Transcriptomic responses of bat cells to European bat lyssavirus 1 infection under conditions simulating euthermia and hibernation.

BACKGROUND: Coevolution between pathogens and their hosts decreases host morbidity and mortality. Bats host and can tolerate viruses which can be lethal to other vertebrate orders, including humans. Bat adaptations to infection include localized immune response, early pathogen sensing, high interferon expression without pathogen stimulation, and regulated inflammatory response. The immune reaction is costly, and bats suppress high-cost metabolism during torpor. In the temperate zone, bats hibernate in winter, utilizing a specific behavioural adaptation to survive detrimental environmental conditions and lack of energy resources. Hibernation torpor involves major physiological changes that pose an additional challenge to bat-pathogen coexistence. Here, we compared bat cellular reaction to viral challenge under conditions simulating hibernation, evaluating the changes between torpor and euthermia. RESULTS: We infected the olfactory nerve-derived cell culture of Myotis myotis with an endemic bat pathogen, European bat lyssavirus 1 (EBLV-1). After infection, the bat cells were cultivated at two different temperatures, 37 °C and 5 °C, to examine the cell response during conditions simulating euthermia and torpor, respectively. The mRNA isolated from the cells was sequenced and analysed for differential gene expression attributable to the temperature and/or infection treatment. In conditions simulating euthermia, infected bat cells produce an excess signalling by multitude of pathways involved in apoptosis and immune regulation influencing proliferation of regulatory cell types which can, in synergy with other produced cytokines, contribute to viral tolerance. We found no up- or down-regulated genes expressed in infected cells cultivated at conditions simulating torpor compared to non-infected cells cultivated under the same conditions. When studying the reaction of uninfected cells to the temperature treatment, bat cells show an increased production of heat shock proteins (HSPs) with chaperone activity, improving the bat's ability to repair molecular structures damaged due to the stress related to the temperature change. CONCLUSIONS: The lack of bat cell reaction to infection in conditions simulating hibernation may contribute to the virus tolerance or persistence in bats. Together with the cell damage repair mechanisms induced in response to hibernation, the immune regulation may promote bats' ability to act as reservoirs of zoonotic viruses such as lyssaviruses.

Species-Specific Molecular Barriers to SARS-CoV-2 Replication in Bat Cells.

Bats are natural reservoirs of numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction between coronaviruses and bat cells is sparse. We investigated the ability of primary cells from Rhinolophus and Myotis species, as well as of established and novel cell lines from Myotis myotis, Eptesicus serotinus, Tadarida brasiliensis, and Nyctalus noctula, to support SARS-CoV-2 replication. None of these cells were permissive to infection, not even the ones expressing detectable levels of angiotensin-converting enzyme 2 (ACE2), which serves as the viral receptor in many mammalian species. The resistance to infection was overcome by expression of human ACE2 (hACE2) in three cell lines, suggesting that the restriction to viral replication was due to a low expression of bat ACE2 (bACE2) or the absence of bACE2 binding in these cells. Infectious virions were produced but not released from hACE2-transduced M. myotis brain cells. E. serotinus brain cells and M. myotis nasal epithelial cells expressing hACE2 efficiently controlled viral replication, which correlated with a potent interferon response. Our data highlight the existence of species-specific and cell-specific molecular barriers to viral replication in bat cells. These novel chiropteran cellular models are valuable tools to investigate the evolutionary relationships between bats and coronaviruses. IMPORTANCE Bats are host ancestors of several viruses that cause serious disease in humans, as illustrated by the ongoing SARS-CoV-2 pandemic. Progress in investigating bat-virus interactions has been hampered by a limited number of available bat cellular models. We have generated primary cells and cell lines from several bat species that are relevant for coronavirus research. The various permissivities of the cells to SARS-CoV-2 infection offered the opportunity to uncover some species-specific molecular restrictions to viral replication. All bat cells exhibited a potent entry-dependent restriction. Once this block was overcome by overexpression of human ACE2, which serves at the viral receptor, two bat cell lines controlled well viral replication, which correlated with the inability of the virus to counteract antiviral responses. Other cells potently inhibited viral release. Our novel bat cellular models contribute to a better understanding of the molecular interplays between bat cells and viruses.

Uncovering the endemic circulation of rabies in Cambodia.

