humans, pets and wild
animals. Currently, there is an emergence of acaricide-resistant tick vectors, with limited treatment options
available for babesiosis, therefore development of efficient drugs against babesiosis is an urgent issue. Major
facilitator superfamily (MFS) is one of the largest families of membrane transporters known to transportcompounds like sugars, drugs, and amino acids. In another apicomplexan parasite, Plasmodium falciparum,
MFS is known to transport sugars and has been studied as a potential drug target. Since mfs genes are conserved
among various Babesia species such as B. ovata, B. bovis and B. bigemina, we characterized Babesia MSF
using B. ovata. Localization of MFS was analysed by indirect immunofluorescence microscopy, andfluorescence was observed inside the parasite and nearby the surface of infected red blood cells. To elucidate
the transporter function of MFS, blastisidin S (BS) resistant parasites were produced. Expression of mfs wascompared between parental parasite and 2 strains of BS-resistant parasites by RNA-seq analysis. From thisanalysis, a reduction of mfs expression was observed on both BS-resistant parasites. This result suggests that
MFS plays a role in the transportion of BS and therefore the parasites have downregulated mfs expression toprotect themselves from adverse effects of BS. To prove this hypothesis, currently, we are overexpressing MFS
on the BS-resistant parasites. Susceptibility to BS will be compared between the BS-resistant parasites and the
MFS overexpressed BS-resistant parasites.

55

P2-7

Investigating Otopetrin-1 (Otop1) Function in Mouse Brown Adipose Tissue
Md Mominul ISLAM (1), Saori YANO-NASHIMOTOYAMAGUCHI (2)(2), Yuko

OKAMATSU-OGURA (3), Soichiro(1)Laboratory of Physiology, Department of Basic Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University,Japan(2)Laboratory of Physiology, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University, Japan(3)Laboratory of Biochemistry, Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University, Japan

Otopetrin-1 (OTOP1) has recently been discovered as a proton (H+)-selective ion channel responsible fordetecting extracellular acidic stimuli in sour taste receptor cells. As stated in BioGPS (a mRNA expression
database), mouse Otop1 (mOtop1) is markedly expressed in brown adipose tissue (BAT). An important functionof brown adipocytes (BAs), the functional unit of BAT, is to burn lipids and produce heat through the activationof Uncoupling protein 1 (UCP1), a transmembrane protein located in the inner mitochondrial membrane of BA.
However, the functional expression of OTOP1 in brown adipocytes remains unknown. Therefore, we aimed toreveal whether OTOP1 is functionally expressed in BA and the possible mechanism of how OTOP1 regulates
the function of BAs. Firstly, the intracellular pH measurement result revealed that the basal pH of the primarycultured differentiated BAs of wild-type (WT) mice was significantly lower than the BAs of Otop1 knock-out(KO) mice, and OTOP1 induces the entry of protons inside the cells in an extracellular acidic environment.
Next, we measured the inward proton (H+) currents of primary cultured differentiated WT BAs and KO BAs
using the whole-cell patch clamp electrophysiology. We found that the inward H+ currents were elicited by an
extracellular acidic solution of pH 5.0 whereas the H+ currents were diminished in KO BAs using the same
extracellular solution. These results suggest that Otop1 is functionally expressed in mouse BAT. However,through the oxygen consumption measurement of freshly isolated WT BAs and KO BAs, the difference between
WT BAs and KO BAs was found insignificant in the basal oxygen consumption rate. The difference between
WT BAs and KO BAs remained negligible following the administration of Norepinephrine for the maximal
effect. We also did not find any body weight and BAT weight difference between WT and KO mice at 5 weeks
old. Together these results suggest that OTOP1 does not play a major role in the oxygen consumption rateincreased by norepinephrine although OTOP1 is functionally expressed in BAs.

56

P2-8

Serological evidence of exposure to hantaviruses among residents of three different climatic
zones in Sri Lanka
Pavani Senarathne (1) (2), Devinda S. Muthusinghe (3), Nipun S. Rathnayake (4), Rydhnieya Vijeyakumaran(4), Videesha Bandara (4), Themali Herath (4), Chandika Gamage (4), Kumiko Yoshimatsu (1) (2)(1)Graduate School of Infectious Diseases, Hokkaido University(2)Laboratory of Animal Experimentations, Institute for Genetic Medicine, Hokkaido University(3)National Research Center for the Control and Prevention of Infectious Diseases, Nagasaki University(4)Department of Microbiology, Faculty of Medicine, University of Peradeniya, Sri Lanka

Introduction:
Human pathogenic hantaviruses are naturally maintained in rodents and transmitted to humans via inhalation ofvirus-contaminated aerosols from infected rodent excreta. Human exposure to this infection was first reported
in Sri Lanka in 1988 and appears to continue to date. Recent genetic evidence from field rodents suggested thepresence of two Thailand orthohantavirus (THAIV)-related viruses in the dry zone of Sri Lanka, i.e., Musbooduga-borne Lanka virus (LNKV) and Rattus rattus-borne Anjozorobe virus (ANJZV). Kegalle, Matale, andKurunegala districts are located in the wet, intermediate, and dry climatic zones of Sri Lanka, and there is no or
limited previous data on the evidences of hantavirus infection among the residents.
Objective:
To explore the exposure to hantavirus infection among residents of selected areas of Kegalle, Matale, andKurunegala districts in Sri Lanka
Methods:
Blood samples and demographic data were collected from randomly recruited 608 individuals living in selectedadministration areas of Kegalle, Matale and Kurunegala districts from January to December 2022. Initially,
serum samples were screened using an Indirect Immunofluorescent Antibody assay (IFA) based on Vero E6 cell
antigens transiently expressing THAIV recombinant N protein (rN). Positive sera were screened with IFA basedon Lanka virus recombinant glycoprotein (rGP) antigens and rGP positives were subjected to serotyping using
recombinant Gn (rGn) glycoprotein antigens of LNKV and ANJZV.
Results:
Out of the 608 study participants, seropositivity to rN THAIV antigens in Kurunegala, Kegalle, and Matale
districts were 22/200 (11%), 18/208 (8.65%), and 15/200 (7.5%) respectively. Among these anti-hantavirus
positives, 30/608 (4.93%) were having anti-rGP antibodies. Furthermore, serotyping results revealed that in
Kurunegala district 7/200 (3.5%) and in Matale district 4/200 (2%) were positive for LNKV while in Kegalle
district 2/208 (0.9%) were positive for ANJZV and 4/208 (1.9%) were positive for LNKV and the remaining
were inconclusive.
Discussion and conclusions:
The findings reveal that the percentages of hantavirus exposure in communities of study areas are higher than
average percentages in the world. No significant difference in antibody prevalence was observed among thedifferent climatic zones in Sri Lanka(p>0.05). Furthermore, the first definitive serotyping evidence of Lanka
virus and Anjozorobe virus infecting humans in selected administrative areas of districts was revealed. Hence,
we would recommend studies to reveal the seroprevalence of hantavirus exposure, and the distribution of its
reservoir hosts in Sri Lanka.

57

P2-9

Effect of GnRH vaccines and agonists for contraception and aggression suppression in zoo
animals.
Ryusuke Tsushima (1) (2), Yoshitaka Kimura (2), Masae Tamaru (2), Rino Takaba (2), Seiji Katagiri (3), Yojiro
Yanagawa (3)(1)Laboratory of Theriogenology, Graduate School of Veterinary medicine, Hokkaido University, Japan(2)Tokiwa Zoo, Japan(3)Laboratory of Theriogenology, Faculty of Veterinary Medicine, Hokkaido University, Japan

In zoos and aquariums, it is important not only to fertilize endangered species but also to contracept animals
due to maintaining animal welfare levels and/or pedigree management. Synthetic progestin has been used as areversible contraception in females, but alternative contraceptive methods are essential due to the risk of side
effects caused by long-term progestin exposure such as obesity and uterine disease. Suppression of reproductive
and aggression by controlling gonadotropin-releasing hormone (GnRH) action, using GnRH vaccines or agonist
implants has been conducted. GnRH vaccines produce anti-GnRH antibodies which neutralize the GnRH
functions, while long-term release GnRH agonists downregulate GnRH receptors in the pituitary, and suppress
sex hormones secretion. However, appropriate usage/dosage are need to be studied in each species.
In the present study, we used GnRH vaccine or GnRH agonist implant to meerkats and spider monkeys. We
administered GnRH vaccines (40 μg/head Gonadotropin-releasing factor protein conjugate; Improvac, Zoetis)
to two males and one female meerkat kept together. Further, GnRH vaccines (100 μg/head) were administered
to two male spider monkeys and GnRH agonist implant (9.4 mg deslorelin; Suprelorin12, Virvac) wereadministered to one female spider monkey kept in together with males. Two to four dose of GnRH vaccine wasadministered first at one month, then 4-5 month intervals. In male meerkats, blood samples were collected
monthly after administrations, while in the others, fecal samples were collected three times a week. Testosterone
in males and estradiol and progesterone in females were measured in blood and feces. One male meerkat showeda depletion in plasma testosterone concentrations while the other showed clear increase. Fighting frequency of
two males was reduced after treatment. A female meerkat showed no mating behaviors with these two males
and no apparent hormonal cyclicity was observed after administration. Male spider monkeys showed a slightdecrease in fecal testosterone after treatment, but aggression in the dominant male was unchanged afteradministration. A female spider monkey mated less frequently with no pregnancy and the female showed noincrease in progesterone after administration, indicating no ovulation occurred.
Present GnRH vaccination may cause suppress aggression and reproduction in meerkats. However, GnRH
vaccination could not suppress the fighting in male spider monkey, which may be due to social structure of thespecies, so need some modification. GnRH agonist implant may suppress reproductive activity for a certain
period in spider monkey female.

58

P2-10Assessment of human lead exposure through consumption of game and livestock meat productsin Namibia; a better understanding of Pb contamination under the One Health umbrella.
Ilana Rautenbach (1), John Yabe (1), Rio Doya (1), Yoshinori Ikenaka (1), Mayumi Ishizuka (1), Shouta M.M.
Nakayama (1)(1)Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University,
Japan.

Namibia is a developing country in southern Africa whose livelihood is based on hunting of wildlife andsubsistence farming. The country is rich in mineral resources with an extensive mining history with more than
250 abandoned mines scattered throughout the landscape. Although, there is extensive mining activities taking
place throughout the country and considerable hunting of game; there are currently few remediation andrehabilitation efforts in place to reduce contamination of the people and environment with toxic heavy metals(THM). In fact, currently Namibia lacks the scientific backing from research to enforce these remediation efforts
and cannot set regulatory standards without the proper base data. Among these toxic heavy metals is lead (Pb),which falls under the substance priority list according to ATSDR Substance Priority List (2022). Only a few
studies have been done on Pb contamination on animals in Namibia (Midzi, 2012). This is affirmed by Yabe etal. (2010) in that there are limited records on contamination in livestock and other animals in southern Africa.
This study which will evaluate the potential for Pb exposure in the Namibian human populations through theconsumption of contaminated game and livestock meat products. The study will also determine the potential
risk for environmental Pb exposure to wildlife and domestic livestock from historical mining activities.
Additionally, identify the risk factors associated with lead toxicity; specifically hunting (ammunition andhunting techniques) and mining activities (soil samples around mining areas). The rationale lies in the limited
information available on lead contamination in Namibia and, more significantly, the lack of insight regarding
its health impact on these communities. Additionally, the study aims to assess the effectiveness of current
hunting methods in minimizing Pb exposure, and whether there is a need to mitigate the current hunting
techniques to safeguard the well-being of these communities.
Convenience sampling will be utilized for this study where samples will be collected from game, canines,
domestic livestock and humans. In addition, soil samples will also be collected throughout Namibia. The process
of metal extraction will take place at the Central Veterinary Laboratory (CVL), Windhoek, Namibia. Thereafter,
the samples will be shipped to the Laboratory of Toxicology, Graduate School of Veterinary Medicine, Hokkaido
University, Japan for lead analysis.

59

P2-11

Application of AS03-like adjuvant to West Nile virus subunit vaccine
Atsuko Inoue (1) (2), Yukari Itakura (1), Shinji Saito (1), Hirofumi Sawa (1) (3) (4), Katsumi Maenaka (2) (5)(6) (7) (8), Koshiro Tabata (1)(1)Division of Biological Response Analysis, Institute for Vaccine Research and Development, Hokkaido University, Japan(2)Laboratory of Biomolecular Science, Faculty of Pharmaceutical Science, Graduate School of Life Science, Hokkaido University,Japan(3)Division of International Research Promotion, International Institute for Zoonosis Control, Hokkaido University, Japan(4)One Health Research Center, Hokkaido University, Japan(5)Division of Vaccine Development, Institute for Vaccine Research and Development, Hokkaido University, Japan(6)Center for Research and Education on Drug Discovery, Faculty of Pharmaceutical Sciences, Hokkaido University, Japan(7)Division of Pathogen Structure, International Institute for Zoonosis Control, Hokkaido University, Japan(8)Global Station for Biosurfaces and Drug Discovery, Hokkaido University, JapanWest Nile virus (WNV), belonging to the genus Orthoflavivirus is a zoonotic disease transmitted from natural
hosts such as birds to humans and horses via mosquitoes as a vector. Although most WNV-infected individuals
are subclinical, approximately 1% of the infected individuals develop severe symptoms such as fatal encephalitis
or meningitis. However, no vaccine is available for clinical use to prevent the infection in humans. Reasons forthe lack of progress in vaccine development include the limited number of clinical cases, which leads to reduced
cost-effectiveness, and concerns that antibodies induced by WNV-vaccination may enhance the otherorthoflavivirus infections, known as antibody-dependent enhancement (ADE).
Live attenuated-vaccines can be produced at a relatively low cost because the production of vaccine antigens
depends on viral growth and requires only a basic cell culture system. However, since the virions contain several
epitopes for ADE-prone antibodies, the use of live attenuated-vaccines may not overcome safety concerns suchas ADE. On the other hand, recombinant subunit vaccines are more expensive for production but have theadvantage of flexibility in antigenic modification using gene editing techniques. Therefore, this vaccine
formulation minimizes the risk of induction of ADE-prone antibodies by vaccination. This study aims to develop
a low-cost, safe, and protective recombinant subunit vaccine against WNV infection using ADE epitopesmodified recombinant protein combined with an oil-in-water nano-emulsion adjuvant, AddaS03.
Envelope proteins (Env) on the virion surface are the major epitopes of antibodies that contribute to protection
from orthoflavivirus infection. Therefore, we used recombinant Env with or without ADE epitope-modifications
as vaccine antigens. Moreover, to reduce the amount of vaccine antigen for immunization, the AddaS03 was
employed. A similar formulation as this adjuvant, AS03 has already been applied to influenza and SARS-CoV2 vaccines for clinical use. However, whether the adjuvant will work effectively for orthoflavivirus antigens isstill unclear. Therefore, to develop a WNV vaccine, we attempted to evaluate this adjuvant activity by comparing
it with the widely used aluminum hydroxide adjuvant.
After immunization of BALB/c mice with Env antigens combined with these adjuvants, the immunized-micewere infected with WNV at a lethal dose. All experiments are currently ongoing.

