Serpentoviruses: More than Respiratory Pathogens.

In recent years, nidoviruses have emerged as important respiratory pathogens of reptiles, affecting captive python populations. In pythons, nidovirus (recently reclassified as serpentovirus) infection induces an inflammation of the upper respiratory and alimentary tract which can develop into a severe, often fatal proliferative pneumonia. We observed pyogranulomatous and fibrinonecrotic lesions in organ systems other than the respiratory tract during full postmortem examinations on 30 serpentovirus reverse transcription-PCR (RT-PCR)-positive pythons of varying species originating from Switzerland and Spain. The observations prompted us to study whether this not yet reported wider distribution of lesions is associated with previously unknown serpentoviruses or changes in the serpentovirus genome. RT-PCR and inoculation of Morelia viridis cell cultures served to recruit the cases and obtain virus isolates. Immunohistochemistry and immunofluorescence staining against serpentovirus nucleoprotein demonstrated that the virus infects not only a broad spectrum of epithelia (respiratory and alimentary epithelium, hepatocytes, renal tubules, pancreatic ducts, etc.), but also intravascular monocytes, intralesional macrophages, and endothelial cells. With next-generation sequencing we obtained a full-length genome for a novel serpentovirus species circulating in Switzerland. Analysis of viral genomes recovered from pythons showing serpentovirus infection-associated respiratory or systemic disease did not reveal sequence association to phenotypes; however, functional studies with different strains are needed to confirm this observation. The results indicate that serpentoviruses have a broad cell and tissue tropism, further suggesting that the course of infection could vary and involve lesions in a broad spectrum of tissues and organ systems as a consequence of monocyte-mediated viral systemic spread.IMPORTANCE During the last years, python nidoviruses (now reclassified as serpentoviruses) have become a primary cause of fatal disease in pythons. Serpentoviruses represent a threat to captive snake collections, as they spread rapidly and can be associated with high morbidity and mortality. Our study indicates that, different from previous evidence, the viruses do not only affect the respiratory tract, but can spread in the entire body with blood monocytes, have a broad spectrum of target cells, and can induce a variety of lesions. Nidovirales is an order of animal and human viruses that comprises important zoonotic pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. Serpentoviruses belong to the same order as the above-mentioned human viruses and show similar characteristics (rapid spread, respiratory and gastrointestinal tropism, etc.). The present study confirms the relevance of natural animal diseases to better understand the complexity of viruses of the order Nidovirales.

Investigations into the presence of nidoviruses in pythons.

BACKGROUND: Pneumonia and stomatitis represent severe and often fatal diseases in different captive snakes. Apart from bacterial infections, paramyxo-, adeno-, reo- and arenaviruses cause these diseases. In 2014, new viruses emerged as the cause of pneumonia in pythons. In a few publications, nidoviruses have been reported in association with pneumonia in ball pythons and a tiger python. The viruses were found using new sequencing methods from the organ tissue of dead animals. METHODS: Severe pneumonia and stomatitis resulted in a high mortality rate in a captive breeding collection of green tree pythons. Unbiased deep sequencing lead to the detection of nidoviral sequences. A developed RT-qPCR was used to confirm the metagenome results and to determine the importance of this virus. A total of 1554 different boid snakes, including animals suffering from respiratory diseases as well as healthy controls, were screened for nidoviruses. Furthermore, in addition to two full-length sequences, partial sequences were generated from different snake species. RESULTS: The assembled full-length snake nidovirus genomes share only an overall genome sequence identity of less than 66.9% to other published snake nidoviruses and new partial sequences vary between 99.89 and 79.4%. Highest viral loads were detected in lung samples. The snake nidovirus was not only present in diseased animals, but also in snakes showing no typical clinical signs. CONCLUSIONS: Our findings further highlight the possible importance of snake nidoviruses in respiratory diseases and proof multiple circulating strains with varying disease potential. Nidovirus detection in clinical healthy individuals might represent testing during the incubation period or reconvalescence. Our investigations show new aspects of nidovirus infections in pythons. Nidoviruses should be included in routine diagnostic workup of diseased reptiles.

Isolation and genetic analysis of a nidovirus from crucian carp (Carassius auratus).

