bluuebunny/crossref_metadata_2025 huggingface dataset:
Rhythmic Wave Patterns on Ambient Pressure Tympanometry in Patients With Objective Tinnitus-associated Pathologies
Authors: Sayyid Zahra N. , Thai Anthony , Swanson Austin , Hosseini Davood K. , Fitzgerald Matthew B. , Ma Yifei , Vaisbuch Yona
Published: December 2019

Objective:

To introduce the concept of ambient pressure tympanometry (APT) and its association with pathologies that may present with objective tinnitus.

Study Design:

Retrospective case series.

Setting:

Tertiary referral center.

Subjects and Methods:

Audiologists performed APT on adult patients as part of routine audiological testing. Ears with myoclonus and patulous Eustachian tube (PET) were identified via review of patient history and physical examination. All other conditions were verified via computed tomography (CT) temporal bone imaging. Ears with conditions that could impair tympanic membrane compliance, such as otosclerosis or tympanic membrane perforation, were excluded. APT findings were analyzed via a novel algorithm.

Results:

A radiographic finding associated with objective tinnitus was confirmed in 67 ears that underwent CT imaging; 45 (67%) of these ears displayed rhythmic APT wave patterns. These included 28 ears with superior semicircular canal dehiscence, 4 ears with sigmoid sinus dehiscence, 6 ears with internal carotid artery dehiscence, 4 ears with glomus tumor, and 3 ears with encephalocele. In addition, we identified three ears with myoclonus and one ear with PET. In a subset of 30 ears with objective tinnitus symptoms that underwent CT imaging, 22 displayed rhythmic waves; of these 22 ears, 20 (91%) had a radiographic finding associated with objective tinnitus.

Conclusions:

Rhythmic APT wave patterns are common and may be associated with numerous temporal bone pathologies that may present with objective tinnitus. APT is a simple, rapid, and widely available tool that warrants further study to determine its value in screening of these otologic conditions.
Cognitive Functions in Adults Receiving Cochlear Implants: Predictors of Speech Recognition and Changes After Implantation
Authors: Zhan Kevin Y. , Lewis Jessica H. , Vasil Kara J. , Tamati Terrin N. , Harris Michael S. , Pisoni David B. , Kronenberger William G. , Ray Christin , Moberly Aaron C.
Published: December 2019

Hypotheses:

Significant variability in speech recognition outcomes is consistently observed in adults who receive cochlear implants (CIs), some of which may be attributable to cognitive functions. Two hypotheses were tested: 1) preoperative cognitive skills assessed visually would predict postoperative speech recognition at 6 months after CI; and 2) cochlear implantation would result in benefits to cognitive processes at 6 months.

Background:

Several executive functioning tasks have been identified as contributors to speech recognition in adults with hearing loss. There is also mounting evidence that cochlear implantation can improve cognitive functioning. This study examined whether preoperative cognitive functions would predict speech recognition after implantation, and whether cognitive skills would improve as a result of CI intervention.

Methods:

Nineteen post-lingually deafened adult CI candidates were tested preoperatively using a visual battery of tests to assess working memory (WM), processing speed, inhibition-concentration, and nonverbal reasoning. Six months post-implantation, participants were assessed with a battery of word and sentence recognition measures and cognitive tests were repeated.

Results:

Multiple speech measures after 6 months of CI use were correlated with preoperative visual WM (symbol span task) and inhibition ability (stroop incongruent task) with moderate-to-large effect sizes. Small-to-large effect size improvements in visual WM, concentration, and inhibition tasks were found from pre- to post-CI. Patients with lower baseline cognitive abilities improved the most after implantation.

Conclusions:

Findings provide evidence that preoperative cognitive factors contribute to speech recognition outcomes for adult CI users, and support the premise that implantation may lead to improvements in some cognitive domains.
Prospective Multicentric Follow-up Study of Cochlear Implantation in Adults With Single-Sided Deafness: Tinnitus and Audiological Outcomes
Authors: Poncet-Wallet Christine , Mamelle Elisabeth , Godey Benoit , Truy Eric , Guevara Nicolas , Ardoint Marine , Gnansia Dan , Hoen Michel , Saaï Sonia , Mosnier Isabelle , Lescanne Emmanuel , Bakhos David , Vincent Christophe
Published: December 2019

Objective:

This study investigated the audiological and tinnitus outcomes of cochlear implantation (CI) in adults with single-sided deafness (SSD) and tinnitus.

Study Design:

Multicentered prospective, non-randomized intervention study.

Setting:

Six French CI centers.

Patients:

Twenty-six patients with SSD and incapacitating tinnitus (Tinnitus Handicap Inventory [THI] >58) underwent cochlear implantation.

Interventions:

First, CIs delivered only masking white noise stimulation for 1 month and then standard CI stimulation.

Main Outcome Measures:

Before and after CI surgery, patients completed the THI, Tinnitus Reaction Questionnaire (TRQ), Subjective Tinnitus Severity Scale (STSS), and two visual analogue scales quantifying tinnitus loudness and annoyance. Speech perception in spatialized noise was tested at 13 months.