60

P2-12

Prevalence and Genomic Characterization of Rotavirus A from Domestic Pigs in Zambia:
Evidence for Possible Porcine-Human Interspecies Transmission
Joseph Ndebe (1) (2), Hayato Harima (3), Herman Moses Chambaro (4), Michihito Sasaki (5), Junya
Yamagishi (6), Annie Kalonda (1) (7), Misheck Shawa (1), Yongjin Qiu (8), Masahiro Kajihara (1),Ayato Takada (2) (9), Hirofumi Sawa (2) (10), Ngonda Saasa (2), Edgar Simulundu (2)(1)Division of International Research Promotion, International Institute for Zoonosis Control, Hokkaido University, Japan(2)Department of Disease Control, School of Veterinary Medicine, University of Zambia, Zambia(3)Laboratory of Veterinary Public Health, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Japan(4)Central Veterinary Research Institute, Ministry of Fisheries and Livestock, Zambia(5)Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University, Japan(6)Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Japan(7)Department of Biomedical Sciences, School of Health Sciences, University of Zambia, Lusaka 10101, Zambia(8)Laboratory of Parasitology, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(9)Division of Global Epidemiology, International Institute for Zoonosis Control, Hokkaido University, Japan(10)Institute for Vaccine Research and Development, Hokkaido University, Japan

Rotavirus A (RVA) is a segmented virus known to cause acute gastroenteritis globally in the young populationof animals and children under the age of 5 years. Economic losses to pig farmers and mortality in children are
the significant concerns associated with RVA infections. However, the low efficacy of current vaccines has beenreported in developing countries, and no known vaccine is used for RVA in the animal population in Zambia.
One of the proposed hypotheses for the observed low vaccine efficacy in these countries, besides nutrient
deficiencies (zinc, vitamins A and D), breast milk antibodies, and environmental enteropathy, is the genetic
diversity of RVA, which is associated mainly with reassortment and interspecies transmission between animals
and humans. A total of 148 samples were conveniently collected and screened by RT-PCR targeting viral protein
6 (VP6) genes. Further, the VP6 positives were screened with the viral protein 7 (VP7) gene and Sanger
sequenced to determine the outer glycoprotein genotype (G-type). By next-generation sequencing, the fulllength genome sequences and the genetic constellations of the representative strains were determined andphylogenetically analyzed. The VP6 gene was detected in 34 samples (22.9%). Among them, the VP7 gene was
detected in 12 samples. BLASTn search based on the VP7 sequences demonstrated that the detected RVAs
comprised three G-types, G4 (n=1), G5 (n=5), and G9 (n=6) which showed the highest nucleotide identities toporcine and human rotavirus strains ranging from 87.5% to 97.1%. Importantly, RVA infections weresignificantly associated with the rainy season but not with diarrhea or age. A Wa-like genetic constellation of
the representative strains, G4-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1 and G9-P[13]-15-R1-C1-M1-A8-N1-T1E1-H1, was identified by full-genome sequences. Phylogenetic analysis revealed that the two representative
strains may have their ancestral origin in pigs. However, some of the segments were related to humans,suggesting genetic reassortment between human and pig-origin RVAs. The study provided the first evidence ofreassortment and possible interspecies transmission of RVA between pigs and humans in Zambia. To mitigate
RVA infections in the pig population, enhanced measures should be implemented during the rainy season. The
study also showed that pigs might be among the sources of the unusual RVA emerging in humans in Africa.
Therefore, the One Health surveillance approach for RVA in animals and humans is recommended to design
effective control measures in developing countries.

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P2-13

Avian influenza virus surveillance and generation of vaccine strain against infection with H5high pathogenicity avian influenza virus circulating in Vietnam
Linh Bao Nguyen (1), Norikazu Isoda (1) (2) (3) (4), Yik Lim Hew (1), Loc Tan Huynh (1), Daiki
Kobayashi (1), Dang
Nguyen Hoang (5), Duc-Huy Chu (6), Diep Thi Nguyen (6), Long Van
Nguyen (6), Takahiro Hiono (1) (2) (3) (4), Yoshihiro Sakoda (1) (2) (3) (4)(1)Laboratory of Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)One Health Research Center, Hokkaido University, Japan(3)International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Japan(4)Hokkaido University Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Japan(5)National Center for Veterinary Diagnosis, Vietnam(6)Department of Animal Health, Vietnam[Objectives]
Vaccination in poultry is an optional measure to control high pathogenicity avian influenza (HPAI) in its
endemic situation. Demand for HPAI vaccines that are more effective in prevention and have higher production
efficiency is increasing in Vietnam. The vaccine strain must be constantly reviewed to respond to changing viral
antigenicity and epidemiology. This study aims to develop a candidate vaccine strain in Vietnam by matching
the antigenicity to the field HPAI viruses (HPAIVs) and to continue monitoring circulating viruses in the field.
[Materials and methods]
A representative strain was selected among HPAIVs isolated in Vietnam in 2023. The sequence at the cleavage
site of H5 HA gene was modified from polybasic amino acids to monobasic amino acid by site-directedmutagenesis. By reverse genetics, a candidate vaccine strain bearing mutant H5 HA and intact NA of therepresentative field strain, and the internal genes from A/Puerto Rico/8/1934 (H1N1) were generated. The
pathogenicity and growth potentials of the candidate vaccine strain in eggs were investigated to assess
effectiveness and safety in vaccine production. The efficacy of the vaccine candidate will be evaluated in the
animal experiment.
Following the 2023 avian influenza surveillance program in Vietnam, in the summer of 2024, swab samples
continued to be collected from poultry at live bird markets (LBMs) in Lang Son province, northern Vietnam.
Samples will be inoculated into 10-day-old embryonated chicken eggs to isolate avian influenza viruses.
Sequencing will be conducted to confirm their subtypes and pathotypes. H5 HPAIVs will be genetically andantigenically analyzed.
[Results and discussion]
An A/duck/Vietnam/HU16-DD3/2023 (H5N1) strain was selected as a representative strain for vaccine
development. This strain was classified into clade 2.3.4.4b and was antigenically close to other viruses in its
clade. The candidate vaccine strain was successfully generated from the field strain, satisfying the requirementof a vaccine strain with high growth potential and low pathogenicity in eggs.
[Expected results]
Blood will be collected from chickens immunized with candidate vaccine and subjected to the antibody
detection. These chickens will be challenged with a wild-type H5 HPAIV strain. Vaccine efficacy will be
evaluated based on the survival rate of chickens and the amount of virus shedding.
Circulating viruses in poultry at LBMs in summer of 2024 will be identified and characterized. These results
will provide evidence of viruses circulating in the field in Vietnam and their characteristics, as well as a basis
for evaluating vaccine candidate efficacy.
62

P2-14

Virome analysis of lizard organs collected in Zambia
Chilekwa Frances Kabamba (1), Koshiro Tabata (1), Yukari Itakura (1), Shinji Saito (1), Bernard M. Hang'ombe(2), Hirofumi Sawa (1), Gabriel Gonzalez (3)(1)Institute for Vaccine Research and Development, Division of Biological Response Analysis, Hokkaido University, Japan(2)Department of Microbiology, University of Zambia, Lusaka(3)Division of Research Support, Institute for Vaccine Research and Development, Hokkaido University, Sapporo, Japan

Introduction: Virome studies in lizards aim to characterize the viral diversity present in these reptilian hosts.
The species of skinks belong to the family Scincidae in the order Squamata. They live on land and are commonly
found near human residences; therefore, representing a potential vector for transmission of pathogens to humans.
To the best of our knowledge, this represents the first study done on Zambian lizards. Their diverse ecologies,
evolutionary histories, and physiological adaptations make them intriguing subjects for virome research. Lizard
viromes may harbour novel viral species or variants, given the unique ecological niches and evolutionary
trajectories of these hosts. Understanding lizard viromes could also reveal potential viral reservoirs or zoonotic
threats, contributing to wildlife conservation and public health efforts.
Objectives: Identification of viruses in Zambian lizards, determination of their prevalence and distribution, as
also assessing their pathogenicity and zoonotic risk.
Materials and methods: Liver and spleen samples (n=200) were collected from lizards in Zambia from Siavonga,
Sinazongwe and Livingstone districts in August 2022. RNA was extracted from the tissue samples and thenpooled into 39 pools according to sampling locations. The species were the same in all 3 locations. RT PCR was
conducted targeting different viral families with specific primers. Sequencing of Alphavirus positive samples
was attempted with primers for pan-alpha virus amplicons; however, performed Sanger sequencing generated
no viral sequences. To further explore the possibility of virus, present in the samples, Next Generation
Sequencing was performed in the RNA pools. Ribosomal RNA depletion using ribominus kit was performed toenable viral RNA enrichment. Then, cDNA synthesis using Prime script II followed and library preparation for
Next Generation Sequencing with the Illumina platform. Data analysis is currently on-going using different
bioinformatics software. Sequence reads were compared with those in the NCBI database using Blastn and
DIAMOND. The taxonomic content of the sequences was computed using Kraken.
Preliminary results: Some positive signals for Lymphocystic disease virus and Murine leukemia virus weredetected and assembled into partial sequences using SPAdes, a de novo sequence assembler. Data analysis andconfirmatory steps are still on going.
Conclusion: Virome studies involve the comprehensive identification and analysis of viral sequences present ina host organism or environmental setting using metagenomic sequencing approaches; however, discerning false
positive from weak signals remains a challenge. From the results, it appears that some of the detected viruses
were integrated into the host genome63

P2-15

Analysis of membrane fission related protein VPS4A/B on West Nile virus replication
Kaito Maeda (1), Keisuke Maezono (1), Passawat Thammahakin (1), Ngoc Thuy Ti Duong (1), Haruto Eguchi(1), Hiroaki Kariwa (1), Shintaro Kobayashi (1) (2)(1)Laboratory of Public Health, Faculty of Veterinary Medicine, Hokkaido University(2)Institute for Vaccine Research and Development(IVReD), Hokkaido University

West Nile virus (WNV), belongs to orthoflavivirus, causes severe encephalitis in humans and horses. WNVparticle is 40-50 nm in diameter and has an envelope derived from host organelle membrane. Deformation oforganelle membrane for producing and budding viral particles was observed in WNV infected cells, but themolecular mechanisms of the deformation is still unknown. Membrane fission related protein VPS4A/B isrelated to production of transport vesicles from organelle membrane. The transport vesicles play a role inintracellular and extracellular protein trafficking and are 50-100 nm in diameter. The morphological similarity
between WNV particles and transport vesicles suggests the involvement of VPS4A/B in production of WNVparticle. In this study, we analyzed the relation between VPS4A/B and WNV.
Intracellular localization of WNV Capsid (C) protein and VPS4A/B was examined in Human neuroblastoma
SH-SY5Y cells infected with WNV (NY99 strain) or expressing C protein. The interaction between C protein
and VPS4A/B in human embryonic kidney 293T cells co-expressing C protein and VPS4A/B was analyzed byimmunoprecipitation. Viral titer was measured in WNV infected cells with inhibition of VPS4A/B byintroducing siRNAs.
VPS4A/B, which was localized throughout the cytoplasm in control cells, was aggregated with C protein in thecells infected with WNV or expressing C protein. VPS4A/B immunoprecipitated with C protein was detectedin the cells expressing both C protein and VPS4A/B. The WNV titer of the cells inhibited VPS4A/B was reduced
compared with that of control cells.
The results suggested that the interaction between VPS4A and C protein in the cytoplasm and that thisinteraction leads to promote WNV production.