A nidovirus was isolated from crucian carp (Carassius auratus). The complete genome of the crucian carp nidovirus (CCNV) is 25,971 nt long and has five open reading frames, encoding the polyprotein 1ab (pp1ab), spike glycoprotein (S), membrane protein (M), and nucleocapsid protein (N). CCNV has the highest similarity to Chinook salmon nidovirus (CSNV). However, the CCNV HB93 pp1ab protein sequence has three long fragment deletions compared with the CSNV. Phylogenetic analysis based on the complete genome sequence showed that CCNV HB93 clusters with CSNV, indicating that CCNV represents a second species in the new genus Oncotshavirus within the new family Tobaniviridae in the order Nidovirales.

Retail Baitfish in Michigan Harbor Serious Fish Viral Pathogens.

Indigenous small cyprinid fish species play an important role in Great Lakes ecosystems and also comprise the backbone of a multimillion-dollar baitfish industry. Due to their widespread use in sport fisheries of the Laurentian Great Lakes, there are increasing concerns that baitfish may introduce or disseminate fish pathogens. In this study, we evaluated whether baitfish purchased from 78 randomly selected retail bait dealers in Michigan harbored fish viruses. Between September 2015 and June 2016, 5,400 baitfish divided into 90 lots of 60 fish were purchased. Fish were tested for the presence of viral hemorrhagic septicemia virus (VHSV), spring viremia of carp virus (SVCV), golden shiner reovirus (GSRV), fathead minnow nidovirus (FHMNV), fathead minnow picornavirus (FHMPV), and white sucker bunyavirus (WSBV). Using the epithelioma papulosum cyprini cell line and molecular confirmation, we demonstrated the presence of viruses in 18 of the 90 fish lots (20.0%) analyzed. The most prevalent virus was FHMNV, being detected in 6 of 30 lots of Fathead Minnow Pimephales promelas and 3 of 42 lots of Emerald Shiners Notropis atherinoides. We also confirmed GSRV in two fish species: the Golden Shiner Notemigonus crysoleucas (5 of 11 lots) and Fathead Minnow (3 of 30 lots). Two VHSV (genotype IVb) isolates were recovered from a single lot of Emerald Shiners. No SVCV, FHMPV, or WSBV was detected in any of the fish examined. Some of the infected fish exhibited clinical signs and histopathological alterations. This study demonstrates that live baitfish are a potential vector for the spread of viral pathogens and underscores the importance of fish health certifications for the Great Lakes baitfish industry.

Isolation of the Fathead Minnow Nidovirus from Muskellunge Experiencing Lingering Mortality.

In 2011, the Fathead Minnow nidovirus (FHMNV; Genus Bafinivirus, Family Coronaviridae, Order Nidovirales) was isolated from pond-raised juvenile Muskellunge Esox masquinongy suffering from lingering mortality at the Wild Rose Hatchery in Wild Rose, Wisconsin. Moribund Muskellunge exhibited tubular necrosis in the kidneys as well as multifocal coalescing necrotizing hepatitis. The FHMNV was also isolated from apparently healthy juvenile Muskellunge at the Wolf Lake State Fish Hatchery in Mattawan, Michigan. The identity of the two syncytia-forming viruses (designated MUS-WR and MUS-WL from Wild Rose Hatchery and Wolf Lake State Fish Hatchery, respectively) as strains of FHMNV was determined based on multiple-gene sequencing and phylogenetic analyses. The pathogenicity of the MUS-WL FHMNV strain was determined by experimentally infecting naive juvenile Muskellunge through intraperitoneal injection with two viral concentrations (63 and 6.3 × 10(3) TCID50/fish). Both doses resulted in 100% mortality in experimentally infected fish, which exhibited severely pale gills and petechial hemorrhaging in eyes, fins, and skin. Histopathological alterations in experimentally infected fish were observed mainly in the hematopoietic tissues in the form of focal areas of necrosis. Phylogenetic analysis of concatenated partial spike glycoprotein and helicase gene sequences revealed differences between the MUS-WL FHMNV, MUS-WR FHMNV, and two other FHMNV originally isolated from moribund Fathead Minnows Pimephales promelas including the index FHMNV strain (GU002364). Based on a partial helicase gene sequence, a reverse transcriptase PCR assay was developed that is specific to FHMNV. These results give evidence that the risks posed to Muskellunge by FHMNV should be taken seriously. Received May 1, 2015; accepted February 8, 2016.

Discovery of a novel nidovirus in cattle with respiratory disease.