Results:

The first month of white noise stimulation triggered a significant improvement in THI scores (72 ± 9 to 55 ± 20, p < 0.05). No change was observed for the other measures. After 1 year of standard CI stimulation, 23 patients (92%) reported a significant improvement in tinnitus. This improvement started 1 to 2 months after CI and exceeded 40% improvement for 14 patients (54%). Average speech-in-noise perception after 1 year significantly improved for the 23 patients who completed these measures.

Conclusions:

CI is efficacious to reduce the handicap of patient with SSD and incapacitating tinnitus, leading to a decrease in reported tinnitus and partial restoration of binaural hearing abilities.
A Retrospective Analysis of Intervention for Testicular Torsion: Searching for a Hallmark of High Reliability
Authors: Calder Jennifer K. , Calder Bennett W. , Arbra Chase A. , Cina Robert A.
Published: December 2019

Introduction:

Consistent delivery of high-quality care is a marker of health-care system reliability. Although clinically abstracted outcome databases have revolutionized surgical quality improvement efforts for many high-volume procedures, their utility in aiding the improvement of time-sensitive processes is less clear. The purpose of this study was to determine whether process measures surrounding the delivery of timely surgical care could delineate the variability in the outcome of patients with testicular torsion. Our secondary aim was to use the data to drive quality improvement efforts locally.

Methods:

We performed a retrospective review of encounters for testicular torsion in patients less than 18 years. Characteristics of patients undergoing detorsion/orchiopexy and orchiectomy were compared. We evaluated orchiectomy as a function of age, insurance status, time of presentation, duration of symptoms, time to ultrasound, and time to surgery.

Results:

Over 10 years, we identified 46 patients, of whom 21 met inclusion criteria. Twelve patients (57.1%) underwent detorsion/orchiopexy, whereas 9 patients underwent orchiectomy. After-hours presentation and age were not predictive of orchiectomy, whereas the duration of symptoms was predictive of orchiectomy. Differences in time to surgery for orchiopexy and orchiectomy groups approached but did not reach significance (P = 0.07).

Conclusions:

Significant variability persists in the timely delivery of comprehensive and coordinated care for children suffering from time-sensitive surgical conditions. Pediatric surgical programs would realize significant benefit from a surgical quality improvement program that incorporates validated process metrics, along with outcome measures, to help drive efficiencies and integrate care more effectively.
Soft eSkin: distributed touch sensing with harmonized energy and computing
Authors: Soni Mahesh , Dahiya Ravinder
Published: December 2019

Inspired by biology, significant advances have been made in the field of electronic skin (eSkin) or tactile skin. Many of these advances have come through mimicking the morphology of human skin and by distributing few touch sensors in an area. However, the complexity of human skin goes beyond mimicking few morphological features or using few sensors. For example, embedded computing (e.g. processing of tactile data at the point of contact) is centric to the human skin as some neuroscience studies show. Likewise, distributed cell or molecular energy is a key feature of human skin. The eSkin with such features, along with distributed and embedded sensors/electronics on soft substrates, is an interesting topic to explore. These features also make eSkin significantly different from conventional computing. For example, unlike conventional centralized computing enabled by miniaturized chips, the eSkin could be seen as a flexible and wearable large area computer with distributed sensors and harmonized energy. This paper discusses these advanced features in eSkin, particularly the distributed sensing harmoniously integrated with energy harvesters, storage devices and distributed computing to read and locally process the tactile sensory data. Rapid advances in neuromorphic hardware, flexible energy generation, energy-conscious electronics, flexible and printed electronics are also discussed.

This article is part of the theme issue ‘Harmonizing energy-autonomous computing and intelligence’.
A semi-holographic hyperdimensional representation system for hardware-friendly cognitive computing
Authors: Serb A. , Kobyzev I. , Wang J. , Prodromakis T.
Published: December 2019

One of the main, long-term objectives of artificial intelligence is the creation of thinking machines. To that end, substantial effort has been placed into designing cognitive systems; i.e. systems that can manipulate semantic-level information. A substantial part of that effort is oriented towards designing the mathematical machinery underlying cognition in a way that is very efficiently implementable in hardware. In this work, we propose a ‘semi-holographic’ representation system that can be implemented in hardware using only multiplexing and addition operations, thus avoiding the need for expensive multiplication. The resulting architecture can be readily constructed by recycling standard microprocessor elements and is capable of performing two key mathematical operations frequently used in cognition, superposition and binding, within a budget of below 6 pJ for 64-bit operands. Our proposed ‘cognitive processing unit’ is intended as just one (albeit crucial) part of much larger cognitive systems where artificial neural networks of all kinds and associative memories work in concord to give rise to intelligence.