64

P2-16

Characterization of WQ3034 activity on DNA-Gyrase of fluoroquinolone resistant
Mycobacterium tuberculosis
David Barnes (1), Yuki Ouchi (2), Sasini Jayaweera (1), Pondpan Moss (1), Jeewan Thapa (1), Chie
Nakajima (1) (2) (3), Yasuhiko Suzuki (1) (2) (3)(1)Division of Bioresources, International Institute for Zoonosis Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)2 International Collaboration Unit, Hokkaido University International Institute for Zoonosis Control, Sapporo, Japan;(3)3 Division of Research Support, Hokkaido University Institute for Vaccine Research and Development, Kita 20 Nishi 10, Kita-ku,
Hokkaido 001-0020, Japan

Tuberculosis (TB) is a global disease caused by Mycobacterium tuberculosis (Mtb), with around 10 million newcases and 1.6 million deaths annually. Standard treatment involves multidrug therapy with rifampicin (RIF),
isoniazid (INH), pyrazinamide, and ethambutol, targeting essential enzymes to inhibit transcription, fatty acidsynthesis, and cell wall synthesis. However, multidrug-resistant TB (MDR-TB) due to mutations in the drug
resistance-determining region (DRDR) of rpoB and katG, responsible for RIF and INH resistance, respectively,
has risen significantly and necessitating second-line drugs like fluoroquinolones (FQs).WQ-3034 (delafloxacin),a new synthetic FQ approved in 2017 for Acute Bacterial Skin and Skin Structure Infections (ABSSSIs), has
shown potency against quinolone-susceptible and quinolone-resistant gram-positive bacteria and similar activity
to ciprofloxacin against certain Enterobacteriaceae.
In this study, we examined the efficacy of WQ-3034 against Mtb using recombinant Mtb DNA gyrases throughDNA supercoiling and inhibitory assays. The DNA supercoiling assay monitors ATP-dependent supercoiling
activity of recombinant DNA gyrases in the presence of increasing drug concentrations, specificallylevofloxacin, moxifloxacin, and WQ-3034. Additionally, we evaluated the in vitro antimycobacterial activity of
WQ-3034 against recombinant Mtb var. bovis Bacille Calmette-Guerin (BCG) strains, where gyrase-coding
genes were replaced with Mtb variants containing resistance-conferring mutations. The minimum inhibitory
concentrations (MICs) of the selected drugs were determined using the broth microdilution method, conductedin duplicate with a 40-fold dilution of a bacterial culture at OD 600nm = 0.15.
The results revealed that the IC50 of WQ-3034 against wild-type (WT) DNA gyrase was nearly equivalent tomoxifloxacin (MFX), while levofloxacin (LVX)'s IC50 was about 1.4 times higher. WQ-3034 showed lower
IC50 values for most mutants compared to MFX, except for the D94A mutant of DNA gyrase A subunit. MFX's
IC50 values increased by 1.2-fold to 5.2-fold, with the highest increase in the G88C mutant. LVX had
significantly higher IC50 values for both WT and mutant DNA gyrases, ranging from 1.4-fold to 75-fold higher
than WQ-3034, with the highest resistance in D94N, D94Y, and G88C mutants. WQ-3034 demonstrated potent
activity, with the lowest MIC (0.03125 ug/mL) against the wild-type strain and higher efficacy against strains
with mutations at position 94 of GyrA and G88C compared to other drugs. MICs of MFX and LVX ranged from0.25 ug/mL to 16 ug/mL and from 0.0625 ug/mL to 8 ug/mL, respectively.
In conclusion, WQ3034 appears to be an effective drug against the tested recombinant BCG strains, including
those with mutations commonly associated with fluoroquinolone resistance.

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P3-2

Investigation of Yersinia species carriage in pigs at a slaughterhouse in Kagoshima Prefecture
Masahiro Toda (1), Yoshiyuki Tomino (2), Aya Komuta (2), Keishiro Akasaka (2), Seiji Uto (2)(1)Division of Global Epidemiology, Graduate School of Infectious Diseases, Hokkaido University, Japan(2)Akune Meat Inspection Center, Health and Welfare Department, Kahoshima Prefecture, Japan

Yersinia species are Gram-negative rod-shaped bacteria. Two Yersinia species, Yersinia enterocolitica (Y.e) andYersinia pseudotuberculosis (Y.p) are known to cause foodborne illnesses. There have been reports of outbreaks
in the past, and Yersinia has been detected in raw pork. Yersinia is a psychrophilic bacterium capable ofproliferating at 4°C, thus able to grow under refrigerated conditions. Many mammals carry Yersinia, with pigs
having a particularly high carriage rate. This study aimed to evaluate the contamination risk of Yersinia in pork
by investigating the presence and pathogenicity of Yersinia species in the intestines of pigs transported to aslaughterhouse.
Rectal contents of pigs introduced to the slaughterhouse were used as the isolation material. One gram of rectal
feces was mixed with 9 ml of PBS and subjected to enrichment culture at 4°C for 4 weeks. After alkaline
treatment, the mixture was inoculated on CHROMagar Y. enterocolitica and cultured at 30°C for 24-36h.
Suspicious colonies were purified and identified using Api rapid ID 32E. The identified Y.e isolates were thensubjected to serotyping and biotyping. To investigate the pathogenicity of isolates, auto-agglutination test and
PCR detection of genes associated with pathogenicity were conducted.
From 2022 to 2024, 150 samples were collected from 14 farms, and Y.e was isolated from 34 samples across 7of the 14 farms (isolation rates per positive farm ranged from 20 to 90%). One sample tested positive for either
Y.p, Y. ruckeri (Y.r), which causes redmouth disease in fish, or Pantoea spp. Additionally, Y. frederiksenii (Y.f),known to exhibit pathogenicity in fish, was isolated from 3 samples, and Y. intermedia, which is considered
non-pathogenic to humans, was isolated from 8 samples. All Y.e isolates were identified as serotype O3 andbiotype 4. All Y.e isolates tested positive for auto-agglutination and for genes associated with pathogenicity.
In this survey, all Y.e isolates were identified as serotype 3 and biotype 4 and were found to possess genes
associated with pathogenicity. This indicates that pigs transported to the slaughterhouse harbored pathogenic
Y.e. Therefore, proper trimming procedures are crucial in cases where contamination with intestinal contents isobserved during the butchering process. Moreover, given the disparity in isolates across farms, variations incontamination levels are expected among different farms. The results of this study will be utilized to promote asafer meat production.

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P3-3

Molecular characterization of measles virus in the Democratic Republic of Congo
Francisca Mawete Muyembe (1) (2) (3), Junior Bulabula (2) (3), Meris Matondo (2) (3), Patrick Mukadi (2)(3), Emmanuel Lokilo (3), Princesse Phaku (3), Eddy Kinganda (2) (3), Tony Wawina (2) (3), Placide Mbala(2) (3), Steve Ahuka (2) (3), Jean-Jacques Muyembe (2) (3), Kimihito Ito (1)(1)Division of Bioinformatics, Graduate School of Infectious Diseases, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Laboratory of Microbiology, Faculty of Medicine, Kinshasa University, Democratic Republic of Congo(3)Laboratory of Virology, Institut National de Recherche Biomedicale, Democratic Republic of Congo

Background
Measles virus genotyping is an important tool in the measles elimination program. It allows monitoring of virus
circulation in the community and evaluation of measles elimination interventions by vaccination. The World
Health Organization recommends member countries to regularly genotype the measles virus, especially thoseprone to recurrent epidemics. In the Democratic Republic of Congo (DRC), there has been no assessment ofcirculating strains for over a decade.
Objectives
The present study aimed to characterize the measles virus circulating in the DRC. Specifically, we will 1)
determine the genotype of the measles virus circulating in the DRC, and 2) determine the geographic distributionof circulating measles virus genotypes in the DRC.
Methods
We conducted a cross-sectional study in 13 provinces reporting measles cases in DRC. Among them are
Kinshasa, Kwango, Kongo Central, Tshopo, Bas-Uele, Haut-Uele, Tanganyika, Sankuru, Sud-Kivu, Lomami,
Kasai, Kasai Central, and Mongala. We selected 186 participants who were suspected of having measles
according to the case definition published by the Direction Epidemiologique de Surveillance. We collectedsocio-demographic information of each participant such as the province of origin, health zone, and clinicalsymptoms relating to the history of the disease using investigation forms. An oropharyngeal swab was takenfrom each participant, put in a virus transport media, and transported to the National Institute for Biomedical
Research (INRB) at four Celsius degrees. Samples collected far from the capital city were stored at -20 Celsius
degrees before shipment. The ribonucleic acid extracted was tested by Reverse Transcriptase-Polymerase Chain
Reaction (RT-PCR). Samples with threshold cycle (CT) values below 35 were considered positive. For all
eligible samples (CT30), sequencing was performed on the Oxford Nanopore Technology platform using theNative Barcoding kit after conventional PCR using specific primers targeting the C-terminal of the N gene.
Sequence reads were recorded as Fast5 files and translated into Fastq files using the Guppy algorithm.
Nucleotide sequences were obtained using the "artic measles" platform. Phylogenetic analyses were conductedusing MEGA X software.
Results and conclusion
Out of 186 samples, 131 resulted in positive by RT-PCR. The positivity rate within collected samples was 70%
with a 95% confidence interval from 63% to 77%. Out of the 13 provinces we investigated, 12 provinces had
positive samples and 1 province had not. A total of 94 positive samples had CT below 30. Phylogenetic analysisof these sequences showed the circulation of the B3 genotype in the 12 provinces.

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P3-4

Doxorubicin cytotoxicity and NF-kB signaling pathway in the H9c2 rat cardiomyocyte cell line
induced to differentiate by retinoic acid.
Mawo Shimizu (1), Masami Morimatsu (1), Teppei Nakamura (1)(1)Laboratory of laboratory of animal science and medicine, Department of Applied Veterinary Science, Faculty of Veterinary Medicine,
Hokkaido University, Japan[Background and Purpose] Clinical use of the anthracycline doxorubicin (DOX) increases the risk of serious
cardiovascular disorders such as heart failure, and although the mechanism of DOX-induced myocardial cell
injury has been extensively studied, the details remain unclear. In this study, we focused on the nuclear factor
kappa B (NF-kB) signaling pathway, which regulates inflammation and apoptosis. We performed molecularbiological analysis of DOX-induced cardiomyocyte injury using the rat cardiomyocyte cell line H9c2, which
was differentiated into a myocardial-like phenotype by the addition of all-trans-retinoic acid (RA).
[Methods] H9c2 cells were differentiated into cardiomyocyte-like cells by adding RA. We added various
concentration of DOX , and analyzed cell viability, phosphorylation of NF-kB/p65 by Western blotting, andexpression levels of NF-kB transduction system component genes and their target genes by real-time PCR after
the addition.
[Results] Cell viability in response to DOX was decreased in cardiomyocyte-like H9c2 cells. The expression
levels of inflammatory cytokines and NF-kB target genes were significantly increased in cardiomyocyte-like
H9c2 cells, and phosphorylation of p65, an indicator of NF-kB transduction system activation, was also
observed. In particular, the expression level of NF-kB transduction system component gene Nfkbiz was
significantly increased by DOX. The expression levels of NF-kB target genes were decreased in the presence of
NF-kB inhibitors.
[Discussion]Nfkbiz is a component of the NF-kB transduction system and regulates NF-kB activity in the
nucleus upon LPS stimulation of immune system cells. Since Nfkbiz expression was markedly increased by
DOX stimulation in cardiomyocyte-like differentiated H9c2 cells, it is predicted that Nfkbz and NF-kB pathway
are closely related to the heart injury and might be a major driver of the intracellular signaling pathway during
the cardiotoxicity of DOX.

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P3-5

Analysis of the molecular basis of host specificity of Babesia bovis using an artificial evolutionary
approach
Khesara Sastrin Prasita Negara (1), Junya Yamagishi (1)(1)Division of Collaboration and Education, International Institute for Zoonosis Control, Hokkaido University, Japan

B. bovis is a non-zoonotic protozoan parasite that infects bovine red blood cell (bRBC). Major manifestations
in bovine babesiosis are high fever, anorexia, ataxia, nervous signs, progressive haemolityic anemia, and evendeath which causes severe economic, social and epidemiological impacts. In a preliminary study at International
Institute for Zoonosis Control (IIZC), they have established adapted clones of B. bovis to human RBC (hRBC).
However, it is found that there is no genetic difference between the parent and the adapted clones, implying
involvement of molecular mechanisms other than genomic mutation. Subsequently, comparative transcriptome
has performed, then 199 genes were up regulated in the adapted clone. Merozoite surface antigen 2a2 gene (msa2a2) was one of them and encode membrane proteins involved in erythrocyte invasion. Meanwhile, the multitransmembrane type b family genes (mtmb) encode predicted multi trans membrane integral membrane protein
which are known to be exported and expressed on the surface of infected RBC. Therefore, msa 2a2 and mtmb
are the promising candidates which determine host specificity in B. bovis. To prove the hypothesis, recombinantparasites will be constructed. On the other hand, we observed change in transcriptome among the parent andadapted clone without changes in genome, suggesting involvement of epigenome. DNA methylations is one of
the implementation of epigenome and no conclusion has been reached as to whether it exists in apicomplexanparasites such as Toxoplasma and Plasmodium. We will examine involvement of DNA methylations in theadaptation using nanopore sequencing which can discriminate unmethylated, 5-methylcytosine (5-mc) and 5hydroxymethylcytosine (5-hmc). Chromatin remodeling is also a part of epigenome. We will apply ATAC-seq
to examine its involvement as well. Moreover, additional deferences in phenotype between the parent andadapted organism will be examined. Infectivity of the hRBC adapted parasites to bRBC is included. The
particular step which hinder infection of the non-adapted B.bovis to hRBC would additionally be investigatedby microscopic observation. This study will demonstrate how versatile the host specificity of Babesia spp. andmolecular mechanism behind them which closely related to evolution and zoonotic risk of the pathogen.