In epidemiology, endemicity characterizes sustained pathogen circulation in a geographical area, which involves a circulation that is not being maintained by external introductions. Because it could potentially shape the design of public health interventions, there is an interest in fully uncovering the endemic pattern of a disease. Here, we use a phylogeographic approach to investigate the endemic signature of rabies virus (RABV) circulation in Cambodia. Cambodia is located in one of the most affected regions by rabies in the world, but RABV circulation between and within Southeast Asian countries remains understudied. Our analyses are based on a new comprehensive data set of 199 RABV genomes collected between 2014 and 2017 as well as previously published Southeast Asian RABV sequences. We show that most Cambodian sequences belong to a distinct clade that has been circulating almost exclusively in Cambodia. Our results thus point towards rabies circulation in Cambodia that does not rely on external introductions. We further characterize within-Cambodia RABV circulation by estimating lineage dispersal metrics that appear to be similar to other settings, and by performing landscape phylogeographic analyses to investigate environmental factors impacting the dispersal dynamic of viral lineages. The latter analyses do not lead to the identification of environmental variables that would be associated with the heterogeneity of viral lineage dispersal velocities, which calls for a better understanding of local dog ecology and further investigations of the potential drivers of RABV spread in the region. Overall, our study illustrates how phylogeographic investigations can be performed to assess and characterize viral endemicity in a context of relatively limited data.

First Case of Lethal Encephalitis in Western Europe Due to European Bat Lyssavirus Type 1.

BACKGROUND: Inaccurate diagnosis of encephalitis is a major issue as immunosuppressive treatments can be deleterious in case of viral infection. The European bat lyssavirus type 1 (EBLV-1), a virus related to rabies virus, is endemic in European bats. No human case has yet been reported in Western Europe. A 59-year-old patient without specific past medical history died from encephalitis. A colony of bats lived in an outbuilding of his house. No diagnosis was made using standard procedures. METHODS: We used a next generation sequencing (NGS) based transcriptomic protocol to search for pathogens in autopsy samples (meninges and brain frontal lobe). Results were confirmed by polymerase chain reaction (PCR) and by antibody testing in serum. Immunochemistry was used to characterize inflammatory cells and viral antigens in brain lesions. Cells and mice were inoculated with brain extracts for virus isolation. RESULTS: The patient's brain lesions were severe and diffuse in white and gray matter. Perivascular inflammatory infiltrates were abundant and rich in plasma cells. NGS identified European bat lyssavirus type 1a in brain, which was confirmed by PCR. A high titer of neutralizing antibodies was found in serum. No viral antigen was detected, and the virus could not be isolated by cell culture or by mouse inoculation. CONCLUSIONS: The patient died from European bat lyssavirus type 1a infection. NGS was key to identifying this unexpected viral etiology in an epidemiological context that did not suggest rabies. People exposed to bats should be strongly advised to be vaccinated with rabies vaccines, which are effective against EBLV-1.

2022 taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Comparison of intra- and inter-host genetic diversity in rabies virus during experimental cross-species transmission.

The development of high-throughput genome sequencing enables accurate measurements of levels of sub-consensus intra-host virus genetic diversity and analysis of the role played by natural selection during cross-species transmission. We analysed the natural and experimental evolution of rabies virus (RABV), an important example of a virus that is able to make multiple host jumps. In particular, we (i) analyzed RABV evolution during experimental host switching with the goal of identifying possible genetic markers of host adaptation, (ii) compared the mutational changes observed during passage with those observed in natura, and (iii) determined whether the colonization of new hosts or tissues requires adaptive evolution in the virus. To address these aims, animal infection models (dog and fox) and primary cell culture models (embryo brain cells of dog and fox) were developed and viral variation was studied in detail through deep genome sequencing. Our analysis revealed a strong unidirectional host evolutionary effect, as dog-adapted rabies virus was able to replicate in fox and fox cells relatively easily, while dogs or neuronal dog cells were not easily susceptible to fox adapted-RABV. This suggests that dog RABV may be able to adapt to some hosts more easily than other host variants, or that when RABV switched from dogs to red foxes it lost its ability to adapt easily to other species. Although no difference in patterns of mutation variation between different host organs was observed, mutations were common following both in vitro and in vivo passage. However, only a small number of these mutations also appeared in natura, suggesting that adaptation during successful cross-species virus transmission is a complex, multifactorial evolutionary process.