The family Coronaviridae represents a diverse group of vertebrate RNA viruses, all with genomes greater than 26,000 nt. Here, we report the discovery and genetic characterization of a novel virus present in cattle with respiratory disease. Phylogenetic characterization of this virus revealed that it clusters within the subfamily Torovirinae, in the family Coronaviridae. The complete genome consists of only 20,261 nt and represents the smallest reported coronavirus genome. We identified seven ORFs, including the canonical nidovirus ORF1a and ORF1b. Analysis of polyprotein 1ab revealed that this virus, tentatively named bovine nidovirus (BoNV), shares the highest homology with the recently described python-borne nidoviruses and contains several conserved nidovirus motifs, but does not encode the NendoU or O-MT domains that are present in other viruses within the family Coronaviridae. In concert with its reduced genome, the atypical domain architecture indicates that this virus represents a unique lineage within the order Nidovirales.

Novel divergent nidovirus in a python with pneumonia.

The order Nidovirales contains large, enveloped viruses with a non-segmented positive-stranded RNA genome. Nidoviruses have been detected in man and various animal species, but, to date, there have been no reports of nidovirus in reptiles. In the present study, we describe the detection, characterization, phylogenetic analyses and disease association of a novel divergent nidovirus in the lung of an Indian python (Python molurus) with necrotizing pneumonia. Characterization of the partial genome (>33 000 nt) of this virus revealed several genetic features that are distinct from other nidoviruses, including a very large polyprotein 1a, a putative ribosomal frameshift signal that was identical to the frameshift signal of astroviruses and retroviruses and an accessory ORF that showed some similarity with the haemagglutinin-neuraminidase of paramyxoviruses. Analysis of genome organization and phylogenetic analysis of polyprotein 1ab suggests that this virus belongs to the subfamily Torovirinae. Results of this study provide novel insights into the genetic diversity within the order Nidovirales.

Identification and characterization of genetically divergent members of the newly established family Mesoniviridae.

The recently established family Mesoniviridae (order Nidovirales) contains a single species represented by two closely related viruses, Cavally virus (CavV) and Nam Dinh virus (NDiV), which were isolated from mosquitoes collected in Côte d'Ivoire and Vietnam, respectively. They represent the first nidoviruses to be discovered in insects. Here, we report the molecular characterization of four novel mesoniviruses, Hana virus, Méno virus, Nsé virus, and Moumo virus, all of which were identified in a geographical region in Côte d'Ivoire with high CavV prevalence. The viruses were found with prevalences between 0.5 and 2.8%, and genome sequence analyses and phylogenetic studies suggest that they represent at least three novel species. Electron microscopy revealed prominent club-shaped surface projections protruding from spherical, enveloped virions of about 120 nm. Northern blot data show that the four mesoniviruses analyzed in this study produce two major 3'-coterminal subgenomic mRNAs containing two types of 5' leader sequences resulting from the use of different pairs of leader and body transcription-regulating sequences that are conserved among mesoniviruses. Protein sequencing, mass spectroscopy, and Western blot data show that mesonivirus particles contain eight major structural protein species, including the putative nucleocapsid protein (25 kDa), differentially glycosylated forms of the putative membrane protein (20, 19, 18, and 17 kDa), and the putative spike (S) protein (77 kDa), which is proteolytically cleaved at a conserved site to produce S protein subunits of 23 and 57 kDa. The data provide fundamental new insight into common and distinguishing biological properties of members of this newly identified virus family.

Mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range.

BACKGROUND: The family Mesoniviridae (order Nidovirales) comprises of a group of positive-sense, single-stranded RNA ([+]ssRNA) viruses isolated from mosquitoes. FINDINGS: Thirteen novel insect-specific virus isolates were obtained from mosquitoes collected in Indonesia, Thailand and the USA. By electron microscopy, the virions appeared as spherical particles with a diameter of ~50 nm. Their 20,129 nt to 20,777 nt genomes consist of positive-sense, single-stranded RNA with a poly-A tail. Four isolates from Houston, Texas, and one isolate from Java, Indonesia, were identified as variants of the species Alphamesonivirus-1 which also includes Nam Dinh virus (NDiV) from Vietnam and Cavally virus (CavV) from Côte d'Ivoire. The eight other isolates were identified as variants of three new mesoniviruses, based on genome organization and pairwise evolutionary distances: Karang Sari virus (KSaV) from Java, Bontag Baru virus (BBaV) from Java and Kalimantan, and Kamphaeng Phet virus (KPhV) from Thailand. In comparison with NDiV, the three new mesoniviruses each contained a long insertion (180 - 588 nt) of unknown function in the 5' region of ORF1a, which accounted for much of the difference in genome size. The insertions contained various short imperfect repeats and may have arisen by recombination or sequence duplication. CONCLUSIONS: In summary, based on their genome organizations and phylogenetic relationships, thirteen new viruses were identified as members of the family Mesoniviridae, order Nidovirales. Species demarcation criteria employed previously for mesoniviruses would place five of these isolates in the same species as NDiV and CavV (Alphamesonivirus-1) and the other eight isolates would represent three new mesonivirus species (Alphamesonivirus-5, Alphamesonivirus-6 and Alphamesonivirus-7). The observed spatiotemporal distribution over widespread geographic regions and broad species host range in mosquitoes suggests that mesoniviruses may be common in mosquito populations worldwide.