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P3-6

Pharmacokinetic analysis of baloxavir marboxil in rare bird species to establish the treatmentof high pathogenicity avian influenza virus infection
Yo Shimazu (1), Mariko Miki (2), Ryo Obara (2), Yumiko Nakaya (3), Takashi Nagamine (3), Yuto Yoshimoto(4), Izumi Ide (5), Takahiro Hiono (1) (6) (7) (8), Norikazu Isoda (1) (6) (7) (8), Takao Shishido (9), Yoshinori
Ikenaka (6) (10) (11), Yoshihiro Sakoda (1) (6) (7) (8)(1)Laboratory of Microbiology, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Pharmaceutical Research Center, Shionogi & Co., Ltd., Japan(3)Okinawa Wildlife Federation, Japan(4)Tokyo Sea Life Park, Tokyo Zoological Park Society, Japan(5)Toyama Wildlife Conservation Center, Toyama Outdoor Nature Museum, Japan(6)One Health Research Center (OHRC), Hokkaido University, Japan(7)Institute for Vaccine Research and Development (IVReD), Hokkaido University, Japan(8) International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Japan(9)Drug Discovery & Disease Research Laboratory, Shionogi & Co., Ltd., Japan(10)Laboratory of Toxicology, Graduate School of Veterinary Medicine, Hokkaido University, Japan(11)Translational Research Unit, Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Hokkaido University, Japan[Background and aim]
High pathogenicity avian influenza virus (HPAIV) infections in captive rare birds have reported during 20222023 seasons. Since no vaccine against HPAIV is available for birds in Japan, antiviral drug administration has
been considered for treatment against HPAIV infection in rare birds from the perspective of conservation
medicine. Previous research showed that oral administration of the human anti-influenza drug, baloxavir
marboxil (BXM) at 2.5 mg/kg, can prevent chickens from the onset of HPAIV. BXM is a prodrug that exerts anantiviral effect by being metabolized to the active form, baloxavir acid (BXA), and the efficiency of thismetabolism was varied among bird species. In this study, the pharmacokinetics of BXM and BXA in four bird
species were analyzed aiming to apply BXM to the treatment of HPAIV infection in captive rare birds. Besides,
supposing the difficulty in oral administration of the drugs to birds infected with HPAIV, the availability of
subcutaneous administration of BXM was assessed by its irritation and pharmacokinetics following
administration.
[Materials and methods]
BXM was administered orally to nine Okinawa rails, four Humboldt penguins, two peregrine falcons and 14
chickens, and plasma was collected 2, 8, 24, and 48 hours post-administration. BXM was administeredsubcutaneously to four chickens, and plasma was collected 2, 8, 24, 72, 168, and 336 hours post-administration.
Concentrations of BXM and BXA in the plasma were measured by liquid chromatography-mass spectrometry
to analyze the pharmacokinetics. 2 weeks after the administration, the chickens administered subcutaneouslywith BXM were euthanized to perform the histopathological search at the injection site.
[Results and discussion]
No side effects were observed in all the birds orally administrated. After the oral administration of BXM at 2.5
mg/kg, the plasma concentrations of BXA increased to 328 ng/mL in Humboldt penguins, 302 ng/mL inchickens, but only 1.8 ng/mL in Okinawa rails, and 12.3 ng/mL in peregrine falcons. After the subcutaneousadministration of BXM at 2.5 mg/kg to chickens, the maximum plasma concentration of BXA was 1189 ng/mL,
approximately 3.9 times that of oral administration. Subcutaneous administration of BXM demonstrated alonger half-life of BXA than oral administration. Inflammation at the injection site of these chickens was notsevere, ensuring the practicality of subcutaneous administration instead of oral administration. Similaradministration studies in other bird species will be conducted to establish the protocol for the therapeutic
administration of BXM.

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Effect of Fuzapladib Sodium Hydrate on expression of adhesion molecule in canine endothelial
cell
Kanittha Darawiroj (1), Takafumi Sunaga (1), Masahiro Okumura (1)(1)Laboratory of veterinary surgery, Faculty of Veterinary Medicine, Hokkaido University, Japan

Inflammation is a fundamental defense mechanism in response to harmful stimuli, involving complex processes
including changes in vascular permeability, leukocyte recruitment and accumulation, and the release of
inflammatory mediators. These processes are critical for restoring tissue balance and resolving acuteinflammation. However, uncontrolled activation and infiltration of immune cells can lead to organ damage anddysfunction. The regulation of neutrophil recruitment depends on endothelial adhesion molecules such as
ICAM-1, P-selectin, E-selectin, and CXCL1. Fuzapladib sodium hydrate effectively inhibits neutrophil
extravasation by targeting the leukocyte function-associated molecule 1 (LFA-1). Nonetheless, the expressionof LFA-1 is governed by a bidirectional signaling process involving both inside-out and outside-in signals,
which directly interact with endothelial adhesion molecules, resulting in a conformational transformation froma low-affinity state to a high-affinity state. The primary aim of this study is to evaluate the impact of fuzapladib
sodium hydrate on the expression of endothelial adhesion molecules, providing insights into its potential
mechanism of action.
This research was conducted in vitro model using primary canine aortic endothelial cell (CaAEOC) to evaluate
the effect of flazapladib sodium hydrate on adhesion molecules including ICAM-1, P-selectin, E-secectin, andCXCL-1. The CaAOEC was incubated with either cytokine (LPS, cTNFa, or cIL-8) alone or combined with
Fuzapladib in a variety of dosages. Fuzapladib impact on the gene expression of candidate adhesion molecules,
including E-selectin, P-selectin, and ICAM-1, was investigated. The results demonstrate that Fuzapladib has no
significant effect on down-regulating E-selectin and ICAM-1 expression in LPS, cTNFa, and cIL-8-induced
CaAOEC. However, Fuzapladib significantly down-regulated P-selectin gene expression in a dose-dependent
manner in cTNFa-induced CaAOEC. The effect of fuzapladib on P-selectin protein will be evaluated by theflow cytometry method. Additionally, Fuzapladib had no significant effect on decreasing CXCL-1 chemokine
in LPS, cTNFa, and cIL-8-induced CaAOEC. The effect of fuzapladib effect on NFkB pathway will be
determine by immunocytochemistry method. As well as JNK and p38 pathways will be identified by the western
blot method. The neutrophil adhesion on endothelial cells will be determined by adhesion assay.
The expected outcome of this research is to contribute to the understanding of the fuzapladib mechanism ofaction, establish the appropriate dose and duration of treatment, and provide recommendations for clinicalpractice. This research may also be applied to other inflammatory disease conditions or used in further
inflammation research studies.

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P3-9

Influence of environmental toxicants on avian gut microbiome
Kohei Ogasawara (1), Rintaro Yano (2), Kazuaki Ito (2), Ayuko Morita (1), Masashi Koide (1), Yurika Tokunaga(1), Yamato Mashimo (1), Mizuki Maruyama (1), Yoshinori Ikenaka (3), Ryo Nakao (4), Naoki Fukuma (5),Shouta M.M. Nakayama (1), Mayumi Ishizuka (1)(1)Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Faculty of Veterinary Medicine, Hokkaido University,Japan(2)Graduate school of Animal and Veterinary Sciences and Agriculture, Obihiro University of Agriculture and Veterinary Medicine(3)One Health Research Center, Hokkaido University(4)Laboratory of Parasitology, Department of Disease Control, Hokkaido University(5)Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine[Background] Gut microbiome is closely related to the host in many aspects. In addition, gut microbiome issensitive to changes in the gut environment and rapidly changes their composition depending on what hostanimals ingest. By focusing on this sensitivity and analyzing changes in the gut microbiome, this study aims toevaluate the effects of environmental toxicants on the host.
[Methods] Chickens (Julia, 6-weeks-old) were divided into 6 groups (DW, corn oil, lead, imidacloprid, warfarin,
and nanoplastic) and each group was administered orally one time. The animals were kept in isolation for one
day prior to administration, and fresh feces were collected daily until three days after administration. All
chickens were euthanized at the final day, and organs (brain, heart, intestine, colon, liver, kidney, spleen, andlung) were collected. Bacterial DNA was extracted from each feces and analyzed using a next generation
sequencer targeting the V3-V4 region of 16S rRNA. For gene expression analysis, qPCR was performed
targeting cytokines (INFα, TGFβ, IL-17F) and toll-like receptors (TLR3, TLR4, TLR5).
[Results and Discussion] Significant reductions in alpha diversity were observed in the lead, imidacloprid, andwarfarin groups. The relative abundance of Lachnospirales and Oscillospirales increased in the lead group, andthe abundancer of Bacillales increased after 3 days of treatment. In the imidacloprid group, the composition of
the main flora did not differ from that of the control group, but the number of bacterial species decreased fromthe day of administration and was comparable to that before administration 3 days later. The warfarin group also
showed a decrease in the number of bacterial species, which did not recover even after 3 days. In the nanoplastic
group, the abundance of Lachnospirales and Oscillospirales increased after administration as in the lead group,
but the abundance of Lactobacillales increased after 3 days of administration. From the gene expression analysis,
however, host immune response was limited. Although most genes did not show significant change amongorgans, the lead, warfarin and nanoplastics groups showed the decrease of TLR4 and TLR5 level in colon.
Moreover, IL-17F level in liver was lower in the all groups than control group. As described above, characteristic
fluctuations in gut microbiome were observed for each environmental toxicant even after a single administration.
The results of gene expression analysis suggested that the change of microbiome occurred prior to host immune
response.

72

P3-10Non-target metabolomics and lipidomics analysis and biomarker search of the mouse brain
following neonicotinoid administration by using LC/Q-TOF/MS
Tomoya Koike (1), Anri Hirai (1), Kei Nomiyama (2), Akihumi Eguchi (3), Nobuhiko Hoshi (4), Tetsushi
Hirano (5), Collins Nimako (1), Yared B. Yohannes (1), Mai Tamba (1), Shouta M.M. Nakayama (1), Mayumi
Ishizuka (1), Yoshinori Ikenaka (6)(1)Laboratory of Toxicology, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Center for Marine Environmental Studies,Division of Environmental Chemistry and Ecotoxicology, Ehime University, Japan(3)Department of Sustainable Health Science, Center for Preventive Medical Sciences, Chiba University, Japan(4)Department of Animal Science, Laboratory of Animal Molecular Morphology, Graduate School of Agricultural Science, Kobe
University, Japan(5)Faculty of Pharmaceutical Sciences, University of Toyama, Japan(6)One Health Research Center, Hokkaido University, Japan

In recent years, psychiatric disorders such as attention deficit hyperactivity disorder (ADHD), developmental
disorders, depression and anxiety disorders are thought to be caused by exposure to certain chemicals. However,current toxicity testing methods are limited in their focus on effects on higher brain function, making it difficult
to accurately and objectively assess neurotoxicity. Therefore, it is necessary to establish more sensitive, specific,
objective, and high-throughput testing methods.
The neonicotinoid insecticides (NNs) used in this experiment are widely used pesticides worldwide; they weredeveloped as insecticides with high selective toxicity to insects as agonists of insect nicotinic acetylcholine
receptors (nAChRs). On the other hand, it has recently been reported that central nervous system (CNS)
symptoms have developed in mammals such as humans by acting not only on insect nAChRs but also on
mammalian nAChRs.
In this experiment, NNs, substances that cause neurotoxicity, were administered to mice as model compounds,
and their effects on the CNS and potential biomarker compounds were evaluated using a mass spectrometer.
In this experiment, acetamiprid (ACE), one of the NNs, was administered orally to C57BL/6 mice atconcentrations of 65 (Group L) and 130 mg/kg (Group H), respectively, and distilled water containing 5 %DMSO was administrated as a control (Group C). 30 minutes after administration, these mice were euthanized.
These concentrations were set at values that would reliably produce neurotoxicity. Subsequently, metabolomes
and lipidomes in the cerebral cortex of mice were analyzed non-targeted by using LC/Q-TOF/MS (Agilent 6546).
Time alignment, peak extraction, and multivariate analysis were performed using agilent software.
Even within 30 minutes after administration, ACE and its metabolites were detected in the cerebral cortex ofGroups L and H and both groups showed significant differences from the control group (Steel test p 0.05).
Nearly 400 compounds were detected as significantly different between groups C and L, C and H, and L and H,
respectively. Firstly, PCA analysis, one of the multivariate analyses, was performed and unfortunately any
characteristic principal components were not detected among the groups. Next, the compounds with significant
differences were analyzed individually, and significant increases were detected for inflammatory mediators,
primarily arachidonic acid. There were also several other compounds that were not annotated but showedsignificant differences and large fold changes.

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P3-11

Prevalence and antimicrobial resistance of Campylobacter spp. in chicken and cattle in Mongolia
Erdenebat Bulgan (1), Zolzaya Byambajav (2), Narantuya Ayushjav (2), Yuji Hirai (1), Momoko Nakayama(1), Misaki Tanaka (1), Nyam-Osor Purevdorj (3), Akio Suzuki (1), Toyotaka Sato (1) (4), Motohiro Horiuchi(1) (4)(1)Laboratory of Veterinary hygiene, Department of Infectious Disease, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Laboratory of Veterinary Sanitation and Hygiene, Ulaanbaatar Veterinary Department, Ulaanbaatar, Mongolia(3)School of Veterinary Medicine, Mongolian University of Life Science, Ulaanbaatar, Mongolia(4)One Health Research Center, Hokkaido University

Background:Campylobacter spp. is the most prevalent foodborne bacteria in the world, with chicken and cattle being primarysources of human infection. Antimicrobial resistance (AMR) of bacteria increasing every year, poses significant
public health concerns. According to WHO report, Mongolia had the highest consumption of antibiotics among
65 countries in 2015. However, there is a lack of epidemiological data on Campylobacter spp. prevalence andAMR in livestock. Thus, we aimed to isolate and identify Campylobacter spp. from chicken and cattle in
Mongolia.
Methods:
Cloacal (71) and rectal swabs (100) were collected from 5 chicken (4 layer :1 broiler) and 10 cattle farms in
Mongolia. Isolation of Campylobacter spp. was performed using Preston broth were incubated for 24 h at 42°C
under microaerobic condition. Then inoculated to mCCDA agar and CHROMAgar-Campylobacter incubatedfor 48 h at 37°C under microaerobic conditions. Campylobacter species were identified using multiplex PCR,MALDI Biotyper and nucleotide sequencing of 16srDNA.. Antimicrobial resistance profiles were determinedby Clinical and Laboratory Standards Institute (CLSI) guideline using E-tests.
Results:
Campylobacter spp. isolation ratio were in layer farms 55.3% (31/56), and in cattle farms 34% (34/100) but notisolated in a broiler farm (0/15). Among the isolates, 25 C. jejuni (80.6%) and 6 C. coli (19.4%) were found inchicken, while 22 C. jejuni (64%), 7 C. hyointestinalis (20.5%), 4 C. fetus (11.7%), and 1 C. lari (2.9%) were
found in cattle, with all species confirmed by MALDI Biotyper. Chicken isolates high resistance to tetracycline(100%) and fluoroquinolone (51.6%) and half of C. coli isolates showed multi-drug (50) resistance. On thecontrary, almost all cattle isolates were sensitive to ciprofloxacin, tetracycline and erythromycin except for one
C. jejuni isolate. All isolates of C. hyointestinalis (7), C. fetus (4), and C. lari (1) were resistant to nalidixic acid,
which is a natural characteristic of these species.
Conclusion:
This is the first report of Campylobacter spp. presence in livestock and their antimicrobial resistance inMongolia. Campylobacter spp. were widely prevalent in both chicken (43.6%) and cattle (34%) farms in
Mongolia. High antibiotic resistance was found in chicken isolates, while it was lower in cattle isolates,suggesting potential differences in antimicrobial usage and resistance profiles between these livestock species.
Further studies in animal-derived food products and human patients are essential to understand their relationshipand transmission routes.