This article is part of the theme issue ‘Harmonizing energy-autonomous computing and intelligence’.
Neural network design for energy-autonomous artificial intelligence applications using temporal encoding
Authors: Mileiko Sergey , Bunnam Thanasin , Xia Fei , Shafik Rishad , Yakovlev Alex , Das Shidhartha
Published: December 2019

Neural networks (NNs) are steering a new generation of artificial intelligence (AI) applications at the micro-edge. Examples include wireless sensors, wearables and cybernetic systems that collect data and process them to support real-world decisions and controls. For energy autonomy, these applications are typically powered by energy harvesters. As harvesters and other power sources which provide energy autonomy inevitably have power variations, the circuits need to robustly operate over a dynamic power envelope. In other words, the NN hardware needs to be able to function correctly under unpredictable and variable supply voltages. In this paper, we propose a novel NN design approach using the principle of pulse width modulation (PWM). PWM signals represent information with their duty cycle values which may be made independent of the voltages and frequencies of the carrier signals. We design a PWM-based perceptron which can serve as the fundamental building block for NNs, by using an entirely new method of realizing arithmetic in the PWM domain. We analyse the proposed approach building from a 3 × 3 perceptron circuit to a complex multi-layer NN. Using handwritten character recognition as an exemplar of AI applications, we demonstrate the power elasticity, resilience and efficiency of the proposed NN design in the presence of functional and parametric variations including large voltage variations in the power supply.

This article is part of the theme issue ‘Harmonizing energy-autonomous computing and intelligence’.
Understanding extraction principles underpinning historical antimicrobial drug discovery for improving rediscovery and reproducibility
Authors: Keulen Geertje van , Luft Diana , Jenkins Rowena E.
Published: December 2019

Area of expertise: Microbiology, biochemistry, antimicrobials, medieval Welsh texts exploration.

Future goals: Medieval and other historical Welsh texts are newly being translated into English, which include the Red Book of Hergest used by the Physicians of Myddfai, the famous Welsh medical practitioners of the 12th century. The previous translations have so far only been studied for Welsh Language (esp. poetry) and cultural aspects, while these books also contain a wealth of information on historical approaches to societal, environmental and medical problems. We aimed to explore the translated texts for the recovery of (bio)chemical, public health and environmental knowledge of the ancient Welsh environment and society.

The newly translated medieval Welsh antimicrobial recipes were reviewed for plant names/parts and other sources, medical indication of application, and materials or processes related to extraction and formulation of the remedy in order to better understand principal components and their activities, and to be able to redesign recipes using modern chemical extraction protocols. Statistical analysis showed that a wide range of materials could potentially function for selective extraction or for precipitation of (non-)active components. Clear descriptions of processes and protocols were usually missing. It therefore requires considerable human and material resources to truly assess the merits of medieval recipes.

It would therefore be worthwhile to discuss the merit of a systematic review of historical extraction and purification principles, to be able to suggest common principles aiding effective rediscovery and redesign of antimicrobials from historical texts. Did medieval societies employ “Standard Operating Procedures”? How can modern scientists accurately and efficiently assess the merits of historical treatment alternatives to infection in the age of increasing antimicrobial resistance?
A strong inhibitory effect of heather honey, propolis and medicinal plant extracts on biofilm formation by pathogenic bacteria
Authors: Billah Zara , Attar Baraa , Hughes Lisa , Seidel Veronique , Efthimiou Georgios
Published: December 2019

Introduction:
P. aeruginosa and S. aureus are opportunistic pathogens that cause a wide range of infections. Their increasing resistance to antibiotics is a serious concern and making them susceptible to treatments is now more essential than ever. There is a need to discover new biofilm inhibitors to increase the susceptibility of these bacteria to antibiotics.

Hypothesis and aims: To evaluate the antibiofilm activity of heather honey, propolis and medicinal plant extracts against P. aeruginosa and S. aureus.

Methodology: Determination of optimum biofilm growth was carried out using a time-course assay over 24 h intervals, using P. aeruginosa PA14 and S. aureus NCTC 4135 strains. The inhibitory effects of all extracts were determined by biofilm inhibition assay in 24-well plates, with biofilms stained with crystal violet and de-stained with ethanol:acetone; OD were measured at 550 nm. Planktonic growth was measured at 600 nm and samples from the wells were streaked to determine bactericidal effects.

Results: Heather honey extracts inhibited both P. aeruginosa and S. aureus by 68%. At 60 μg/mL, one of the propolis extracts promoted biofilm growth of both pathogens. Two other propolis extracts also promoted growth in P. aeruginosa but inhibited biofilm formation in S. aureus by 76.5% and 13.8%, respectively. Three plant extracts inhibited S. aureus biofilm by 7.5%, 10.2% and 87.6% and inhibited P. aeruginosa by −34.9%, 34.7% and 19.4%, respectively.

Conclusion: All samples showed varying biofilm inhibition capabilities, but biofilm formation seemed to be more easily inhibited in S. aureus than in P. aeruginosa.
Chikungunya virus recruits the ESCRT trafficking machinery at multiple steps during the intracellular life cycle
Authors: Torii Shiho , Orba Yasuko , Sasaki Michihito , Wada Yuji , Hobson-Peters Jody , Hall Roy , Sawa Hirofumi
Published: December 2019

Chikungunya virus (CHIKV) causes a re-emerging zoonotic disease characterized by fever and joint pain. Since there are no available vaccines nor medicine for CHIKV infection, better understanding of molecular mechanisms underlying CHIKV infection is demanded to develop new treatments. To date, few studies exist describing host factors necessary for intracellular trafficking of CHIKV; thus, the aim of this study is to identify trafficking genes regulating CHIKV infection and to investigate their roles.

We conducted imaging-based siRNA screen targeting 73 trafficking genes, examined the interaction with CHIKV proteins by co-immunoprecipitation and confocal analyses, and then investigated which infection steps of CHIKV were affected by the identified gene(s).