Delving into the Aftermath of a Disease-Associated Near-Extinction Event: A Five-Year Study of a Serpentovirus (Nidovirus) in a Critically Endangered Turtle Population.

Bellinger River virus (BRV) is a serpentovirus (nidovirus) that was likely responsible for the catastrophic mortality of the Australian freshwater turtle Myuchelys georgesi in February 2015. From November 2015 to November 2020, swabs were collected from turtles during repeated river surveys to estimate the prevalence of BRV RNA, identify risk factors associated with BRV infection, and refine sample collection. BRV RNA prevalence at first capture was significantly higher in M. georgesi (10.8%) than in a coexisting turtle, Emydura macquarii (1.0%). For M. georgesi, various risk factors were identified depending on the analysis method, but a positive BRV result was consistently associated with a larger body size. All turtles were asymptomatic when sampled and conjunctival swabs were inferred to be optimal for ongoing monitoring. Although the absence of disease and recent BRV detections suggests a reduced ongoing threat, the potential for the virus to persist in an endemic focus or resurge in cyclical epidemics cannot be excluded. Therefore, BRV is an ongoing potential threat to the conservation of M. georgesi, and strict adherence to biosecurity principles is essential to minimise the risk of reintroduction or spread of BRV or other pathogens.

Co-infecting viruses of species Bovine rhinitis B virus (Picornaviridae) and Bovine nidovirus 1 (Tobaniviridae) identified for the first time from a post-mortem respiratory sample of a sheep (Ovis aries) in Hungary.

In this study, a picornavirus and a nidovirus were identified from a single available nasopharyngeal swab (NPS) sample of a freshly deceased sheep, as the only vertebrate viruses found with viral metagenomics and next-generation sequencing methods. The sample was originated from a mixed feedlot farm in Hungary where sheep and cattle were held together but in separate stalls. Most of the sheep had respiratory signs (coughing and increased respiratory effort) at the time of sampling. Other NPS were not, but additional enteric samples were collected from sheep (n = 27) and cattle (n = 11) of the same farm at that time. The complete/nearly complete genomes of the identified viruses were determined using RT-PCR and Nanopore (MinION-Flonge) / Dye-terminator sequencing techniques. The results of detailed genomic and phylogenetic analyses indicate that the identified picornavirus most likely belongs to a type 4 genotype of species Bovine rhinitis B virus (BRBV-4, OR885914) of genus Aphthovirus, family Picornaviridae while the ovine nidovirus (OvNV, OR885915) - as a novel variant - could belong to the recently created Bovine nidovirus 1 (BoNV) species of genus Bostovirus, family Tobaniviridae. None of the identified viruses were detectable in the enteric samples using RT-PCR and generic screening primer pairs. Both viruses are well-known respiratory pathogens of cattle, but their presence was not demonstrated before in other animals, like sheep. Furthermore, neither BRBV-4 nor BoNVs were investigated in European cattle and/or sheep flocks, therefore it cannot be determined whether the presence of these viruses in sheep was a result of a single host species switch/spillover event or these viruses are circulating in not just cattle but sheep populations as well. Further studies required to investigate the spread of these viruses in Hungarian and European sheep and cattle populations and to identify their pathogenic potential in sheep.

Characterization of Dak Nong virus, an insect nidovirus isolated from Culex mosquitoes in Vietnam.