74

P3-12

Effect of polymorphisms in the basic region of Meq protein of Mareks disease virus, an avian
oncogenic herpesvirus, on virulence
Jumpei Sato (1), Murata Shiro (1) (2), Yoshinosuke Motai (1), Shwe Yee Win (1), Shunsuke Yamagami (1),Fumiya Horio (1), Naoya Maekawa (2), Tomohiro Okagawa (2), Satoru Konnai (1) (2) (3), Kazuhiko Ohashi(1) (2) (4)(1)Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan(2)Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan(3)Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo, Japan(4)International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan

Mareks disease virus (MDV) is an alphaherpesvirus that causes Mareks disease (MD) characterized bymalignant lymphomas in infected chickens. MDV field strains exhibit increased virulence, and sporadic
outbreaks are still reported despite vaccination. The meq oncogene of MDV encodes a 339 amino-acid protein
and is essential for the transformation of chicken CD4+ T cells. A recent study using recombinant MDV (rMDV)
revealed that polymorphisms in Meq are involved in the enhanced virulence of MDV in vivo. Most MDV strains
isolated in Japan display glutamic acid and tyrosine at positions 77 and 80 in the basic region of Meq, whilethese amino acid residues in MDV strains in the United States are lysine and glutamic acid, respectively. In thisstudy, we aimed to investigate whether the differences in the amino acid residues in the basic region of Meq
contribute to its functions and MDV virulence. To analyze the effect of polymorphisms in the basic region on
the transcriptional regulation, we introduced amino acid substitutions observed in Japanese isolates at positions
77 and 80 in Meq (Meq(77-80)) of the US isolate, RB-1B. Reporter assays revealed that the amino acidsubstitutions in the basic region decreased both transactivation activity and transcriptional repression.
Additionally, we generated an RB-1B-based recombinant MDV encoding Meq or Meq(77-80) (rMDV_Meq orMeq(77-80), respectively) and analyzed T cell dynamics in chickens infected with rMDVs by flow cytometry.
Compared to rMDV_Meq, rMDV_Meq(77-80) showed mild disease progression, but exhibited different clinicalmanifestations, such as open-mouth behavior and body weight loss. In chickens infected with rMDV_Meq, theproportions of CD4+ T cells, which are targets for the transformation by MDV, were increased, whereas anincrease in the percentage of CD8+ T cells was observed in rMDV_Meq(77-80)-infected chickens. Thesefindings suggest that polymorphisms in the basic region of Meq alter the pathogenesis of MDV.

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P3-13

Genetic insight into the viral epidemic patterns in clade 2.3.4.4b H5N1 high pathogenicity avian
influenza virus in Japan during 2021-2024
Lim Yik Hew (1), Norikazu Isoda (1) (2) (3) (4), Takahiro Hiono (1) (2) (3) (4), Daiki Kobayashi (1),
Loc Tan Huynh (1), Linh Bao Nguyen (1), Yoshihiro Sakoda (1) (2) (3) (4)(1)Laboratory of Microbiology, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)One Health Research Center, Hokkaido University, Japan(3)International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Japan(4)Hokkaido University Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Japan[Objectives]
High pathogenicity avian influenza viruses (HPAIVs) infection has continued to impact poultry and wild birds
globally. Since the introduction of clade 2.3.4.4b H5N1 HPAIVs to Japan in late 2021, the situation has
worsened due to the continuous introduction of the viruses in this clade in each subsequent years, leading tovirus spillover to local birds, poultry, and even mammals. Therefore, H5 HPAIVs isolated in Hokkaido and thesouthern part of Japan between 2021-2024 were subjected to the phylodynamic analysis based on the geographic
locations and collection dates to understand the origin, evolution, and spread of H5 HPAIVs in Japan.
[Materials and methods]
Fecal samples collected from the migratory birds, tracheal or cloacal swabs obtained from deceased birds, andorgan homogenates from deceased mammals were inoculated into 10-day-old embryonated chicken eggs toisolate viruses. The isolated viruses were serologically subtyped through the hemagglutination inhibition test
and pathotyped by nucleotide sequences encoding the cleavage site of the hemagglutinin (HA) gene.
Furthermore, all eight gene segments of HPAIVs were analyzed phylogenetically after determining the fullgenome sequences by next-generation sequencing. Bayesian phylodynamic analysis was performed on the
concatenated eight gene segments of Japan H5 HPAIVs to investigate the virus transmission between thegeographic locations.
[Results and discussions]
Phylogenetic analysis of the HA gene has revealed that all HPAIVs isolated in 2022-2024 were categorized into
clade 2.3.4.4b, classified into four subgroups: G2a, G2b, G2c, and G2d, and most HPAIVs was clustered in thesubgroup of G2d. Isolates in the subgroups of G2b and G2c were mainly composed of isolates circulated in
Honshu Island of Japan in 2021-2023. All isolates in the subgroup of G2a were genetically very close to H5N5HPAIVs circulated in Northern Europe since 2022. Further genetic analysis revealed that seven different geneconstellations were detected during this period. Phylodynamic analysis suggested multiple incursions of
HPAIVs with various characteristics to Japan during 2021-2024 via bird migration. Introducing these viruses toJapan has further led to the persistence of HPAIV in wild bird populations in Japan in each subsequent winterseason, allowing the generation of HPAIV with genetically distinct lineage and further geographic spread due
to bird migration throughout Japan. The current event highlights the importance of continuous monitoring andrapid information sharing, which are crucial to understanding the global dynamic of HPAIV.

76

P3-14Isolation of novel bat parainfluenza virus from Egyptian fruit bat in Zambia
Tomoya Tsubo (1), Naganori Nao (1) (2), Katendi Changula (3), Hayato Harima (4), Yongjin Qiu (5), Akina
Mori-Kajihara (1), Yoshiki Eto (1), Michihito Sasaki (1), Edgar Simulundu (3) (6), Daniel Mwizabi (7), Bernard
M Hangombe (3) (8), Hirofumi Sawa (1) (2) (9) (3), Ayato Takada (1) (2) (3), Masahiro Kajihara (1)(1)International Institute for Zoonosis Control, Hokkaido University, Japan(2)One Health Research Center, Hokkaido University, Japan(3)School of Veterinary Medicine, the University of Zambia, Zambia(4)Faculty of Agriculture, Tokyo University of Agriculture and Technology, Japan(5)Faculty of Veterinary Medicine, Hokkaido University, Japan(6)Macha Research Trust, Choma, Zambia(7)Department of National Parks and Wildlife, the Ministry of Tourism and Arts, Lusaka, Zambia(8)Africa Center of Excellence for Infectious Diseases of Humans and Animals, University of Zambia, Zambia(9)Institute for Vaccine Research and Development, Hokkaido University, Japan

Recently bats have been recognized as an important carrier of known and potential zoonotic viruses, including
paramyxoviruses. To understand the distribution and genetic diversity of bat paramyxoviruses in Zambia, 180
Egyptian fruit bats were captured in 2015, and the colon homogenates were inoculated onto Vero E6 cells
expressing transmembrane serine protease 2 (Vero E6/TMPRSS2 cells) and ZFBK15-137RA cells derived from
Egyptian fruit bat kidney. Vero E6/TMPRSS2 cells inoculated with one of the samples showed mild cytopathic
effects such as cell detachment and syncytium formation. Through deep sequencing analysis and subsequentbioinformatics, a 15,630 nt sequence similar to the human parainfluenza virus 2 (HPIV2) genome was detectedin the supernatant. The whole-genome analysis of this bat parainfluenza virus (BPIV) indicated that the virus
genome encodes N, V/P/I, M, F, HN, and L proteins as HPIV2 does. The homology of each gene between HPIV2and the isolated BPIV is 69-77% and 75-91% in nucleotide and amino acid sequences, respectively.
Phylogenetic analysis demonstrated that this BPIV showed the closest relationship with HPIV2 among all
known paramyxoviruses. To assess the pathogenic potential of the isolated BPIV strain in animal models, mice,
rats, and hamsters were infected with the virus and clinically monitored for 14 days after infection. Only
hamsters showed mild body weight loss upon BPIV infection, and the other animals did not manifest clinicalsigns. These findings indicate that BPIV isolated in the present study has the potential for cross-species infection.
Since the pathogenicity and infectivity of BPIV in humans are totally unclear, further characterization of thevirus is required to assess the risk of BPIV as an agent for emerging diseases.

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P3-15

Establishment of comprehensive molecular detection system for rodent/shrew-borne
henipaviruses and screening in rodent and shrews in Japan
Jingshu Li (1), Isshu Kojima (2), Keita Mizuma (1), Yuma Ohari (1), Mebuki Ito (1), Hayato Torisu (3), Satoshi
Ohdachi (4), Izumi Kida (1), Keita Matsuno (1)(1)Division of Risk Analysis and Management, International Institute for Zoonosis Control, Hokkaido University, Japan(2)Faculty of Veterinary Medicine, Okayama University of Science, Japan(3)Faculty of Environmental Earth Science, Hokkaido University, Japan(4)Institute of Low Temperature Science, Hokkaido University, Japan

Background: The Henipavirus genus (family Paramyxoviridae) is mainly constituted with bat-borne viruses
including highly pathogenic Hendra and Nipah viruses. Recently, a number of henipaviruses that are geneticallydistinct from the bat-borne viruses has been reported from rodents and shrews especially in East Asia; Mojiangand Langya viruses found in China and Gamak and Daeryong viruses detected from shrews in South Korea.
Notably, in China, Langya virus infection was confirmed in patients showing fever and respiratory symptoms,suggesting zoonotic infections of rodent/shrew-borne henipaviruses. However, there is currently no reports ofhenipavirus detection in Japan. Thus, we aim to establish a comprehensive detection method for therodent/shrew-borne henipaviruses for applying to the genetic screening in Japan.
Method and material: We designed primer sets capable to detect all the reported rodent/shrew-borne henipavirus
sequences on NCBI, based on the primers previously reported for a pan-paramyxovirus RT-PCR system(Vanmechelen B et al., 2022). The designed primer sets were initially screened using SYBR Green RT-qPCR
with a plasmid containing Langya virus L gene as a template. Then, the most specific and sensitive primer set
was used for establishing a conventional one-step RT-PCR system. Rodents and shrews were captured using
sherman traps and panchu traps in different locations in Japan, mainly in Hokkaido. After the morphological
identification of species, rodents and shrews were euthanized, and their kidneys and oral swabs were collected.
RNAs extracted from the kidney and oral swab samples, we performed the RT-PCR for the screening ofhenipaviruses.
Results: In total, 6 primers were newly designed based on the henipavirus sequences, and 24 primer
combinations together with the paramyxovirus primers were tested. Screening using SYBR Green RT-qPCR,
we use following two criteria to choose a primer set for following analysis ; Lower Ct value and a single peakin the melting curve throughout the different template concentration. As a result, one primer set was used forscreening samples by conventional PCR. From June 2023 to March 2024, we collected 192 rodent and shrew
samples in Hokkaido, mainly, 47 samples are Apodemus speciosus, 35 samples are Sorex unguiculatus, and 33
samples are Sorex gracillimus. Currently, we are screening samples for rodent/shrew-borne henipaviruses using
the newly established RT-PCR system, and metagenome analysis of these samples is also ongoing.
Conclusion: A comprehensive detecting system for rodent/shrew-borne henipaviruses was established, and wewill apply it for exploring the potential infection of rodent/shrew borne henipavirus in Japan.

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P4-1

Unveiling the Potential of WQ-3810 Assessing its Efficacy Against Fluoroquinolone-Resistant
Mycobacterium avium
Sasini Jayaweera (1), Jeewan Thapa (1), Chie Nakajima (1) (2), Yasuhiko Suzuki (1) (2)(1)Division of Bioresources, International Institute for Zoonosis Control, Graduate School of Infectious Diseases, Hokkaido University(2)Institute for Vaccine Research and Development, Hokkaido University, Sapporo, Japan

Mycobacterium avium, a member of the Mycobacterium avium complex (MAC) within non-tuberculous
mycobacteria, poses a significant threat as an opportunistic pathogen causing MAC lung diseases. Macrolideresistant MAC infections are typically treated with fluoroquinolones (FQs), but their extensive use canprecipitate resistance, necessitating effective alternatives. WQ-3810, a novel fluoroquinolone, has demonstrated
substantial efficacy against FQ-resistant pathogens; however, its activity against M. avium remains
underexplored. This study aimed to evaluate WQ-3810's inhibitory effects on recombinant wild-type (WT) DNA
gyrase, along with four mutant gyrase with amino acid substitutions, Ala91Val, Asp95Ala, Asp95Gly, Asp95Tyr
in A subunit, using a DNA supercoiling inhibitory assay to determine the drug concentration inhibiting half ofthe enzyme activity (IC50). Additionally, the Minimum Inhibitory Concentration (MIC) of WQ-3810 was
assessed against 11 clinical M. avium isolates, comprising 7 WT strains and 2 each of Asp95Gly and Asp95Tyr
mutants. The comparative analysis included three established FQs (ciprofloxacin (CIP), moxifloxacin (MFX),
and levofloxacin) from previous studies. Results indicated that WQ-3810 exhibited dose-dependent inhibition
against WT and mutant gyrase A proteins. Specifically, all four mutant M. avium DNA gyrase A variants
displayed higher IC50 values (2.60 to 4.47 fold) than the WT, indicating reduced susceptibility to WQ-3810.
Notably, WQ-3810 demonstrated lower IC50 values than CIP for three out of four mutant strains, underscoring
its superior inhibitory potency against mutant gyrase A forms. MIC assessments revealed WQ-3810 exhibited
MICs comparable to MFX for WT strains, and half the MICs for mutant strains, suggesting favorable inhibitory
activity against both WT and mutant M. avium DNA gyrase A. In conclusion, WQ-3810 shows promisingpotential as a therapeutic option against fluoroquinolone-resistant M. avium infections, supported by its robust
inhibitory effects on WT and mutant gyrase A enzymes. These findings advocate for further clinical studies tovalidate its efficacy and safety profile in treating MAC lung diseases, addressing the critical need for effective
treatments amidst rising resistance issues.