The siRNA screen showed that endosomal sorting complexes required for transport (ESCRT) proteins were involved in CHIKV infection. Co-immunoprecipitation analyses between ESCRT proteins and CHIKV proteins revealed that both structural and non-structural proteins of CHIKV interacted with HGS, a component of ESCRT-0 complex. Confocal analyses demonstrated colocalization of HGS with CHIKV E2 and dsRNA, a marker for the replicated CHIKV genome. Gene knockdown analyses using CHIKV replicon and CHIKV-like particle system demonstrated that HGS facilitated both genome replication and post-translational steps of CHIKV infection, as well as other ESCRT factors.

Here, we propose for the first time that CHIKV requires ESCRT factors at multiple steps during its intracellular life cycle.
Modulation of arbovirus infection by mosquito saliva
Authors: Lefteri Daniella A , Pondeville Emilie , Bryden Steven R , Pingen Marieke , McKimmie Clive S
Published: December 2019

Arboviruses constitute a major public health problem; in particular mosquito-borne arboviruses that continuously emerge and re-emerge. Arbovirus infection of mammals is enhanced by the presence of a mosquito-bite at the inoculation site, or by the co-inoculation of extracted mosquito saliva alongside virus, in comparison to virus experimentally administered by needle inoculation in the absence of bite/saliva. Inflammatory responses elicited to saliva appear to be key in facilitating this enhancement. As such, we have studied the mechanistic basis for these observations by investigating mosquito-bite factors, as well as host responses, involved in facilitating viral enhancement. We have studied whether saliva from different mosquito species successfully enhance virus infection. Interestingly, while saliva from Aedes genus enhanced virus infection, An. gambiae saliva did not. This could partly explain why An. gambiae mosquitos are unsuitable vectors for transmitting most arboviruses. By comparing the effects that saliva from these different species have at the inoculation-site, we have further specified which inflammatory responses modulate arbovirus infection in the skin. Using an in vivo mouse-model we demonstrate that An. gambiae causes significantly less oedema than Ae. aegypti and that histamine induced oedema in the absence of salivary-factors also enhances infection. Also, measuring cytokine responses to Aedes and Anopheles saliva, showed that several key anti-viral chemokines such as CCL5 were significantly more upregulated by Anopheles. Hence, we’re providing important insights into how mosquito saliva modulates infection. A better understanding of this will aid the development of anti-viral treatments targeting factors within the mosquito bite that are common to many distinct infections.
Virus-host interactions for Bluetongue virus and characterization of a new NS3 function
Authors: POURCELOT Marie , Kundlacz Cindy , Caignard Grégory , Fablet Aurore , Moraes Rayane Amaral Da Silva , Léger Thibaut , Rijn Piet A. van , Zientara Stephan , Vitour Damien
Published: December 2019

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of ruminants. Infections by this virus result in variable clinical outcomes depending on host and viral factors. We know that these variances largely rely on virus-host molecular interactions. To investigate these networks, our lab has developed since few years an interactomic research programme based on the yeast two-hybrid approach.To date, we have carried out the complete BTV interactome against two complementary DNA libraries, from bovine and culicoides cell lines, that allowed to the identification of about one hundred new putative cellular partners for BTV. We are now validating these interactions through biochemical and functional studies. To complete this interactomic programme, we now also combine affinity purification approach coupled to mass spectrometry. Upon immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified new cellular interactors including the serine/threonine kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway. We showed that BTV-NS3 enhances the MAPK/ERK pathway and this activation is BRAF-dependent. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate this pathway is conserved throughout multiple serotypes but appears to be specific to BTV compared to other orbiviruses. Inhibition of MAPK/ERK pathway activation with the pharmacological molecule U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.
Orthobunyavirus recruits host ESCRT machinery to Golgi compartments to achieve efficient viral particle production
Authors: Barbosa Natalia , Dias Marcos , Mendonca Leila , Schindler Michael , Arruda Eurico , Silva Luis da
Published: December 2019

Oropouche virus (OROV) is the etiological agent of Oropouche fever, a febrile illness frequent in South America. Despite its importance, the replicative cycle of this Orthobunyavirus is only starting to emerge. In this study we described the assembly pathway of OROV in Hela cells. After a thorough description of OROV one-step replication cycle, we observed by Immunofluorescence large vesicle-like structures enriched in OROV proteins termed viral factories (Vfs). These vesicles were associated with cis-Golgi (Giantin), trans-Golgi network (TGN46) and endosomal (HRS) proteins. Immuno-EM analysis of these Vfs revealed a resemblance with multivesicular body structure, which also contained viral particles. Interestingly, no leakage of late endosomal proteins (CD63 and Lamp1) was observed associated with Vfs. Therefore, we looked if ESCRT (Endosomal Sorting Complexes required for Transport) machinery could play a role in OROV replication. Depletion of Tsg101 (ESCRT-I) and Alix by siRNA compromised Vfs formation (∼40% in both cases) that contained a significantly lower number of intra-luminal viral-like particles (∼50% in both cases). Additionally, depletion of Alix led to a strong reduction in viral production (∼80%). Moreover, the overexpression of a dominant negative form of the AAATPase Vps4A (Vps4E/Q), which disrupts the MVB pathway, led to an enlargement in Vfs’ area (∼119%), where Vps4E/Q accumulated. Importantly, we also detected a colocalization between Vps4E/Q/TGN46 and Alix/TGN46 in viral factories, suggesting an involvement of ESCRT machinery, which is re-localized to the TGN in virus assembly. Therefore, our data represents an unprecedented mechanism of how viruses hijack host cell components for coordinated morphogenesis.
A salivary protein of Aedes aegypti promotes dengue-2 virus replication and transmission
Authors: Shiao Shin-Hong
Published: December 2019