Large-Scale Phylogenomic Analysis Reveals the Complex Evolutionary History of Rabies Virus in Multiple Carnivore Hosts.

The natural evolution of rabies virus (RABV) provides a potent example of multiple host shifts and an important opportunity to determine the mechanisms that underpin viral emergence. Using 321 genome sequences spanning an unprecedented diversity of RABV, we compared evolutionary rates and selection pressures in viruses sampled from multiple primary host shifts that occurred on various continents. Two major phylogenetic groups, bat-related RABV and dog-related RABV, experiencing markedly different evolutionary dynamics were identified. While no correlation between time and genetic divergence was found in bat-related RABV, the evolution of dog-related RABV followed a generally clock-like structure, although with a relatively low evolutionary rate. Subsequent molecular clock dating indicated that dog-related RABV likely underwent a rapid global spread following the intensification of intercontinental trade starting in the 15th century. Strikingly, although dog RABV has jumped to various wildlife species from the order Carnivora, we found no clear evidence that these host-jumping events involved adaptive evolution, with RABV instead characterized by strong purifying selection, suggesting that ecological processes also play an important role in shaping patterns of emergence. However, specific amino acid changes were associated with the parallel emergence of RABV in ferret-badgers in Asia, and some host shifts were associated with increases in evolutionary rate, particularly in the ferret-badger and mongoose, implying that changes in host species can have important impacts on evolutionary dynamics.

The shift in rabies epidemiology in France: time to adjust rabies post-exposure risk assessment.

The epidemiology of rabies in France and western Europe has changed during the past 22 years. In France, rabies in non-flying terrestrial mammals was declared to be eliminated in 2001, and the risk of rabies is now limited to contact with bats, rabid animals illegally imported from rabies-enzootic countries and traveller exposure in enzootic areas. We analysed the epidemiology of rabies in France from 1995 to 2016, describing and analysing data on human rabies surveillance as well as data on post-exposure prophylaxis (PEP) collected from the network of French antirabies clinics. Over the study period, seven individuals were diagnosed with rabies in France, all of whom were infected outside mainland France. PEP data analysis revealed an expected overall decrease in PEP administration for individuals exposed in mainland France, but there was still overuse of anti-rabies drugs, given the very low epidemiological risk. On the other hand, a significant increase in PEP delivered to individuals exposed abroad was evidenced. These epidemiological trends indicate that clear guidelines should be provided to support physicians' efforts to adjust rabies risk assessment to the evolution of the epidemiological situation.

Genetic Diversity within Alphaherpesviruses: Characterization of a Novel Variant of Herpes Simplex Virus 2.

UNLABELLED: Very low levels of variability have been reported for the herpes simplex virus 2 (HSV-2) genome. We recently described a new genetic variant of HSV-2 (HSV-2v) characterized by a much higher degree of variability for the UL30 gene (DNA polymerase) than observed for the HG52 reference strain. Retrospective screening of 505 clinical isolates of HSV-2 by a specific real-time PCR assay targeting the UL30 gene led to the identification of 13 additional HSV-2v isolates, resulting in an overall prevalence of 2.8%. Phylogenetic analyses on the basis of microsatellite markers and gene sequences showed clear differences between HSV-2v and classical HSV-2. Thirteen of the 14 patients infected with HSV-2v originated from West or Central Africa, and 9 of these patients were coinfected with HIV. These results raise questions about the origin of this new virus. Preliminary results suggest that HSV-2v may have acquired genomic segments from chimpanzee alphaherpesvirus (ChHV) by recombination. IMPORTANCE: This article deals with the highly topical question of the origin of this new HSV-2 variant identified in patients with HIV coinfection originating mostly from West or Central Africa. HSV-2v clearly differed from classical HSV-2 isolates in phylogenetic analyses and may be linked to simian ChHV. This new HSV-2 variant highlights the possible occurrence of recombination between human and simian herpesviruses under natural conditions, potentially presenting greater challenges for the future.

Severe ketoalkalosis as initial presentation of imported human rabies in France.

We report a patient with an unusual initial metabolic presentation of imported human rabies who became symptomatic within 2 weeks of returning from Mali to France. This is the single case of imported human rabies identified in France within the past 11 years and the first report of viral RNA in bronchial secretions.

Prospects for dog rabies elimination in Nigeria by 2030.