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P4-2

Studies on sexual dimorphism in the development of brown adipose tissue
Zibo Yang (1), Bongsoo Seok (1), Shucheng Xu (1), Anju Tsukada (1), Khaled Mahmud Sujan (1), Yukino
Tamakoshi (1), Mohamed Elfeky (1), Mira Kato-Suzuki (1), Yuko Okamatsu-Ogura (1)(1)Laboratory of Biochemistry, Faculty of Veterinary Medicine, Hokkaido University, Japan

Brown adipose tissue (BAT) is a site of energy expenditure where lipid energy is burned for non-shivering
thermogenesis through the activity of Uncoupling Protein 1 (UCP1). It has attracted attention as a therapeutictarget for the treatment of obesity and related metabolic diseases. BAT activity and quantity is reported to bediffer between males and females in both mice and humans. This sexual dimorphism is explained by the actionsof sex hormones: estrogen enhances brown adipocyte differentiation, whereas androgen suppresses it. However,
it is not known whether sex differences exist in the fetal period or before sexual maturity. In this study, weevaluated the sexual dimorphism during BAT development in mouse embryos.
Weights of whole body and BAT were measured in mouse embryos at embryonic day 16 (E16) and E18.
Genotyping PCR determined the sex of the embryos. Body weights were similar between male and female
embryos at both E16 and E18. In contrast, BAT weights were significantly higher in males than females at E18,
being 44.6% higher in males, while they were similar between sexes at E16. mRNA analysis of UCP1, a BATspecific protein involved in non-shivering thermogenesis, showed no sex differences at E16 and E18.
Additionally, males at E18 exhibited a higher tendency to expression of genes related to brown adipocyte
function, such as Cell death-inducing DFFA-like effector-A (CIDE-A), Peroxisome proliferator-activatedreceptor-gamma coactivator (PGC)-1alpha, and Fatty acid binding protein 4 (FABP4, aP2). The expression of
Iodothyronine deiodinase 2 (DIO2), an enzyme that converts thyroxine (T4) to triiodothyronine (T3) andenhances UCP1 expression, was significantly higher in male than in female mice at E18. These results suggestthat the function and quantity of BAT are higher in males compared to females during the late developmental
period. Next, single-cell RNA-seq data from a public database (accession number GSE189088) using isolated
Myf5-expressing cells from E14 mouse embryos were analyzed. Myf5-expressing cells give rise to both brown
adipocytes and skeletal muscle cells. The analysis suggested the expression of the Androgen receptor in Myf5expressing progenitor cells.
In conclusion, male embryos showed higher BAT weight and a tendency for higher expression of genes related
to brown adipocyte function at E18. As the androgen receptor is expressed in Myf5-expressing progenitor cells,
testosterone may be involved in BAT development in embryos.

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P4-3

Enhancement of vaccine-induced T-cell responses by feeding probiotics in calves
Mari Ikehata (1), Tomohiro Okagawa (2), Hayato Nakamura (1), Naoya Maekawa (2), Shiro Murata (1) (2),Kazuhiko Ohashi (1) (2) (3), Satoru Konnai (1) (2) (4)(1)Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University(3)International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University(4)Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University

Neonatal animals, such as calves, have immature immune systems, hence immunization with vaccines isessential to protect them from infectious diseases. However, there are significant individual differences in
response to vaccines, and they do not always provide adequate protection in all individuals. Therefore, it isnecessary to develop strategies to enhance protective efficacy of vaccines, particularly targeting low responders
to vaccines.
Probiotics have attracted attention because of their beneficial immunomodulatory effects on humans. Various
strains of bacteria, including butyric acid- and lactic acid-producing bacteria are currently used as probiotics infood supplement for humans and animals including calves. Although feeding probiotics has been suggested to
improve the immunity of calves, the effect of probiotics on enhancing the immune responses to vaccination in
calves is still unknown. Thus, this study aimed to investigate whether the immune responses, especially T-cell
responses, to vaccination are enhanced in calves when received the combination of probiotics feeding withvaccination, by using a large number of animals.
Two-week-old calves were divided into the following 3 feeding groups (20, 19, and 18 calves): (1) negative
control feed; (2) butyric acid bacteria feed (BAB); (3) live bacteria-mixed feed (Zeosapo). All calves wereadministrated with a commercial live-attenuated viral vaccine (Calfwin6) twice at four and eight weeks after
the start of the feeding. T-cell responses to one of the vaccine antigens, bovine respiratory syncytial virus(BRSV), were then assessed by analyzing the expression of lymphocyte activation markers CD25 and CD69
and the production of Th1 cytokines in PBMC culture assays. T-cell responses to BRSV were observed after theprime and booster vaccinations in all feeding groups. The ratio of CD25+CD69+ cells in CD4+ T cells weresignificantly increased after the booster vaccination in BAB-fed and Zeosapo-fed groups. In particular, asignificant increase in CD25 expression was observed in CD4+ T cells in those groups. The productions of Th1
cytokines, such as IFN-γ and TNF-α, in the culture supernatant were increased after the booster vaccination in
BAB-fed group. In addition, significant weight gain was observed in calves fed with BAB or Zeosapo.
In conclusion, this clinical study demonstrates that feeding probiotics enhances CD4+ T-cell and Th1 responses
to vaccination in calves. Thus, probiotics are expected to strengthen the protective efficacy of vaccines in calves.
Further studies would be necessary to understand the mechanisms involved in this enhancement of immune
responses to vaccines by feeding probiotics.

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P4-4

The global aspect of mRNA transcriptome in canine mammary gland tumor
Shaohsu Wang (1), Nobuhiro Nozaki (1), Yutaro Ide (1), Mohammad Arif (1), Sarranhyaah Subramaniam (1),
MD. Nazmul Hasan (1), Naoki Miura (1)(1)Joint Graduate School of Veterinary Medicine, Kagoshima University, Japan

Canine mammary gland tumors (MGTs) commonly occur in intact female dogs and have been reported as
malignant in fifty percent of total cases. Aberrantly expressed oncogenes play a pivotal role in tumor
development. Considering the role of oncogenes in canine MGT, in this study, we aimed to characterize thealtered transcriptomic profile in canine MGT using next-generation sequencing (NGS).
This study included benign tumors (benign mixed tumor, adenoma), malignant tumors (adenocarcinoma), andhealthy control samples. Differential expression analysis of NGS reads was performed using the CLC genomic
workbench platform. Differentially expressed genes (DGEs) were investigated in silico to elucidate further the
essential network genes involved in the potential molecular mechanisms of MGT development.
Our study revealed that 50 DGEs were identified in the MGT group (including all subtypes) compared to thehealthy controls. Among them, 37 genes were downregulated, 13 were upregulated, and 15 novel genes wereidentified. Two differentially expressed genes, ENSCAFG00845029941 and FGL2, were selected based on
significant statistical criteria (Fold change ± >2, FDR P-Value 0.05) for qRT-PCR validation. The NGS analysis
found upregulation of ENSCAFG00845029941, and downregulation of FGL2, which was confirmed by qRTPCR validation. This study also uncovered that the location of DGEs on chromosomes in each comparison group
showed a similar trend. DGEs were applied for the Gene Ontology (GO) and Kyoto Encyclopedia Genes andGenomes (KEGG) pathway analysis, showing that most results were related to muscle development andcardiomyopathy.
Considering this, we investigated transcriptome profiles using NGS to identify the DGEs in canine mammary
glands compared with different subtypes of canine MGT. The identified two DGEs could be used to distinguishcanine MGT from healthy control dogs. Further research is necessary to elucidate their functions, associatedtranscription factors, and interacting pathways in the development of MGT.

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P4-5

Research plans to elucidate the relationship between ticks and birds
Yuki OHSUGI (1), Nariaki Nonaka (1), Ryo Nakao (1)(1)Laboratory of Parasitology, Department of Disease Control Faculty of Veterinary Medicine, Hokkaido University, Japan

Ticks are important vector that carry tick-borne pathogens (TBPs) such as viruses, bacteria, and protozoa,
sometimes causing significant health and economic damage. Because many ticks parasitize wildlife and spreadwith their migration, understanding the impact of wildlife on the maintenance and spread of ticks and TBPs isimportant. Birds are especially known to carry ticks to distant places due to their ability to fly. Therefore,clarifying the relationship between ticks, TBPs, and birds is also useful in predicting the expansion of tick-borne
diseases. In this study, we will introduce the three research plans to clarify the relationship between ticks/TBPs
and birds.
1. Identification of bird DNA in ticks
In order to elucidate the relationship between ticks and birds, it is necessary to capture birds and test themagainst ticks and TBPs. However, it is challenging to capture a wide range of migratory birds due to technicaland ethical difficulties. To overcome this problem, we plan to develop the methods which can identify the bird
species from the blood meal source of collected ticks. This study, the DNA of host birds remaining in the tickbody is amplified by PCR and sequenced to detect and identify the birds.
2. Elucidation of the effects of bird feeding on ticks
To elucidate how feeding of bird blood affects the ticks, we will develop experimental models of tick infestation
on birds. Ticks will experimentally suck blood to chickens and rabbits, after engorgement and molting, the bodysize and morphology will be compared among both groups of ticks. In the adult female ticks, the number of
eggs and hatching rate will be compared among both groups after laying eggs.
3. Field sampling
To clarify the relationship between ticks and birds in the field, tick sampling is conducted in Teuri Island,
Hokkaido. This Island is known as a breeding site for seabirds and also important stopover site for other birds
during migration. In addition, the absence of medium and large-seized wild mammals makes it easy to exclude
the influence of tick abundance by mammals. In this study, we will survey the seasonal changes of tick in Teuri
Island where they are likely to be transported by birds to determine the dispersal of ticks by birds.

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P4-6

Depression-like behavior in pups induced by the administration of corticosterone to pregnant
mice.
Aya KONDO (1), Ryota EGUCHI (1), Ken-ichi OTSUGURO (1)(1) Laboratory of Pharmacology, Faculty of Veterinary Medicine, Hokkaido University, Japan[Background and Purpose] In several human epidemiological studies, it has been reported that children of
mothers who are exposed to stress during pregnancy are more likely to develop depression in the future. The
pathological mechanism of depression is thought to differ from that of depression in adults, but the details are
unknown. Excessive stress leads to overproduction of stress hormones. Stress hormones are degraded in thenormal placentas, but it has been reported that the expression of the enzyme degrading stress hormones
decreases in the placentas of stressed mothers. Thus, stress hormones produced excessively in the mother due
to stress may transfer to the fetus and cause depression. Therefore, we examined the effects of increased stresshormones in pregnant mice on the behavior of the pups.
[Materials and Methods] Female C57BL6/J mice (11-13 weeks old) were mated with males. The stress hormone
corticosterone (CORT, 40 ug/ml) was administered via drinking water from day 0 (day 0-) or day 14 (day 14-)of pregnancy until birth. The control group was administered the solvent hydroxypropyl-β-cyclodextrin. To
measure spontaneous activity and depressive behavior, the open field test and forced swimming test were
performed in the pups every week from 4 weeks old, and in the mothers after weaning. Body weights were
measured in pups and mothers.
[Results] In the forced swimming test, immobility time was significantly increased in pups born from CORTtreated (day 0-) mothers compared to pups born from solvent-treated ones. The immobility time prolongation
continued from 6 to 13 weeks old. Meanwhile, there were no significant differences in open field test and bodyweight between these two groups. However, there were no changes in immobility time in pups born from CORTtreated (day 14-) mothers compared to pups born from solvent-treated ones. In addition, no behavioral changes
were observed in the mother mice.
[Discussion] The present results indicate that pups born from CORT-treated mothers exhibit depressive-likebehavior, and that these changes continue at least until 13 weeks old. We will use this model in the future toinvestigate the details of the pathological mechanism.