Although dengue is the most prevalent arthropod-borne viral disease in humans, no effective medication or vaccine is presently available. Previous studies suggested that mosquito salivary proteins influence infection by the dengue virus (DENV) in the mammalian host. However, the effects of salivary proteins on DENV replication within the Aedes aegypti mosquito remain largely unknown. In this study, we investigated the effect of a specific salivary protein (named AaSG34) on DENV serotype 2 (DENV2) replication and transmission. We showed that transcripts of AaSG34 were upregulated in the salivary glands of Aedes aegypti mosquitoes after a meal of blood infected with DENV2. Transcripts of the dengue viral genome and envelop protein in the salivary glands were significantly diminished after an infectious blood meal when AaSG34 was silenced. The effect of AaSG34 on DENV2 transmission was investigated in Stat1-deficient mice. The intradermal inoculation of infectious mosquito saliva induced hemorrhaging inthe Stat1-deficient mice; however, saliva from the AaSG34-silenced mosquitoes did not induce hemorrhaging, suggesting that AaSG34 enhances DENV2 transmission. This is the first report to demonstrate that the protein AaSG34 promotes DENV2 replication in mosquito salivary glands and enhances the transmission of the virus to the mammalian host.
The piRNA pathway in host-pathogen interaction: Aedes albopictus and arboviruses
Authors: Marconcini Michele , Houé Vincent , Valerio Federica , Palatini Umberto , Pischedda Elisa , Ometto Lino , Forneris Federico , Failloux Anna-Bella , Bonizzoni Mariangela , Chávez Alejandro Nabor Lozada
Published: December 2019

Aedes albopictus is an aggressive invasive species and a competent vector for over 20 arboviruses, including Chikungunya, Dengue and Zika viruses. Understanding the molecular and cellular interactions between viruses and vectors is key to implement transmission-blocking strategies to prevent viral outbreaks. However, the mechanisms that shape vector competence are poorly understood. Recent evidence reveals that the genomes of Aedes spp. harbour fragmented viral sequences which produce PIWI-interacting RNAs (piRNAs), suggesting a role in vector competence. Current knowledge of the piRNA pathway in Ae. albopictus is limited, and its possible role in the establishment of persistent infections widely unknown.

We combined cutting-edge bioinformatic analyses based on next-generation sequencing data with molecular biology and virology techniques to characterise the main genes of the piRNA pathway in this mosquito species, assess their polymorphisms and analyse their expression throughout mosquito development and following infection with the Chikungunya and Dengue-1 viruses.

We identified seven piwi genes which displayed high levels of polymorphism across populations and signs of adaptive evolution. Superposition of protein homology models indicate high structure similarity among all Piwi proteins, with high levels of amino acid conservation in the inner regions devoted to RNA binding. On the contrary, solvent-exposed surfaces showed low conservation, with sites under positive selection. Infection experiments indicated specific responses depending on viral species, time of infection and mosquito tissue, highlighting distinct roles for specific Piwi proteins.

In conclusion, this work helps define the role of the piRNA pathway in persistent arboviral infections and understand the evolutionary divergence among piwi proteins.
Assessment of tick-borne flavivirus host factors through genome-scale screens
Authors: Flather Dylan P. , Marceau Caleb D. , Mlera Luwanika , Grabowski Jeffrey M. , Bloom Marshall E.
Published: December 2019

Powassan virus (POWV) is the only tick-borne flavivirus (TBFV) known to circulate in North America. Although there are relatively few documented cases of POWV disease, investigations into POWV are justified due to the increasing incidence of infection and significant case fatality rate associated with this virus. To better describe the molecular biology of the POWV replication cycle in mammalian cells, we performed genome-scale screens to uncover host factors required for viral replication. Putative proviral host factors were identified by infecting pools of cells containing knockout mutations in non-essential genes with POWV, followed by analysis of cells resistant to virus-induced cell death. Many endoplasmic reticulum membrane complex proteins were revealed in these screens, suggesting that TBFVs share some common host cell hijacking strategies with mosquito-borne flaviviruses. Candidate proteins that function in cell-matrix adhesion, glycosylation, or RNA binding in uninfected cells were the focus of validation studies. We used single-gene knockout cell lines to investigate possible proviral roles for specific proteins in the replication cycle of either POWV or Langat virus (LGTV), a non-pathogenic and model TBFV. Proteins identified in the POWV screens were not necessarily critical for LGTV replication. These results suggest that our screens were able to identify both pan-flaviviral, as well as POWV-specific, host gene products exploited during virus replication. Ongoing work is focused on characterizing distinct host-cell requirements of diverse flaviviruses. This genetic assessment of POWV replication factors, in combination with ensuing mechanistic studies, will provide possible avenues for the development of host-targeting countermeasures.
Molecular determinants of dengue virus infection in Aedes aegypti midgut
Authors: Siriphanitchakorn Tanamas , Choy Milly M. , Ng Wy Ching , Ng Dorothy , Tan Hwee Cheng , Zhang Lixin Summer , Kini R. Manjunatha , Ooi Eng Eong
Published: December 2019

