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.

A nidovirus perspective on SARS-CoV-2.

Two pandemics of respiratory distress diseases associated with zoonotic introductions of the species Severe acute respiratory syndrome-related coronavirus in the human population during 21st century raised unprecedented interest in coronavirus research and assigned it unseen urgency. The two viruses responsible for the outbreaks, SARS-CoV and SARS-CoV-2, respectively, are in the spotlight, and SARS-CoV-2 is the focus of the current fast-paced research. Its foundation was laid down by studies of many corona- and related viruses that collectively form the vast order Nidovirales. Comparative genomics of nidoviruses played a key role in this advancement over more than 30 years. It facilitated the transfer of knowledge from characterized to newly identified viruses, including SARS-CoV and SARS-CoV-2, as well as contributed to the dissection of the nidovirus proteome and identification of patterns of variations between different taxonomic groups, from species to families. This review revisits selected cases of protein conservation and variation that define nidoviruses, illustrates the remarkable plasticity of the proteome during nidovirus adaptation, and asks questions at the interface of the proteome and processes that are vital for nidovirus reproduction and could inform the ongoing research of SARS-CoV-2.

Genetic, Morphological and Antigenic Relationships between Mesonivirus Isolates from Australian Mosquitoes and Evidence for Their Horizontal Transmission.

The Mesoniviridae are a newly assigned family of viruses in the order Nidovirales. Unlike other nidoviruses, which include the Coronaviridae, mesoniviruses are restricted to mosquito hosts and do not infect vertebrate cells. To date there is little information on the morphological and antigenic characteristics of this new group of viruses and a dearth of mesonivirus-specific research tools. In this study we determined the genetic relationships of recent Australian isolates of Alphamesonivirus 4 (Casuarina virus-CASV) and Alphamesonivirus 1 (Nam Dinh virus-NDiV), obtained from multiple mosquito species. Australian isolates of NDiV showed high-level similarity to the prototype NDiV isolate from Vietnam (99% nucleotide (nt) and amino acid (aa) identity). Isolates of CASV from Central Queensland were genetically very similar to the prototype virus from Darwin (95-96% nt and 91-92% aa identity). Electron microscopy studies demonstrated that virion diameter (≈80 nm) and spike length (≈10 nm) were similar for both viruses. Monoclonal antibodies specific to CASV and NDiV revealed a close antigenic relationship between the two viruses with 13/34 mAbs recognising both viruses. We also detected NDiV RNA on honey-soaked nucleic acid preservation cards fed on by wild mosquitoes supporting a possible mechanism of horizontal transmission between insects in nature.

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2.

The present outbreak of a coronavirus-associated acute respiratory disease called coronavirus disease 19 (COVID-19) is the third documented spillover of an animal coronavirus to humans in only two decades that has resulted in a major epidemic. The Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for developing the classification of viruses and taxon nomenclature of the family Coronaviridae, has assessed the placement of the human pathogen, tentatively named 2019-nCoV, within the Coronaviridae. Based on phylogeny, taxonomy and established practice, the CSG recognizes this virus as forming a sister clade to the prototype human and bat severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus, and designates it as SARS-CoV-2. In order to facilitate communication, the CSG proposes to use the following naming convention for individual isolates: SARS-CoV-2/host/location/isolate/date. While the full spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined, the independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying viruses at the species level to complement research focused on individual pathogenic viruses of immediate significance. This will improve our understanding of virus­host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.

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.

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.

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.

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.

Description and initial characterization of metatranscriptomic nidovirus-like genomes from the proposed new family Abyssoviridae, and from a sister group to the Coronavirinae, the proposed genus Alphaletovirus.

Transcriptomics has the potential to discover new RNA virus genomes by sequencing total intracellular RNA pools. In this study, we have searched publicly available transcriptomes for sequences similar to viruses of the Nidovirales order. We report two potential nidovirus genomes, a highly divergent 35.9 kb likely complete genome from the California sea hare Aplysia californica, which we assign to a nidovirus named Aplysia abyssovirus 1 (AAbV), and a coronavirus-like 22.3 kb partial genome from the ornamented pygmy frog Microhyla fissipes, which we assign to a nidovirus named Microhyla alphaletovirus 1 (MLeV). AAbV was shown to encode a functional main proteinase, and a translational readthrough signal. Phylogenetic analysis suggested that AAbV represents a new family, proposed here as Abyssoviridae. MLeV represents a sister group to the other known coronaviruses. The importance of MLeV and AAbV for understanding nidovirus evolution, and the origin of terrestrial nidoviruses are discussed.

Insight into the evolution of nidovirus endoribonuclease based on the finding that nsp15 from porcine Deltacoronavirus functions as a dimer.

Nidovirus endoribonucleases (NendoUs) include nonstructural protein 15 (nsp15) from coronaviruses and nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E, and MHV nsp15 indicate that it mainly forms a functional hexamer, whereas nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine Deltacoronavirus (PDCoV) nsp15 primarily exists as dimers and monomers in vitro Biological experiments reveal that a PDCoV nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV nsp15. This result may explain why PDCoV nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells, that NendoU from arterivirus gained the ability to form dimers, and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses.