The attainment of the global target of zero dog-mediated human rabies by 2030 depends on functional rabies programmes. Nigeria, a rabies-endemic country, and the most populous country in Africa has a very poor rabies control strategy with a score of 1.5 out of 5 based on the Stepwise Approach towards Rabies Elimination (SARE). In this article, we report a scoping review that we conducted to highlight the strengths, weaknesses, opportunities and threats as well as situational analysis of rabies control in Nigeria and suggest a timeline for key activities that are needed to ensure zero by 30. Our findings reveal that rabies is grossly under-reported as only 998 human and 273 dog-suspected rabies cases were reported across Nigeria between 2017 and 2022. Our literature review also demonstrates a paucity of information on rabies in both human and animal health sectors. A total of 49 studies on dog rabies in Nigeria, with a predominance of reports from the North Central geopolitical region (48%, n = 23) were therefore included in this study. Currently, only 16.2% (n = 6/37) of Nigerian states have available data related to the estimated dog populations, the dog ownership rates, the vaccination status of dogs or the incidence of dog bites. Based on a dog-to-human ratio of 1:16.3, we estimated that the dog population in Nigeria was 12,969,368 (95% CI: 12,320,900-13,617,836). Thus, to attain herd immunity and dog rabies control in Nigeria, at least 9.1 million dogs must be vaccinated annually. Our review reveals that, despite the strengths and available opportunities to achieve rabies control in Nigeria by 2030, the weaknesses and challenges will make the attainment of zero by 30 very difficult or impossible. Nigeria's best-case scenario by the year 2030 is SARE stage 3-4 (control-elimination) out of 5. Otherwise, the rabies control programme might not surpass SARE stages 2-3. To attain zero by 30, Nigeria must re-strategize its current rabies control programme by funding and implementing the national strategic plan for rabies control, creating a rabies desk office in the 37 states (FCT inclusive), rigorously conducting mass vaccination campaigns, providing post-exposure prophylaxis, prioritizing mass enlightenment with a focus on responsible pet ownership and conduct baseline national rabies surveillance in the animal and human health sectors.

Surveillance and control of rabies in La Reunion, Mayotte, and Madagascar.

Mayotte and La Reunion islands are currently free of animal rabies and surveillance is performed by the French Human and Veterinary Public Health Services. However, dog rabies is still enzootic in Madagascar with 4 to 10 confirmed human cases each year. The number of antirabies medical centres in Madagascar is still scarce to provide easy access to the local population for post-exposure rabies prophylaxis. Furthermore, stray dog populations are considerable and attempts to control rabies by mass campaigns of dog vaccination have not received sufficient attention from the national health authorities. To address these challenges, an expanded program to control rabies needs to be initiated by the Malagasy authorities.

[Rabies]. / La rage.

Rabies virus, a neurotropic lyssavirus responsible for unavoidable fatal encephalitis, is transmitted by saliva of infected animals through bite, scratch or licking of broken skin or a mucous membrane. Infection can be prevented by timely prevention (wash for several minutes, antisepsis and vaccination completed by antirabies immunoglobulins [Ig] according to the severity of exposure). The 55,000 human deaths estimated annually worldwide result mainly from uncontrolled canine rabies in enzootic countries (particularly in Africa and in Asia), attributable to a lack of resources or interest for this disease. Bat rabies, henceforth first cause of human's rabies in many countries in America, affects a very small number of individuals but seems more difficult to control. Shortened vaccine protocols, rationalized use of Ig and development of products of substitution should enhance access of exposed patients to prevention. Finally, research on the biological cycle, the pathogeny and on escape of virus-induced mechanisms from the immune system should continue to pave the way for presently unknown treatments of clinical rabies.

[Bats and viruses: Balancing infection control and immune tolerance]. / Des chauves-souris et des virus - Entre contrôle de l'infection et tolérance immunitaire.

In recent decades, bats have been associated with numerous viral pandemics. Bats harbor a large variety of viruses, some of which have a high zoonotic potential for humans. While infection with these viruses can be fatal in other mammals, bats are often infected asymptomatically. It is hypothesized that a balanced immune response would enable them to maintain homeostasis during infection, thus limiting viral replication while avoiding the impact of excessive inflammation. Deciphering these mechanisms, using adapted in vitro models, will help assess and avoid the potential zoonotic risk of these animals, while paving the way for the development of therapeutics for infectious and inflammatory diseases.