To be successfully transmitted to another susceptible human host by Aedes aegypti, dengue virus (DENV) must first successfully infect the mosquito midgut. The virus-host interactions that enable successful midgut infection, however, is not well understood. To understand the important interactions for successful midgut infection, we took advantage of the wild-type DENV2 16681 and its attenuated derivative PDK53, which has been shown to be refractory in mosquito infection. Using oral infectious-blood feeding, we observed that PDK53 failed to produce infectious progenies in the midgut, despite detectable viral genome replication. Furthermore, we found that the foci of PDK53 infection in the midgut, detected by immunofluorescence staining, was limited in both size and number, compared to its parent, 16681. Transcriptional analysis of the mosquito midgut revealed increased expression of genes in multiple innate immune pathways upon PDK53 infection but not 16681. To pinpoint the mutation responsible for this phenotype, we constructed an infectious clone of 16681 and used site-directed mutagenesis to substitute each of the known mutations in PDK53 into the 16681 genomic backbone. This approach pinpointed the NS1 G53D mutation as the single most important attenuating mutation in PDK53 in engendering refractoriness to mosquito midgut infection. Mechanistically, our data also suggests that this mutation affected the virus-ER-resident protein interactions that impacted the efficiency of DENV replication and hence induction of the innate immune response. Our findings reveal insights into the pathogenesis of dengue and adds to the body of knowledge on critical virushost interactions that govern epidemiological fitness of DENV.
National Collection of Pathogenic Viruses: A Repository for Well Characterised and Authenticated Viruses
Authors: Onianwa Okechukwu , Russell Julie , Alexander Sarah
Published: December 2019

The National Collection of Pathogenic Viruses (NCPV) is one of four repositories making up Public Health England Culture Collections. It was established two decades ago to offer safe and secure facilities for storage of patents and deposits as well as to supply standardised virus products to the wider scientific community. Within the collection are strains of both medical and veterinary importance, with representation from a wide variety of virus families, classified as Hazard Groups 2, 3 and 4 by the Advisory Committee for Dangerous Pathogens (ACDP). These include arboviruses such as Dengue, Yellow-fever, Oropouche and Sandfly fever Naples.

Throughout the year, the collection receives virus deposits. The depositing process is free and simple. The quality of every virus material received in the collection is assessed through a series of tests to confirm viability and absence of microbial contamination (including mycoplasmas). Virus identity is confirmed by nucleic acid profiling. Once standards are met, the virus products are made available to the wider scientific community. In addition to facilitating access to viruses and their associated products as well as secure storage for patents, NCPV also undertakes collaborative projects and contract research/developmental work with academic and commercial partners, by providing expertise in the handling of viral pathogens.

A repository for well-characterised, authenticated viruses is a useful resource for the scientific community, aiding in the understanding of circulating viral pathogens and providing access to quality-assured reference reagents for the identification and development of vaccines and therapeutics in the event of global viral outbreaks.
Pan-antivirals to combat re-emerging alphaviruses
Authors: Jacobs Sofie , Abdelnabi Rana , Lambin Dominique , Marchand Arnaud , Chaltin Patrick , Neyts Johan , Delang Leen
Published: December 2019

Mosquito-transmitted alphaviruses are distributed globally and include human pathogens that can cause severe long-term arthritogenic or neurological complications. There are currently no market-approved antivirals or vaccines to treat or prevent these infections. Outbreaks are difficult to predict as they can emerge spontaneously in susceptible human populations. Furthermore, it is challenging to determine the causative pathogen due to great similarities in clinical features of alphavirus-associated diseases. Therefore, drugs with broad-spectrum anti-alphavirus activity could serve as a fast first-line therapy in case of an outbreak. We have performed high-throughput screenings of broad-chemical space libraries and identified a series of small molecules with antiviral activity against different chikungunya virus lineages. Interestingly, we demonstrated that this series exerts broad-spectrum anti-alphavirus activity against a range of arthritogenic alphaviruses. Time-of-addition studies showed that this series has in vitro antiviral activity early in the viral RNA replication stage. Furthermore, these molecules were not cross-resistant with favipiravir-resistant chikungunya virus, a compound that inhibits the viral RNA polymerase. To investigate the mechanism of action in more detail, in vitro resistance selection is currently ongoing. Elucidation of the specific antiviral target of this newly identified series could reveal a new target in the alphavirus proteome, which could be valuable for the development of broad-spectrum anti-alphavirus drugs.
Chikungunya virus resistant to the antiviral favipiravir is severely attenuated in mosquitoes
Authors: Langendries Lana , Jacobs Sofie , Abdelnabi Rana , Neyts Johan , Delang Leen
Published: December 2019

It is currently unclear whether antiviral drug-resistant arboviruses can be transmitted by their mosquito vectors. We showed previously that the dissemination and the transmission of a chikungunya virus (CHIKV) resistant to the antiviral favipiravir was markedly decreased as compared to WT (Delang et al, mSphere 2018). The attenuated phenotype of this resistant virus was confirmed in Aag2 and C6/36 mosquito cells. Here, we aimed to study the mechanism of the attenuated phenotype in more detail.