Genomes of viral isolates derived from different mosquitos species.

Eleven viral isolates derived mostly in albopictus C6/36 cells from mosquito pools collected in Southeast Asia and the Americas between 1966 and 2014 contained particles with electron microscopy morphology typical of reoviruses. Metagenomics analysis yielded the near complete genomes of three novel reoviruses, Big Cypress orbivirus, Ninarumi virus, and High Island virus and a new tetravirus, Sarawak virus. Strains of previously characterized Sathuvarachi, Yunnan, Banna and Parry's Lagoon viruses (Reoviridae), Bontang virus (Mesoniviridae), and Culex theileri flavivirus (Flaviviridae) were also characterized. The availability of these mosquito virus genomes will facilitate their detection by metagenomics or PCR to better determine their geographic range, extent of host tropism, and possible association with arthropod or vertebrate disease.

Isolation and characterization of a novel mesonivirus from Culex mosquitoes in China.

A new insect nidovirus (named Yichang virus) from the family Mesoniviridae was isolated, identified, and characterized from Culex mosquitoes in Hubei, China. Results showed a high number of viral RNA copies (up to 1011 copies/ml) within 48h in C6/36 cells. In addition, the titers of the Yichang virus reached maximal levels of 107 PFU/mL at 6 d post-infection (dpi). The virus produced moderate cytopathic effects when the multiplicity of infection ranged from 0.001-0.1 at 6 dpi, but did not replicate in mammalian cells. Under electron microscopy, the virion of the Yichang virus appeared as spherical particles with diameters of ∼80nm and large club-shaped projections. Although subsequent genomic sequence analysis revealed that the Yichang virus had similar protein patterns as those of other mesoniviruses, the nucleotide acids shared less than 20% BLAST query coverage with known viruses in the family Mesoniviridae, and showed a maximum sequence identity of 67% for RNA-dependent RNA polymerase (RdRp). The putative protein sequences showed slightly higher identity (28%-68%), and the most conserved domain was RdRp. Based on the phylogenetic and pairwise evolutionary distance analyses, the Yichang virus should be considered a new species belonging to a currently unassigned genus within the family Mesoniviridae.

Invertebrate RNA virus diversity from a taxonomic point of view.

Invertebrates are hosts to diverse RNA viruses that have all possible types of encapsidated genomes (positive, negative and ambisense single stranded RNA genomes, or a double stranded RNA genome). These viruses also differ markedly in virion morphology and genome structure. Invertebrate RNA viruses are present in three out of four currently recognized orders of RNA viruses: Mononegavirales, Nidovirales, and Picornavirales, and 10 out of 37 RNA virus families that have yet to be assigned to an order. This mini-review describes general properties of the taxonomic groups, which include invertebrate RNA viruses on the basis of their current classification by the International Committee on Taxonomy of Viruses (ICTV).

Discovery and Partial Genomic Characterisation of a Novel Nidovirus Associated with Respiratory Disease in Wild Shingleback Lizards (Tiliqua rugosa).

A respiratory disease syndrome has been observed in large numbers of wild shingleback lizards (Tiliqua rugosa) admitted to wildlife care facilities in the Perth metropolitan region of Western Australia. Mortality rates are reportedly high without supportive treatment and care. Here we used next generation sequencing techniques to screen affected and unaffected individuals admitted to Kanyana Wildlife Rehabilitation Centre in Perth between April and December 2015, with the resultant discovery of a novel nidovirus significantly associated with cases of respiratory disease according to a case definition based on clinical signs. Interestingly this virus was also found in 12% of apparently healthy individuals, which may reflect testing during the incubation period or a carrier status, or it may be that this agent is not causative in the disease process. This is the first report of a nidovirus in lizards globally. In addition to detection of this virus, characterisation of a 23,832 nt segment of the viral genome revealed the presence of characteristic nidoviral genomic elements providing phylogenetic support for the inclusion of this virus in a novel genus alongside Ball Python nidovirus, within the Torovirinae sub-family of the Coronaviridae. This study highlights the importance of next generation sequencing technologies to detect and describe emerging infectious diseases in wildlife species, as well as the importance of rehabilitation centres to enhance early detection mechanisms through passive and targeted health surveillance. Further development of diagnostic tools from these findings will aid in detection and control of this agent across Australia, and potentially in wild lizard populations globally.

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.

Fathead minnow nidovirus infects spotfin shiner Cyprinella spiloptera and golden shiner Notemigonus crysoleucas.