Title: Des chauves-souris et des virus - Entre contrôle de l'infection et tolérance immunitaire. Abstract: Durant les dernières décennies, les chauves-souris ont été associées à de nombreuses pandémies virales. Ces animaux hébergent en effet une diversité importante de virus, certains à potentiel zoonotique pour l'homme. Alors que ces virus peuvent être mortels chez d'autres mammifères, les chauves-souris sont souvent infectées de façon asymptomatique. La mise en place d'une réponse immunitaire équilibrée leur permettrait de maintenir l'homéostasie lors de l'infection, en limitant la réplication virale tout en évitant l'impact d'une inflammation trop importante. Le décryptage de ces mécanismes, à l'aide de modèles in vitro adaptés, devrait contribuer à évaluer et à éviter le risque zoonotique potentiel de ces animaux, tout en ouvrant la voie au développement de thérapeutiques pour les maladies infectieuses et inflammatoires.

Revised Taxonomy of Rhabdoviruses Infecting Fish and Marine Mammals.

The Rhabdoviridae is a large family of negative-sense (-) RNA viruses that includes important pathogens of ray-finned fish and marine mammals. As for all viruses, the taxonomic assignment of rhabdoviruses occurs through a process implemented by the International Committee on Taxonomy of Viruses (ICTV). A recent revision of taxonomy conducted in conjunction with the ICTV Rhabdoviridae Study Group has resulted in the establishment of three new subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae) within the Rhabdoviridae, as well as three new genera (Cetarhavirus, Siniperhavirus, and Scophrhavirus) and seven new species for viruses infecting fish or marine mammals. All rhabdovirus species have also now been named or renamed to comply with the binomial format adopted by the ICTV in 2021, comprising the genus name followed by a species epithet. Phylogenetic analyses of L protein (RNA-dependent RNA polymerase) sequences of (-) RNA viruses indicate that members of the genus Novirhabdovirus (subfamily Gammarhabdovirinae) do not cluster within the Rhabdoviridae, suggesting the need for a review of their current classification.

Complete Genome Sequences of Five Rabies Virus Strains Obtained from Domestic Carnivores in Liberia.

As in other African countries, canine rabies is endemic in Liberia. However, data concerning the genetic diversity of rabies virus isolates circulating in this country remain limited. We report here the complete genome sequences of five rabies viruses obtained from domestic animals. All of them belonged to subgroup H within the Africa 2 clade.

Nearly Complete Genome Sequences of Eight Rabies Virus Strains Obtained from Domestic Carnivores in the Democratic Republic of the Congo.

In this report, we describe eight nearly complete genome sequences of rabies virus strains collected in the Democratic Republic of the Congo from domestic carnivores in 2017 and 2018. All of them clustered into a specific phylogroup among the Africa 1b lineage in the Cosmopolitan clade.

Lyssavirus detection and typing using pyrosequencing.

Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3' terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin.

Evaluation of high-throughput sequencing for identifying known and unknown viruses in biological samples.

High-throughput sequencing furnishes a large number of short sequence reads from uncloned DNA and has rapidly become a major tool for identifying viruses in biological samples, and in particular when the target sequence is undefined. In this study, we assessed the analytical sensitivity of a pipeline for detection of viruses in biological samples based on either the Roche-454 genome sequencer or Illumina genome analyzer platforms. We sequenced biological samples artificially spiked with a wide range of viruses with genomes composed of single or double-stranded DNA or RNA, including linear or circular single-stranded DNA. Viruses were added at a very low concentration most often corresponding to 3 or 0.8 times the validated level of detection of quantitative reverse transcriptase PCRs (RT-PCRs). For the viruses represented, or resembling those represented, in public nucleotide sequence databases, we show that the higher output of Illumina is associated with a much greater sensitivity, approaching that of optimized quantitative (RT-)PCRs. In this blind study, identification of viruses was achieved without incorrect identification. Nevertheless, at these low concentrations, the number of reads generated by the Illumina platform was too small to facilitate assembly of contigs without the use of a reference sequence, thus precluding detection of unknown viruses. When the virus load was sufficiently high, de novo assembly permitted the generation of long contigs corresponding to nearly full-length genomes and thus should facilitate the identification of novel viruses.

Application of broad-spectrum resequencing microarray for genotyping rhabdoviruses.

The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world.