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P4-7

ECOLOGY AND MOLECULAR EVOLUTION OF HOKKAIDO VIRUS, A GENOTYPE OF
ORTHOHANTAVIRUS PUUMALAENSE, AMONG MYODES RODENTS
Thuy Thi Ngoc Duong (1) (2), Michihito Sasaki (3) (4), Yasuko Orba (3) (4) (5), Keisuke Aoshima(6) (7), Passawat
Thammahakin (1), Keisuke
Maezono (1), Shintaro Kobayashi (1) (4), Hiroaki
Kariwa (1)(1)Laboratory of Public Health, Department of Preventive Veterinary Medicine, Faculty of Veterinary Medicine, Hokkaido University,Japan(2)Department of Microbiology and Immunology, Tay Nguyen Institute of Hygiene and Epidemiology, Vietnam(3)Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University, Japan(4)Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Japan(5)International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Japan(6)Laboratory of Comparative Pathology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Japan(7)One Health Research Center, Cancer Research Unit, Hokkaido University, Japan[Background]
Thirty-eight orthohantavirus species have been reported in the genus Orthohantavirus, the family Hantaviridae
worldwide. Multiple orthohantaviruses have been identified as highly pathogenic to humans and represent a
threat to public health. Hokkaido virus (HOKV), a genotype of Orthohantavirus puumalaense (Puumala virus;
PUUV) carried by Myodes rufocanus bedfordiae, was first identified in Tobetsu, Hokkaido, Japan. Although it
is genetically related to the prototype of PUUV, genetic analysis of HOKV is limited.
[Purpose]This study aimed to know the ecology and the evolutionary pathway of HOKV in Myodes species.
[Methods]
A field survey was conducted in a forest in Tobetsu in 2022 and captured 44 rodents. Rodent sera were used totest anti-HOKV IgG antibodies by ELISA and IFA methods. Detection of viral RNA in lung tissue was
performed by RT-PCR. Virus isolation was attempted from lung samples of virus RNA-positive rodents by using
MRK 101 cells (originating from the kidney of M.rufocanus). The viral genome segments and mitochondrial
DNA of rodents were amplified and sequenced. Genetic analyses were carried out based on viral and rodent
mitochondrial DNA sequences.
[Results and Discussions]
Complete HOKV genome sequences of coding regions were obtained from five virus-RNA-positive rodents(four Myodes rufocanus bedfordiae and one Apodemus speciosus). Phylogenetic analysis revealed a close
relationship between the phylogenies and geographical origins of HOKV. Analysis of the D-loop region of
mitochondrial DNA from rodents captured in Tobetsu revealed that the population of M.rufocanus bedfordiae
has been genetically stable in the isolated forest for more than 20 years. The evolutionary rate of HOKV basedon the S segment was estimated to be 1.548 x 10-4 substitutions/site/year. Comparison of the phylogenetic treesof the S segments of orthohantaviruses and the cytochrome b genes of Myodes species suggested that Myodesrelated orthohantaviruses evolved by genetic isolation of Myodes species and host-switching of the virus.
[Conclusions]
HOKV may have evolved by the population separation of M.rufocanus in the wide areas of the Northern
Hemisphere. The evolutionary dynamics of orthohantaviruses within the Myodes rodent population during ageographical timescale will help us illustrate the divergence of genotypes of orthohantavirus.

85

P4-8

Role of Nerve Growth factor and anti-Nerve Growth Factor Monoclonal Antibodies in
Osteoarthritis pathology.
Nomsa Handondo Mulenga (1), Takafumi Sunaga (1), Kanittha Darawiroj (1), Masahiro Okumura (1)(1)Laboratory of veterinary surgery, Faculty of veterinary medicine, Hokkaido University, Japan

The primary goal in managing osteoarthritis (OA) is pain alleviation. Non-steroidal anti-inflammatory drugs(NSAIDs) are widely used to manage osteoarthritic pain; however, growing concerns over their long-term side
effects have created the need for better therapeutics aimed at managing OA pain. Over the past decade, studies
have highlighted that nerve growth factor (NGF) increases following injury, inflammation, and chronic pain.
Consequently, anti-Nerve Growth Factor (anti-NGF) targeted therapies have become a major focus in current
research. Anti-NGF monoclonal antibodies have recently been developed and approved for treating OA pain inveterinary medicine. However, their functional involvement in OA cartilage degeneration remains unclear. This
study aims to investigate the effects of NGF and anti-NGF on cartilage in canine articular chondrocytes. Primarycanine chondrocytes were grown in monolayer culture and subjected to treatments of either NGF, anti-NGF, ora combination of both in the presence or absence of Interleukin-1 beta. Following treatment, Von Kossa andAlizarin red stains were used to evaluate the effects of NGF and anti-NGF on matrix mineralization. Additionally,
RT-qPCR was used to assess the long-term effects of NGF and/or anti-NGF on extracellular matrix production
by profiling the expression of extracellular matrix and cartilage hypertrophy-related genes: type I and II collagen,Sox-9, and Runx2, with GAPDH as a reference gene. Our results showed that treatment with anti-NGF resulted
in higher matrix mineralization within healthy chondrocyte cell cultures. In the cell culture groups exposed toInterleukin-1 beta, treatment with anti-NGF downregulated Sox-9 levels compared to the positive control and
NGF groups. However, these findings were not statistically significant. Therefore, the next phase of ourexperiment will employ the use of small interfering RNA (siRNA) to completely suppress NGF production toconfirm our current findings and better understand the role of NGF/TrkA signaling in articular chondrocytes.
The study will then focus on the effect of combining Pentosan Polysulphate (PPS) and anti-NGF onchondrocytes in vitro by assessing changes in proteoglycan synthesis and extracellular matrix production. The
expected outcomes of this study are to highlight the active role of NGF/TrkA signaling in the pathogenesis ofosteoarthritis and the adverse effects associated with long-term use of anti-NGF therapy. It will also help
determine whether anti-NGF therapy can be combined with disease-modifying osteoarthritic drugs such as PPS
for better treatment outcomes and prognosis.

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P4-9

EVALUATION OF UNINTENTIONAL EFFECTS ON ENDANGERED SPECIES ENDEMIC
TO THE BONIN ISLANDS CAUSED BY DEPLOYMENT OF ANTICOAGULANTRODENTICIDE
Masashi Koide (1), Yusuke KAWAI (2), Mitsuki KONDO (3), Yoshiya ODAYA (4), Kazuo HORIKOSHI (5),Hajime SUZUKI (5), Takashi NAGAMINE (6), Yumiko NAKAYA (6), Yoshinori IKENAKA (1), Mayumi
ISHIZUKA (1), Shouta M.M. NAKAYAMA (1) (7)(1)Hokkaido University, Japan(2)Obihiro University of Agriculture and Veterinary Medicine, Japan(3)National Institute for Environmental Studies, Japan(4)Abiko City Museum of Birds, Japan(5)Institute of Boninology, Japan(6)Conservation & Animal Welfare Trust, Japan(7)The University of Zambia

The Bonin Islands are Japanese island group with a unique ecosystem comprising many endemic plants andanimals. Some animals in the islands such as Bonin fruit bat (Pteropus pselaphon) and Bonin greenfinch (Chloris
sinica kittlitzi) are listed as endangered species by the IUCN. One of the factors threatening the survival of these
species is ecosystem disturbance caused by non-native rodents represented by Black rats (Rattus rattus). In order
to control these rodents in the islands, Diphacinone, one of the anticoagulant rodenticides (ARs), has been
applied. However, the potential effects of the use of rodenticides on non-target animals in this area are still
unclear. ARs inhibit the blood coagulation system by blocking vitamin K epoxide reductase (VKOR), ultimately
causing fatal hemorrhage. When absorbed into the body, ARs are metabolized by Cytochrome P450 (CYP) andexcreted. There are differences between species in sensitivity to ARs, of which the binding affinity to the target
molecule VKOR and the ability of CYP to metabolize ARs are important determinants. In this study, microsomal
fractions were purified from the livers of Oriental greenfinches (Chloris sinica minor), as an alternative to Boningreenfinch, and of Bonin fruit bats. With use of these microsomal fractions, inhibition tests of VKOR activity
using Warfarin and Diphacinone were conducted. In order to assess in vitro susceptibility to rodenticides,
Warfarin metabolic tests were also performed. The IC50 values obtained from the activity inhibition tests using
Warfarin were 0.204, 0.351, and 5.95 uM, respectively for SD rats, Bonin fruit bats, and Oriental greenfinches,
showing higher values for greenfinches compared to rats, while that of bats was comparable to that of rats. The
equivalent values from the Diphacinone tests were 0.131, 0.133, and 0.648 uM, respectively, suggesting a higher
susceptibility in bats than in rats. Vmax/Km was calculated as an indicator of metabolic capacity for eachmetabolite found in the Warfarin metabolic tests, yielding totals of 4.28, 3.23, and 4.40 (mL/min/nmol CYP),
respectively, rats, bats, and greenfinches. These results indicate that both of bats and greenfinches could have
similar Warfarin metabolic capacity as that of rats. Better estimation of the susceptibility requires further studies
such like metabolic assay using Diphacinone, in vivo administration tests, functional analysis of each CYP typeby cloning, risk assessment of misfeeding, or the same kind of tests using samples from the close related species
to Bonin fruit bats.

87

P4-10Feasibility of glutathione S-transferase as an antigen to develop a universal vaccine with broad
efficacy against avian mites
Shwe Yee Win (1), Shiro Murata (1) (2), Fumiya Horio (1), Jumpei Sato (1), Yoshinosuke Motai (1), Takumi
Sato (3), Eiji Oishi (3), Tomohiro Okagawa (2), Naoya Maekawa (2), Satoru Konnai (1) (2) (4), Kazuhiko
Ohashi (1) (2) (5)(1)Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Japan(3)Vaxxinova Japan K.K., Japan(4)Institute of Vaccine Research and Development (GU-IVReD), Hokkaido University, Japan(5)International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Japan

Blood-sucking avian mites, poultry red mites (Dermanyssus gallinae, PRMs), tropical fowl mites(Ornithonyssus bursa, TFMs), and northern fowl mites (O. sylviarum, NFMs) are detrimental pests which
negatively impact on health and welfare of poultry worldwide. Vaccine approach is a promising strategy for
their control. Glutathione S-transferase (GST) is a multifunctional enzyme, which are conserved in variousorganisms and is required for drug metabolism and detoxification. For the development of a universal vaccine,
molecules, which are highly conserved among avian mites and detrimental to their physiology, could be ideal
antigen candidates. In this study, we aimed to evaluate the potential of GST for application as a common vaccine
antigen with broad protective efficacy against avian mites. We identified the GST genes from TFMs and NFMs,
and the nucleotide sequences of the GST genes of TFMs and NFMs were 80% identical to that of PRMs. The
recombinant GSTs (rGSTs) from TFMs, NFMs, and PRMs were prepared, and all rGSTs exhibited catalytic
activity to conjugate glutathione with a substrate. We isolated immune plasmas from chickens immunized witheach rGST, and immune plasma against each rGST was cross-reacted with rGSTs of different avian mites.
Moreover, we observed significant decreases in the survival rates of PRMs fed with immune plasma against
rGSTs of TFMs and NFMs, in addition to PRMs. These results suggest that GST is an antigen candidate fordeveloping a universal vaccine with broad efficacy across avian mites.

88P4-11

Effect of polymorphisms in the meq oncogene of Marek's disease virus distributed in Japan on
the pathogenicity
Yoshinosuke Motai (1), Shiro Murata (1) (2), Jumpei Sato (1), Shunsuke Yamagami (1), Shwe Yee Win (1),Fumiya Horio (1), Naoya Maekawa (2), Tomohiro Okagawa (2), Satoru Konnai (1) (2) (3), Kazuhiko Ohashi(1) (2) (4)(1)Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Japan(3) Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Japan(4)International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Japan

Marek's disease virus (MDV) causes malignant lymphomas and neurological disorders such as leg paralysis andtorticollis (Marek's disease, MD) in infected chickens. MD is currently controlled by vaccination; however,sporadic MD occurrences are still observed due to the emergence of highly virulent strains worldwide. Meq, an
oncoprotein of MDV, serves as a transcriptional factor, and is involved in tumorigenesis by regulating theexpression of host and viral genes. In addition, polymorphisms in the amino acid sequences of Meq among
MDV strains are known to affect the pathogenicity of MDV. In Japan, MD cases are sporadically reported, andunique polymorphisms are present in Meq of Japanese isolates. In this study, we investigated the effect ofpolymorphisms in Meq of Japanese isolates on the MDV pathogenicity. To investigate the pathogenicity of Meq
encoded in Japanese isolates, we generated an RB-1B, a highly virulent strain of MDV, -based recombinant
MDV (rMDV) encoding Meq of Nr-c1 (rRB-1B_Nr-c1-Meq). The sequence of Nr-c1-Meq is widely observedin Japanese isolates. When we compared the mortality and tumor incidence in chickens infected with rRB-1B
and rRB-1B_Nr-c1-Meq, chickens infected with rRB-1B_Nr-c1-Meq showed lower mortality than those
infected with rRB-1B. Neurological disorders and solid tumors were observed in visceral organs in chickens
infected with rRB-1B, while chickens in the rRB-1B_Nr-c1-Meq infected group did not develop these diseases.
Instead, a depression and open-mouth behavior were observed in chickens in the rRB-1B_Nr-c1-Meq infectedgroup. In addition, no increase in weights, viral loads, and proportions of CD4+ T cells was observed in spleensfrom chickens with clinical signs in the rRB-1B_Nr-c1-Meq infected group, although all parameters wereincreased in rRB-1B-infected chickens. These data suggest no signs of tumor cell growth in rRB-1B_Nr-c1Meq infected chickens. On the other hand, a decrease in thymus weight was observed in chickens developed
MD in both groups, suggesting that thymus atrophy associated with cytolytic infection were caused by both ofthe viruses. Thus, rMDV encoding Meq of Japanese isolates displayed different pathology from the parental
virus, RB-1B. These findings suggest that MDV pathogenesis is affected by amino acid sequences of Meq.
Further investigation is required to elucidate the mechanisms underlying the differences in pathogenesis
associated with Meq sequences, and to clarify the occurrences of MD cases with clinical signs such as openmouth behavior in the field.