In this study, we isolated and characterized an insect nidovirus from the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae) in Vietnam, as an additional member of the new family Mesoniviridae in the order Nidovirales. The virus, designated "Dak Nong virus (DKNV)," shared many characteristics with Cavally virus and Nam Dinh virus, which have also been discovered recently in mosquitoes, and these viruses should be considered members of a single virus species, Alphamesonivirus 1. DKNV grew in cultured mosquito cells but could not replicate in the cultured vertebrate cells tested. N-terminal sequencing of the DKNV structural proteins revealed two posttranslational cleavage sites in the spike glycoprotein precursor. DKNV is assumed to be a new member of the species Alphamesonivirus 1, and the current study provides further understanding of viruses belonging to the new family Mesoniviridae.

Genetic analysis of a novel nidovirus from fathead minnows.

A bacilliform virus was isolated from diseased fathead minnows (Pimephales promelas). Analysis of the complete genome coding for the polyprotein (pp1ab), spike (S), membrane (M) and nucleocapsid (N) proteins revealed that the virus was most like white bream virus (WBV), another bacilliform virus isolated from white bream (Blicca bjoerkna L.) and the type species of the genus Bafinivirus within the order Nidovirales. In addition to similar gene order and size, alignment of deduced amino acid sequences of the pp1ab, M, N and S proteins of the fathead minnow nidovirus (FHMNV) with those of WBV showed 46, 44, 39 and 15 % identities, respectively. Phylogenetic analysis using the conserved helicase domain of the replicase showed FHMNV was distinct from WBV, yet the closest relative identified to date. Thus, FHMNV appears to represent a second species in the genus Bafinivirus. A PCR assay was developed for the identification of future FHMNV-like isolates.

Mesoniviridae: a proposed new family in the order Nidovirales formed by a single species of mosquito-borne viruses.

Recently, two independent surveillance studies in Côte d'Ivoire and Vietnam, respectively, led to the discovery of two mosquito-borne viruses, Cavally virus and Nam Dinh virus, with genome and proteome properties typical for viruses of the order Nidovirales. Using a state-of-the-art approach, we show that the two insect nidoviruses are (i) sufficiently different from other nidoviruses to represent a new virus family, and (ii) related to each other closely enough to be placed in the same virus species. We propose to name this new family Mesoniviridae. Meso is derived from the Greek word "mesos" (in English "in the middle") and refers to the distinctive genome size of these insect nidoviruses, which is intermediate between that of the families Arteriviridae and Coronaviridae, while ni is an abbreviation for "nido". A taxonomic proposal to establish the new family Mesoniviridae, genus Alphamesonivirus, and species Alphamesonivirus 1 has been approved for consideration by the Executive Committee of the ICTV.

Vomiting and wasting disease associated with hemagglutinating encephalomyelitis viruses infection in piglets in Jilin, China.

One coronavirus strain was isolated from brain tissues of ten piglets with evident clinical manifestations of vomiting, diarrhea and dyskinesia in Jilin province in China. Antigenic and genomic characterizations of the virus (isolate PHEV-JLsp09) were based on multiplex PCR and negative staining electron microscopy and sequence analysis of the Hemagglutinin-esterase (HE) gene. These piglets were diagnosed with Porcine hemagglutinating encephalomyelitis virus (PHEV).Necropsy was performed on the piglets. Major pathological changes included meningeal hyperemia, meningeal hemorrhage and cortical hemorrhage. Minor changes were also observed in other organs. Histopathological changes included satellitosis and neuronophagia in the cerebral cortex.Mice were infected with the isolated virus. Their histopathological changes were similar to those symptoms observed in the piglets, exhibiting typical changes for non-suppurative encephalitis. Thus, Porcine hemagglutinating encephalomyelitis virus mainly causes damage to the nervous system but also impacts other organs. This viral strain (isolate PHEV-JLsp09) found in the Siping area of Jilin Province in China is evolutionally closest to the HEV-67N stain (North American strain), indicating that this viral strain evolved from the PHEV from North America.

Dianke virus: A new mesonivirus species isolated from mosquitoes in Eastern Senegal.

An increasing number of insect-specific viruses are found around the world. Very recently, a new group of insect-specific viruses, the Mesoniviridae family, was discovered in Africa, Asia, North America and Australia. Here we report the first detection and isolation of a new virus belonging to Mesonivirus genus in Senegal, West Africa. The so-called Dianke virus was detected in 21 species of arthropods trapped in the eastern part of the country. Male individuals were also infected, supporting vertical transmission assertion of insect specific viruses. As described for other mesoniviruses, no viral replication was observed after inoculation of mammalian cells. Viral replication in mosquito cells was blocked at a temperature of 37 °C, highlighting the importance of thermal conditions in Mesonivirus host restriction. Similar to our study, where a diverse range of arthropod vectors were found infected by the new virus, several studies have detected mesonivirus infection in mosquitoes with concerns for human health. It has been shown that dual infections in mosquito can alter viral infectivity. Due to their extensive geographic distribution and host range, as well as their use as potential disease control agents in vector populations, more studies should be done for a better knowledge of arthropod-restricted viruses prevalence and diversity.