Since the initial isolation of the fathead minnow nidovirus (FHMNV), concerns have been raised regarding the risks it may pose to other fish species. In this study, 7 fish species resident to the Laurentian Great Lakes were challenged intraperitoneally with 2 doses of FHMNV: 102.8 and 104.8 median tissue culture infective dose (TCID(50)) ml(-1). Infected spotfin shiner Cyprinella spiloptera and golden shiner Notemigonus crysoleucas suffered morbidity and mortality during the 40 d observation period, while other species, including creek chub Semotilus atromaculatus, rainbow trout Oncorhynchus mykiss, largemouth bass Micropterus salmoides and walleye Sander vitreus, showed no clinical signs or mortality. FHMNV was re-isolated on the epithelioma papulosum cyprini cell line from the tissues of infected spotfin shiner and golden shiner, which harbored high numbers of viral RNA copies as measured by quantitative loop-mediated isothermal amplification. Infected spotfin shiner and golden shiner exhibited external petechiae, exophthalmia, oedematous kidneys, and liver pallor. Histopathological analysis revealed multifocal areas of necrosis in the kidney, spleen and liver of infected fish. Spotfin shiner and golden shiner were then infected with 2 doses of FHMNV (10(3.5) and 10(3.9) TCID(50) ml(-1)) by immersion to mimic more natural modes of infection. Spotfin shiner experienced 60% mortality at both doses, while golden shiner did not experience mortality nor develop any clinical signs following a 40 d observation period. Overall, piscivorous fish tested in this study do not seem to be at risk for infection, while cyprinids appear to vary in their susceptibility to the strain of FHMNV used in this study.

The aetiology of wobbly possum disease: Reproduction of the disease with purified nidovirus.

The objective of this study was to investigate a role of a recently discovered marsupial nidovirus in the development of a neurological disease, termed wobbly possum disease (WPD), in the Australian brushtail possum (Trichosurus vulpecula). Four possums received 1 mL of a standard inoculum that had been prepared from tissues of WPD-affected possums, 4 possums received 1.8 mL (1 × 10(6) TCID50) of a cell lysate from inoculated cultures, and 4 possums received 1 mL (× 10(7) TCID50) of a purified WPD isolate. All but one possum that received infectious inocula developed neurological disease and histopathological lesions characteristic for WPD. High levels of viral RNA were detected in livers from all possums that received infectious inocula, but not from control possums. Altogether, our data provide strong experimental evidence for the causative involvement of WPD virus in development of a neurological disease in infected animals.

[Isolation and Identification of the Nam Dinh Virus from Mosquitoes on the China-Laos-Myanmar Border].

Three strains of an insect nidovirus, the Nam Dinh virus (NDiV), isolated in Yunnan Province, China, have been identified. Aedes albopictus C6/36 cells were used to isolate NDiV from mosquitoes collected in Yunnan Province in 2012.Culture supernatants with a positive cytopathic effect were harvested for virus identification by sequence-independent single primer amplification. Transmission electron microscopy revealed virion structure to be spherical with a diameter of 60~80nm.Reverse transcription-polymerase chain reaction was applied to amplify sequences of RNA-dependent RNA-polymerase (RdRp), HEL1(superfamily 1helicase)and spike protein. The amino-acid sequences of three isolates from Yunnan Province showed>98% homology with NDiV strains. Phylogenetic analyses showed that these three isolates, along with NDiV, could be classified into the family Mesoniviridae.

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.

Ball python nidovirus: a candidate etiologic agent for severe respiratory disease in Python regius.

UNLABELLED: A severe, sometimes fatal respiratory disease has been observed in captive ball pythons (Python regius) since the late 1990s. In order to better understand this disease and its etiology, we collected case and control samples and performed pathological and diagnostic analyses. Electron micrographs revealed filamentous virus-like particles in lung epithelial cells of sick animals. Diagnostic testing for known pathogens did not identify an etiologic agent, so unbiased metagenomic sequencing was performed. Abundant nidovirus-like sequences were identified in cases and were used to assemble the genome of a previously unknown virus in the order Nidovirales. The nidoviruses, which were not previously known to infect nonavian reptiles, are a diverse order that includes important human and veterinary pathogens. The presence of the viral RNA was confirmed in all diseased animals (n = 8) but was not detected in healthy pythons or other snakes (n = 57). Viral RNA levels were generally highest in the lung and other respiratory tract tissues. The 33.5-kb viral genome is the largest RNA genome yet described and shares canonical characteristics with other nidovirus genomes, although several features distinguish this from related viruses. This virus, which we named ball python nidovirus (BPNV), will likely establish a new genus in Torovirinae subfamily. The identification of a novel nidovirus in reptiles contributes to our understanding of the biology and evolution of related viruses, and its association with lung disease in pythons is a promising step toward elucidating an etiology for this long-standing veterinary disease. IMPORTANCE: Ball pythons are popular pets because of their diverse coloration, generally nonaggressive behavior, and relatively small size. Since the 1990s, veterinarians have been aware of an infectious respiratory disease of unknown cause in ball pythons that can be fatal. We used unbiased shotgun sequencing to discover a novel virus in the order Nidovirales that was present in cases but not controls. While nidoviruses are known to infect a variety of animals, this is the first report of a nidovirus recovered from any reptile. This report will enable diagnostics that will assist in determining the role of this virus in the causation of disease, which would allow control of the disease in zoos and private collections. Given its evolutionary divergence from known nidoviruses and its unique host, the study of reptile nidoviruses may further our understanding of related diseases and the viruses that cause them in humans and other animals.