89

P4-12

Receptor binding preference of avian influenza viruses to α2,3 sialoglycans with various local
density
Daiki Kobayashi (1), Nongluk Sriwilaijaroen (2) (3), Ryota Adachi (4), Manabu Igarashi (5) (6), TakahikoMatsushita (4), Norikazu Isoda (1) (6) (7) (8), Koji Matsuoka (4), Yasuo Suzuki (3), Yoshihiro Sakoda (1) (6)(7) (8), Takahiro Hiono (1) (6) (7) (8)(1)Laboratory of Microbiology, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)Department of Preclinical Sciences, Faculty of Medicine, Thammasat University(3)Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka(4)Area for Molecular Function, Division of Material Science, Graduate School of Science and Engineering, Saitama University(5)Division of Global Epidemiology, International Institute for Zoonosis Control, Hokkaido University(6)International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University(7)One Health Research Center (OHRC), Hokkaido University(8)Institute for Vaccine Research and Development (IVReD), Hokkaido University[Objectives]
Avian influenza viruses (AIVs) contain two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), bothof which recognize the same molecule sialoglycans while they have opposing functions; receptor binding by
HA and receptor destroying by NA. The receptor binding specificity of HA has been well studied; for example,
HA of duck-origin AIVs specifically bind to terminal sialic acid linked to penultimate galactose with ﾎｱ 2,3
linkage but prefer branched glycans with extended poly N-acetyl-lactosamine chains. However, the adequate
local density of the target glycans for the receptor binding affinity of AIVs was still unknown under the delicate
balance of HA and NA. In this study, glycan preferences of AIVs were investigated using sialic acid-containing
glycopolymers with various numbers of acrylamide spacers.
[Materials and methods]
The recombinant HA proteins and virus virions of A/duck/Hong Kong/960/1980 (H6N2), whose binding
specificity to branched sialosides had been characterized, were used for solid-phase direct binding assay. In theassay, NA catalytic activity cleaves the sialic acid from the glycopolymers. Therefore, the exposed glycoepitopes
after the NA-mediated cleavage were detected with Erythrina Cristagalli agglutinin (ECA). To explain thereceptor binding specificity of viruses, distances between receptor-binding sites in an HA trimer and between
neighboring glycoepitopes in a polymer were calculated through homology modeling and molecular dynamics
simulations.
[Results and discussion]
The recombinant HAs preferred glycopolymers with higher density but did not bind to the homopolymer withthe highest density. In contrast, virus virions preferred lower-density glycopolymers; which was explained by
the preference of NA activity to higher-density glycan detected with ECA. To understand the functionalsignificance of clustering HAs on the virus virions, the glycan binding analysis was also performed with an NA
inhibitor. Interestingly, virions accepted a broad range of glycopolymers with low to high density except for thehomopolymer in the presence of the NA inhibitor. The in silico analyses suggested higher glycan density
interferes with HA binding due to the steric hindrance, demonstrating that HA has optimal glycan density tofacilitate higher binding affinity. Meanwhile, HA clustering on the virions compensates binding affinity to low
glycan density, and NA cleaves higher-density glycans more effectively. These results suggested that HA andNA showed different preference toward the glycan density and the cumulative effects of receptor binding by
HA and cleaving by NA regulate the preferences of AIV virions.

90

P4-13

Establishment of a rapid PCR-based reverse genetics system for feline coronavirus
Izumi Kida (1), Tomokazu Tamura (2) (3) (4), Takasuke Fukahara (2) (3) (4) (5), Keita Matsuno (1) (3) (4)(1)Division of Risk Analysis and Management, International Institute for Zoonosis Control, Hokkaido University, Japan(2)Department of Microbiology and Immunology, Faculty of Medicine, Hokkaido University, Sapporo, Japan(3)Institute for Vaccine Research and Development, HU-IVReD, Hokkaido University, Sapporo, Japan(4)One Health Research Center, Hokkaido University, Sapporo, Japan(5)Laboratory of Virus Control, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

Background: Feline coronavirus (FCoV) is a causative agent of feline infectious peritonitis (FIP). FCoV isclassified into two pathotypes, feline enteric coronavirus (FECV), which exhibits low pathogenicity andtypically results in asymptomatic or mild enteritis, and highly pathogenetic FIP virus (FIPV), which originatesfrom FECV mutated in the infected cats. The molecular mechanisms of pathotype switch from FECV to FIPV
are still unknown due to the lack of suitable molecular experimental tools of FCoV.
Objectives: For allowing rapid modifications of FCoV genome, we aim to rescue FCoV recombinants (rFCoVs)
using a circular polymerase extension reaction (CPER) method and to generate rFCoVs possessing reporter
genes and desired mutations.
Methods: Serotype-2 feline coronavirus strain WSU-79/1146 was used for construction of a rFCoV. The virus
genome was divided into 10 continuous fragments and amplified by PCR. To distinguish between rFCoVs andthe parental virus, two silent mutations were introduced into ORF1a as genetic markers. The full-length cDNA
clone of FCoV under the control of the CMV promoter obtained by CPER was transfected into Fcwf-4 cells cocultured with HEK293T cells. To construct rFCoVs carrying reporter gene, StayGold gene and Hibit luciferase
gene were inserted in ORF7a and 7b, respectively. After cytopathic effect (CPE) was observed in the transfected
cells, the supernatant (passage 0 (P0) virus) was passaged in Fcwf-4 cells. The supernatant of Fcwf-4 cells (P1virus) was used for further analysis.
Results: Infectious particles of rFCoV WSU-79/1146 were recovered successfully. Virus recovery wasconfirmed by CPE and expression of viral antigen in transfected cells and Fcwf-4 cells inoculated with the P0
viruses. Sequence analysis of recombinant P1 viruses demonstrated that there was no sequence difference
between rFCoVs and the parental virus except for the genetic markers and reporter genes. The growth kinetics
of rFCoVs and the parental virus were similar to each other. The expression of the reporter genes carried byrFCoVs was confirmed in the infected cells after several passages.
Conclusions: The CPER method has been applied to generate FCoV recombinants for the first time to the bestof our knowledge. The present system capable to rapidly introduce desired modifications in FCoV genome will
facilitate the investigation on the molecular mechanism(s) of pathotype switch of FCoV. Furthermore, rFCoVs
carrying reporter genes will be powerful tools for understanding viral life cycles and screening of antiviral drugs.

91

P4-14

Assessing antigenic variation of the rabies virus glycoprotein in South American street strains
compared to vaccine strains
Cassia N Sousa Moraes (1), Gabriel Gonzalez (2), Koshiro Tabata (1), Shinji Saito (1), Hirofumi Sawa (1),Yukari Itakura (1)(1)Division of Biological Response Analysis, Institute for Vaccine Research and Development, Hokkaido University, Japan(2)Division of Research Support, Institute for Vaccine Research and Development, Hokkaido University, Japan[Background]
Rabies is a viral zoonosis that can infect most mammals, including humans. Although preventable byvaccination, it causes over 59,000 deaths annually worldwide. Among the five viral proteins (N, P, M, G, andL) encoded by rabies virus (RABV), the glycoprotein (G) is the major target of neutralizing antibodies and plays
an important role in viral antigenicity. Thus, the antigenic differences in the RABV G between inactivatedvaccine and street-circulating strains could influence the effectiveness of currently available vaccines. RABVG amino acid sequences of South American street strains have been reported to have the lowest similarities withthose of the vaccine strains among the street isolates from other continents, suggesting a significant impact inthe effectiveness of vaccines against the circulating RABV in South America.
[Goal]
Identifying the predominant mutations in the RABV G of street strains in South America over the past 30 years
and evaluating the impact on antigenicity and vaccine effectiveness.
[Materials and methods]
Through bioinformatics analysis, we assessed the diversity of publicly available amino acid sequences of the
RABV G (n=610), derived from South American RABV street strains between 1995 and 2021, alongside
sequences from seven vaccine strains downloaded from the NCBI GenBank database. The phylogenetic
relations and clustering of these sequences were multiple sequence analyzed using MAFFT and MEGA X basedon the amino acid sequences. The amino acid substitution frequency over time was analyzed with a custom R
script.
[Preliminary results]
The dataset comprises RABV G sequences from four South American countries (Argentina, Brazil, French
Guiana, and Peru) obtained from 12 different hosts. These sequences primarily clustered into two groups in aphylogenetic tree: one dog-associated variant and another associated to bats, with vaccine strains clusteringcloser to the dog-related strains. In total, 40 amino acid substitutions were identified in the G ectodomain incomparison to at least one of the vaccines, with two putatively affecting the antigenic sites.
[Expected results]
We aim to provide comprehensive data on the impact of antigenic variation of street RABV on rabies vaccine
efficacy in South America. Bioinformatically-identified amino acid substitutions in the RABV G will be further
assessed for its antigenic changes comparing with vaccine strains by in vitro and in vivo experiments using
neutralization assays. Our study will offer evidence-based recommendations to update the vaccine strains andenhance public health protection against the circulating diverse street strains in South America.

92

P4-15

Establishment of reverse genetic approaches of the Rabies virus street strain belonging to the
SEA4 subclade and its application
Nijiho Kawaguchi (1), Yukari Itakura (2), Naoto Ito (3), Hirofumi Sawa (1) (2), Yasuko Orba (1) (2), Michihito
Sasaki (1) (2)(1)Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University, Japan(2)Institute for Vaccine Research and Development, Hokkaido University, Japan(3)Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, Japan

Rabies virus (RABV) is the causative agent of rabies, a lethal neurological disease in mammals. RABV strainsconsist of two groups: street strains, which are field strains isolated from a human patient or an animal withrabies, and fixed strains, which include laboratory strains and vaccine strains established through multiple
passages of street strains in cultured cells and/or animals. Compared with fixed strains, street strains havedifferent characteristics, such as inconsistent and longer incubation period and higher pathogenicity after
peripheral infections (neuroinvasiveness). However, studies on the molecular mechanisms of RABV
pathogenesis have mainly been carried out by using fixed strains, and little is known about the pathogenic
mechanisms of street strains. RABV Toyohashi strain is a street strain isolated in Japan from an imported casewhich had been bitten by rabid dog in the Philippines. In order to facilitate molecular studies of RABV, we tried
to establish a reverse genetics (RG) system of the Toyohashi strain. The infectious virus was recovered from acDNA clone of Toyohashi strain and exhibited similar growth efficiency as the original virus in cultured celllines. Both the original and recombinant strains showed similar pathogenicity with high neuroinvasiveness in
mice, and the infected mice developed a long and inconsistent incubation period, which is characteristic of street
strains. We also generated a recombinant Toyohashi strain expressing viral phosphoprotein (P protein) fusedwith the fluorescent protein mCherry, and tracked the intracellular dynamics of the viral P protein using livecell imaging. In summary, we successfully established an RG system for RABV street strain belonging to SEA4
subclade circulating only in the Philippines. This RG system will be a powerful tool for manipulating the
genome of the Toyohashi strain to investigate the molecular basis of infection and pathogenesis of the RABV
street strain which is currently prevalent in the Philippines.

93P4-16

Assessment of the safety profile of chimeric marker vaccine against classical swine fever
Loc Tan Huynh (1), Mikihiro Otsuka (1), Maya Kobayashi (1), Hung Dinh Ngo (1), Lim Yik Hew (1), Takahiro
Hiono (1) (2) (3) (4), Norikazu Isoda (1) (2) (3) (4), Yoshihiro Sakoda (1) (2) (3) (4)(1)Laboratory of Microbiology, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Japan(2)International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Japan(3)Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Japan(4)One Health Research Center, Hokkaido University, Japan[Objectives]
Vaccination is a crucial strategy for controlling classical swine fever (CSF), with live attenuated vaccines(LAVs) being commonly used. However, LAVs pose inherent risks, including the potential for reversion tovirulence. Previous studies have confirmed the biological profiles and the capability to differentiate infectedfrom vaccinated animals of two chimeric viruses, vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, which weregenerated based on the backbone of the LAV vGPE- strain. This study aimed to evaluate the safety profile ofthese two chimeric viruses, specifically focusing on their potential for reversion to virulence, in accordance with
the World Organization for Animal Health (WOAH) guidelines.
[Materials and methods]
Each chimeric virus was injected into two 2-week-old pigs, which were monitored daily for clinical signs for 7
days. Tonsil homogenates were collected at the end of the experiment for virus titration, sequencing, and used
as inoculum for the next passage. If no virus was recovered, the experiment ended; otherwise, the recovered
virus was used to inoculate another pair of pigs. This process was repeated for a total of five serial passages.
Also, the viral growth kinetics and interferon-alpha/beta (IFN-α/β) production of two chimeric viruses were
investigated in vitro.
[Results]
Both vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns complied with the test, as no virus was recovered after thesecond and fourth passages, respectively. The vGPE- virus was still recovered until the fifth passage in pigs,
with no clinical abnormalities observed. Sequencing revealed amino acid substitutions at T660 (E1) in
P1/vGPE-/PAPeV Erns, I3842 (NS5B) in P3/vGPE-/PhoPeV Erns and P5/vGPE-, were identical to the passaged
virus and parental CSF virus (CFSV) ALD strain. In vitro investigation indicated comparable viral growthamong two chimeric viruses, yet vGPE-/PAPeV Erns consistently induced higher IFN-α/β levels than vGPE/PhoPeV Erns.
[Discussion]
The attenuation of two chimeric viruses can be affirmed by substituting the CSFV glycoprotein Erns for either
PAPeV Erns or PhoPeV Erns. Meanwhile, the vGPE- strain maintained stable replication without increasing
virulence in pigs. The mechanism underlying the attenuation of two chimeric viruses in pigs remained elusive.
Since the study design only partially adhered to the WOAH recommendations regarding the reversion to thevirulence study protocol, it is essential for future research to address these concerns comprehensively. In
addition, future studies should prioritize investigating the safety of chimeric viruses in breeding stocks (sows orboars), especially on pregnant sows, to address the risk of transplacental transmission effectively.

94

SaSSOH Organizing Commi�ee

Faculty of Veterinary Medicine, Hokkaido University
Nishi-9, Kita-18, Kita-ku, Sapporo 060-0818, Japan
Tel: 011-706-9545
Email: sassoh@vetmed.hokudai.ac.jp
Web: h�ps://sassoh.vetmed.hokudai.ac.jp/