A case report of reptile-associated nidovirus (serpentovirus) in a ball python (Python regius) in Taiwan.

Reptile-associated nidoviruses (serpentoviruses) have been reported to cause proliferative interstitial pneumonia in pythons and other reptile species. A captive, younger than 2 years old, intact female ball python (Python regius) showed increased oral mucus, wheezing, and audible breathing with weight loss. Gross and microscopic examination revealed large amounts of mucus in the esophagus and proliferative interstitial pneumonia. Serpentovirus genes were detected from the lung tissues by polymerase chain reaction. The current serpentoviruses was phylogenetically grouped with the serpentovirus previously identified in the US. No case of serpentovirus infection has been reported in Asia. The present report provides information of complete genome sequence and global distribution of serpentovirus.

Discovery of a novel Piscanivirus in yellow catfish (Pelteobagrus fulvidraco) in China.

A bacilliform virus was isolated from yellow catfish in China. This virus can directly adapt in cultures of EPC cells. The virus particles, which were rod-shaped approximately 120 nm long and 20 nm wide, were visible in the cytoplasm of EPC cells. The full-length genome of this virus is 26,985 nt. The genome contains four open reading frames that encode polyprotein1ab, spike glycoprotein, M protein, and N protein. There was a putative slippery sequence 14861UUUAAAC14867, which could be modeled into an RNA pseudoknot structure. The predicted amino acid sequence of pp1ab, S, M, and N genes shares 8.7%-40.2% homology with those of the two known Bafinivirus strains-WBV and FHMNV. Based on the viral morphology, genome organization, and sequence homology, this newly identified bacilliform virus appears to be Piscanivirus. To the best of our knowledge, this is the first report of Piscanivirus in yellow catfish and Piscanivirus in China.

Endangered wild salmon infected by newly discovered viruses.

The collapse of iconic, keystone populations of sockeye (Oncorhynchus nerka) and Chinook (Oncorhynchus tshawytscha) salmon in the Northeast Pacific is of great concern. It is thought that infectious disease may contribute to declines, but little is known about viruses endemic to Pacific salmon. Metatranscriptomic sequencing and surveillance of dead and moribund cultured Chinook salmon revealed a novel arenavirus, reovirus and nidovirus. Sequencing revealed two different arenavirus variants which each infect wild Chinook and sockeye salmon. In situ hybridisation localised arenavirus mostly to blood cells. Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture.

Identification of a novel nidovirus as a potential cause of large scale mortalities in the endangered Bellinger River snapping turtle (Myuchelys georgesi).

In mid-February 2015, a large number of deaths were observed in the sole extant population of an endangered species of freshwater snapping turtle, Myuchelys georgesi, in a coastal river in New South Wales, Australia. Mortalities continued for approximately 7 weeks and affected mostly adult animals. More than 400 dead or dying animals were observed and population surveys conducted after the outbreak had ceased indicated that only a very small proportion of the population had survived, severely threatening the viability of the wild population. At necropsy, animals were in poor body condition, had bilateral swollen eyelids and some animals had tan foci on the skin of the ventral thighs. Histological examination revealed peri-orbital, splenic and nephric inflammation and necrosis. A virus was isolated in cell culture from a range of tissues. Nucleic acid sequencing of the virus isolate has identified the entire genome and indicates that this is a novel nidovirus that has a low level of nucleotide similarity to recognised nidoviruses. Its closest relatives are nidoviruses that have recently been described in pythons and lizards, usually in association with respiratory disease. In contrast, in the affected turtles, the most significant pathological changes were in the kidneys. Real time PCR assays developed to detect this virus demonstrated very high virus loads in affected tissues. In situ hybridisation studies confirmed the presence of viral nucleic acid in tissues in association with pathological changes. Collectively these data suggest that this virus is the likely cause of the mortalities that now threaten the survival of this species. Bellinger River Virus is the name proposed for this new virus.