First, replication kinetics studies at 32°C for both Vero and mosquito cells confirmed that the attenuated fitness in mosquito cells is associated with the cell line and not with temperature. A passaging experiment of WT CHIKV on Vero cells in the absence of favipiravir showed that the observed attenuation of the resistant CHIKV was not due to passaging on Vero cells during the resistance selection. To identify the molecular mechanism of the attenuated phenotype, the genes encoding for the non-structural proteins (nsP) of the favipiravirres CHIKV were swapped into a WT CHIKV backbone. The replication fitness of these nsP2, nsP3 or nsP4 single swap variants did not differ significantly from the fitness of WT CHIKV in Vero and C6/36 cells, indicating that a combination of mutations in multiple non-structural proteins is responsible for the attenuated phenotype. Double nsP swap variants are therefore constructed and these will provide additional information on the molecular mechanism of the attenuation. Together, our results may provide interesting insights in the mosquito tropism of CHIKV.
Karyotype changes in cultivated tick cell lines
Authors: Kotsarenko Kateryna , Vechtova Pavlina , Sterba Jan
Published: December 2019

Background. Tick cell lines are an easy-to-handle system for the study of viral and bacterial infections and different aspects of tick physiology. However, long-term cultivation of tick cells can influence genome stability. The aim of our work was to analyze and compare the karyotypes of Ixodes scapularis, I. ricinus, and Ornithodoros moubata tick cell lines after long-term been in culture.

Methods. Mitotic spreads were prepared to count the number of chromosomes in ISE6, ISE18, IRE11, IRE/CTVM19, IRE/CTVM20, and OME/CTVM22 cell lines. The genome size of tick cell cultures was estimated by flow cytometry using propidium iodide staining.

Results. The modal chromosome numbers around 22 and around 48 were typical for both I. ricinus and I. scapularis cell lines and differed from the diploid chromosome number in Ixodesticks – 28. In the OME/CTVM22cell line, the modal chromosome number was 33, instead of 20 in Ornithodoros ticks. All tick cell lines had a larger genome size in compare to genomes of parental ticks.

Conclusions.Tick cell lines can be used for research purposes, however, differences in the internal processes between different cell populations should be taken into account.

Acknowledgments: We thank Lesley Bell-Sakyi and the Tick Cell Biobank for provision of the tick cell lines.
Targeting functional RNA structures in CHIKV and ZIKV
Authors: Prosser Oliver , Merits Andres , Stonehouse Nicola J , Tuplin Andrew
Published: December 2019

Emerging arboviruses such as Zika virus (ZIKV) and Chikungunya virus (CHIKV) represent a significant threat to human health and have a high potential to cause outbreaks in the near future. At present, there are no specific antivirals available for either of these important pathogens, despite the wide global prevalence of their vector, Aedes spp. mosquitos.

The positive-strand genomes of ZIKV and CHIKV, members of the flavivirus and alphavirus genera respectively, contain functional, structured cis-acting RNA elements which are essential for virus replication. By specifically targeting such RNA elements using antisense locked nucleic acid oligonucleotides (antisense-LNA), we aim to disrupt their function, and analyse the effect this has on virus replication at different life cycle stages.

Obtaining high quality, single-nucleotide-resolution structural data is essential prior to targeting RNA structures. Consequently, we mapped RNA structural elements within the ZIKV 5’ genome region using a combination of biochemical SHAPE probing, thermodynamic models and phylogenetic analysis. We are currently validating our structural data by analysis of mutant phenotypes in a reverse genetic system.

We demonstrate that functional RNA elements in CHIKV can be specifically targeted - inhibiting replication in both sub-genomic replicon and infectious virus systems. Surface plasmon resonance confirmed that an antisense-LNA binds to a specific stem-loop target with a Kd of 310nM and has an IC50 of 35nM in a sub-genomic replicon system. In future work, we aim to investigate selection of RNA-aptamers against CHIKV and target ZIKV genomic stem-loops using antisense-LNAs.
Development of reverse genetics for Toscana virus
Authors: Alexander Akira , Dunlop James , Brennan Ben , Kuchi Srikeerthana , Mair Daniel , Filipe Ana da Silva , Ratinier Maxime , Arnaud Frédérick , Kohl Alain
Published: December 2019

