NTU-KU Joint Funding
Final Report

Section 1

NTU principle investigator
Name (last name, first name)

Hui-Wen Chen

Position

Professor

Faculty/Department

Department of Veterinary Medicine

KU principle investigator
Name (last name, first name)

Keizo Tomonaga

Position

Professor

Faculty/Department

Department of Virus Research, Institute for
Frontier Life and Medical Sciences

Visiting ECR*
Name (last name, first name)Jing-Yuan Chen/ Ai-Ai Chou

Position

Postdoctoral researcher/Master student

Faculty/Department

Department of Veterinary Medicine*Please complete this section if the KU principal investigators hosted ECRs from NTU.

Host researcher*
Name (last name, first name)

Takehiro Kanda

Position

Assistant professor

Faculty/Department

Department of Virus Research, Institute for
Frontier Life and Medical Sciences*Please complete this section if the host researcher is different from the KU principal
investigator.
Section 2

Project title
International collaboration on epidemiologicalsurveillance and vaccine development of avian
bornaviruses
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Section 3

Period of project
From dd/mm/yy
to dd/mm/yy

13/02/23 – 23/02/23

Section 4

Summary of the project (approx. 100 words)*KU PIs are required to submit a summary of the project in Japanese in addition to theEnglish summary (approx. 200–300 characters).
(Please enter the summary of the project)
⼀本鎖RNAウイルスであるボルナウイルスは、哺乳類から⿃類まで幅広い宿主に重篤な神経疾患を引き起こすが、その詳細な発症機序は不明である。現在、ボルナウイルスに対して認可された治療薬やワクチンは存在していない。本プロジェクトでは、⿃ボルナウイル
スのワクチン開発を⽬標に、組換えボルナウイルス作製技術であるリバースジェネティク
スシステムの確⽴を⽬指した。⽬標達成のため。NTUから参加したECRに⿃ボルナウイル
スの取り扱いと組換えボルナウイルスの作製のための材料作製等に関する共同研究を進めた。NTUとの共同研究より、ボルナウイルス感染の制御に向けた研究を加速させることができると考えられた。

Bornavirus causes severe neurological diseases in a wide range of mammalian andavian species, but the pathogenesis remains unclear. Currently, there is noauthorized treatment or vaccine against bornavirus. Due to the slow replication rateof bornavirus, establishing a reverse genetic system is a quicker approach togenerate recombinant viruses for investigating gene functions and advancing clinicalapplications. In this project, our goal is to establish a reverse genetic system forbornaviruses. Additionally, we aim to determine whether the ratio of RNA-dependent
RNA polymerase helper plasmids transfected into cells impacts the recombinantvirus titer. In conclusion, this project will enable us to generate recombinant viruses
more efficiently and expedite bornavirus-related research.
Section 5 (Please complete this section if ECRs from NTU participated in collaborative research at KU)

Achievements and Outcomes of ECRs’ Stay (approx. 100–250 words)*This section should be filled by each of the ECR(s) (one paragraph per ECR) based on
his/her experience of staying in Japan.
(Please enter the achievements and outcomes for each of the ECR(s).)
Establishing the reverse genetic system of Parrot bornavirus type 7

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1. Outline of the experiment design

2. Experiment schedule

3. Experiment procedures(1) PaBV-7-infected cell preparation and RNA extraction: We seeded PaBV-7infected QT6 cells one day before extracting the viral RNA.
(2) RT-PCR & plasmid construction: After extracting PaBV-7 viral RNA, we used
RTPCR to generate PaBV-7 antigenome fragments and two ribozymes (ahammerhead ribozyme and a hepatitis delta virus ribozyme). Then we assembled thefragments into a full-length antigenome flanked by ribozymes and insert it into thevector.
(3) Transformation and insert sequence confirmation: We transformed theantigenome plasmid into E. coli, then colony PCR and sequence analysis wereconducted to confirm that the insert sequence is correct.
Rescuing rBoDV-1 by reverse genetic
1. Outline of the experiment design
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To find out the best ratio of transfecting RNA-dependent RNA polymerase (L) helperplasmid, we transfected 293T cells with different amounts of L helper plasmid, thenwe determined the recombinant BoDV-1 virus titer by TCID50 assay.
2. Experiment schedule:

3. Experiment procedures:
1. Cell preparation and plasmid transfection: We seeded the 293T cells one day
before transfection. Then we transfected the cells with 2000 ng antigenome, 500 ng
of nucleoprotein helper plasmid, 25 ng of phosphoprotein plasmid, 45 ng of matrix
protein, 15 ng of glycoprotein and different amounts of polymerase plasmid (0, 100,250, 500, 750, 1000 ng).
2. Virus rescue & infection: After three days of incubation, we collected therecombinant virus and then infected Vero cells with ten-fold serial dilution of thevirus in the 96 well plate. We examined the GFP fluorescent signal by microscope
three days post-infection to determine the virus titer.

Research results
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1. Establishing reverse genetic system of bornaviruses
We are still working on generating the right sequence of parrot bornavirus type 7.
2. Rescuing rBoDV-1 by reverse geneticFigure 1. The representative photos showed that when the cells were transfectedwith L helper plasmid, the GFP signals can be observed under the microscope,indicating the recombinant viruses were successfully generated.

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Figure 2. TCID50 assay was conducted to determine the recombinant virus titer.

Figure 3. TCID50 assay showed that the virus titer of transfecting 250 ng of L helperplasmid is the highest among all groups.

Section 6

Photographs with captions

Dr. Tomonaga and Dr. Kanda introduced
the project.

Visiting ECRs toured Dr. Tomonaga’slab.

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Dr. Kanda demonstrated the experiment.

Dr. Hui-Wen Chen gave a seminar inKU.

Dr. Jing-Yuan Chen performed PCRexperiment.

Master student, Ai-Ai Chou, conducted
the RNA extraction.
*Please submit digital files (such as JPEG or GIF files) of the photographs used in yourreport as attachments. The size of each image should be at least 4MB, so that it can be used
for printed materials. Please ensure that none of the photographs submitted will cause any
issues relating to portrait rights.

URL at which project outcomes can be viewed (Optional)*E.g. workshop notifications/programs/reports, evidence of academic papers published orotherwise made available, etc.
URL:
https://www.infront.kyoto-u.ac.jp/event/event-3781/

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