Toscana virus (TOSV) is a Phlebovirus in the Phenuiviridae family, found in the countries surrounding the Mediterranean. It is unusual within the Phlebovirus genus for exhibiting tropism for the central nervous system: TOSV is one of the top causes of seasonal acute meningitis/encephalitis within its range. However, little progress has been made in the study of TOSV, largely due to the lack of a reliable reverse genetics system. We used RNA sequencing to determine the sequence of Toscana strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007). This data was used to construct cDNA plasmids encoding the viral L, M and S antigenomic sequences under control of the T7 RNA promoter to recover recombinant viruses when expressed in BSR-T7/5 CL21 cells. By sequencing amplified viral cDNA, we identified two single base pair mismatches in the original TOSV reference genome (NC_006318, 19, 20), which when corrected restored functionality to the polymerase. We were then able to recover infectious recombinant TOSV (rTOSV) from cDNA clones, as well as establishing a minigenome system. Using reverse genetics, it has been possible to produce the non-structural gene (NSs) deletion mutants (a known interferon antagonist). These strains are in the process of characterisation and are to be used in infection studies in Phlebotomine flies. TOSV vaccine development has been severely hindered by the failure of previous efforts to develop reverse genetics systems for this virus. With a system now in place, it will hopefully be possible to design novel attenuated vaccine candidates.
Mosquito vector factors driving arbovirus infection in Anopheles
Authors: Loreto Raquel , Failloux Anna-Bella , Vernick Kenneth
Published: December 2019

Anopheles mosquitoes are efficient vectors of human malaria, but they are the primary vector of just one arbovirus, O’nyong nyong virus (ONNV). Consequently Anopheles-virus interactions have been relatively unexamined. It is puzzling that almost all arbovirus transmission is mediated by Aedes and its relatives while Anopheles transmit just a single virus. One hypothesis is that antiviral mechanisms are more efficient in Anopheles than Aedes, but ONNV can circumvent them. Here, we combined empirical data and bioinformatic analysis to generate a high-value set of Anopheles candidate genes likely involved in host-virus interactions. Many of the candidate genes are unannotated. We screened our panel of genes through in vitro gene silencing and identified both proviral and antiviral factors in Anopheles mosquitos. So far, the many genes tested have shown an effect on ONNV replication. For instance, the silencing of the unannotated gene AGAP00570 limited 80% of viral replication suggesting its proviral role in the ONNV-Anopheles cells system. Similarly, silencing of the Leucine-Rich repeat IMmune 4 (LRIM4) reduced the viral replication in 78%. On the contrary, preliminary data in another leucine-rich repeat protein, the APL1C, indicates the antiviral activity of this gene in Anopheles cells. ONNV is an emergent virus in Africa with epidemic potential, and these results reveal the host vector factors that influence mosquito-ONNV infection.
Louping Ill Virus Outbreak in North-East Wales
Authors: Dorey-Robinson Daniel , Folly Arran , Phipps Paul , Vidana Beatriz , Holmes Paul , Johnson Nicholas
Published: December 2019

The disease louping ill is an encephalitic viral infection that is fatal in sheep (Oves aries) and grouse (Lagopus lagopus). The disease is caused by the louping ill virus (LIV) and transmitted by sheep ticks (Ixodes ricinus) in upland areas of the United Kingdom. Reported outbreaks are sporadic and prevention is mainly targeted at controlling the vector. Despite the implications for animal welfare and the rural economy, research on the epidemiology and control of LIV has been neglected in recent years. In April of 2019, a group of 200 yearling ewes were moved onto hill grazing at a farm near Oswestry on the English/Welsh border. Within 2-3 weeks, ten had developed clinical signs suggestive of neurological impairment, including torticollis, fitting, head shaking and recumbency. A number of the affected ewes were later found dead. Following post mortem, histopathological investigation of brain tissue from an affected ewe, which had undergone euthanasia, detected glial nodules and perivascular cuffing indicating subacute non-suppurative encephalitis. The haemagglutination inhibition serological test was positive and provided evidence for infection with LIV. Reverse transcription polymerase chain reaction (RT-PCR) gave a positive result for samples of the hind brain and the resulting sequence confirmed the presence of LIV. Phylogenetic analysis indicated that the virus showed the highest identity (>99%) with LIV sequences from Aberystwyth in west Wales and was distinct from other LIV isolates found in the north of England. This case study highlights the ongoing threat to UK sheep from LIV.
Monitoring of Insecticide Resistance on Aedes sp. Mosquitoes in Banyumas Regency, Indonesia
Authors: Wijayanti Siwi Pramatama Mars , Octaviana Devi , Nurlaela Sri , Widiastuti Dyah
Published: December 2019

Background. The continuous use of insecticides for mosquito-borne disease control can cause insecticide resistance, and if left unchecked, this could lead to a substantial increase in disease incidence. The aims of this study are to monitor insecticide insecticides against Malathion and Cypermethrin and identifying the mechanisms underlying the resistance in the area of study.

Method. This is a descriptive study located in Banyumas Regency. Aedes sp. mosquitoes werecollected from three endemic areas (Arcawinangun, Karangpucung, and Purwanegara) by the ovitrap installation to 100 houses each village (total 300 houses). Filial 1 of Aedes were tested their insecticide resistance to Malathion and Cypermethrin by susceptibility test, biochemical assay and molecular by PCR.

Results. The results of the susceptibility test showed the average percentage of mosquito mortality from three villages was 30,67% which were included in the resistance category. However, the results of the biochemical assay showed that 70% of mosquitoes are still very susceptible (AV<0,7). Molecular tests are underway and the results are likely to be obtained in August 2019

Conclusion. The population of Aedes sp. in the study area has been resistant to malathion and cypermethrin, and the mechanism underlying this resistance was not based on a biochemical mechanism. It is necessary to rotate the use of insecticide active substances in DHF vector control by selecting insecticides that have a different mode of action.