Detection of vesivirus in minks (Neovison vison), Italy 2021.

Vesiviruses are important animal pathogens with a broad host range, and they have also been involved in accidental contamination of cells used for the production of drugs for rare and life-threatening human diseases. A vesivirus (family Caliciviridae) was detected in minks (Neovison vison) with respiratory and neurological signs, during syndromic surveillance for SARS-CoV-2 conducted in Italy. The complete genome (8,397 nucleotides in length) of the vesivirus strain ITA/2021/mink/TE (OR130287) was obtained by combining NGS approach with 5' and 3' RACE protocols. The virus was seemingly more related (95.9-97.2% nt identity in the partial RNA-dependent RNA polymerase) to American vesivirus isolates 9/1980/US, 12/1980/US, and 20/1980/US dating back to the early 1980s than to recent mink strains. These results highlight the importance of gathering information on the virome of animals.

Canine caliciviruses of four serotypes from military and research dogs recovered in 1963-1978 belong to two phylogenetic clades in the Vesivirus genus.

BACKGROUND: Vesiviruses (family Caliciviridae) had been shown capable of invading a variety of host species, raising concern of their zoonotic potential. Since the 1980's, several canine caliciviruses (CaCV) isolates have been reported and are phylogenetically related to the vesiviruses with features distinct from both Vesicular exanthema of swine virus (VESV) and Feline calicivirus (FCV) species in phylogeny, serology and cell culture specificities. Etiological studies of canine diseases in dogs used for military services and laboratory studies were conducted in 1963-1978 at the Walter Reed Army Institute of Research. Multiple known and unknown viral pathogens including caliciviruses were recovered. METHODS: Four unidentified isolates were recovered in Walter Reed Canine Cells (WRCC) from respiratory, fecal and penile specimens. Physicochemical tests, electron microscopy, viral cultivation in human and animal cells, antibody neutralization assays, and recently the genome sequencing were used to characterize the isolates. Sera from these dogs and their cohorts were tested with the isolates to determine origin and prevalence of the infections. RESULTS: The viral isolates were small non-enveloped spherical RNA virions, 27 to 42 nm in diameter with cup-like structures, indicating they are caliciviruses. They propagated in WRCC and MDCK cells, not in either other canine cells or human and other animal cells. Each isolate is antigenically distinct and react with dog sera in respective cohorts. The genomes have nucleotide identities ranging from 70.3% to 90.7% and encode the non-structural polyprotein (1810 amino acids), major capsid protein (691 amino acids) and minor structural protein (134 amino acids). They belong to two different phylogenetic clades in Vesivirus genus with close relation with canine calicivirus (CaCV). CONCLUSIONS: These CaCV isolates have restricted cell tropism, antigenic diversity and genetic variation. Further investigation will shed light on antigenic relation to other vesiviruses, and its pathogenicity for dogs and potential infectivity to other animals. Together with the previously reported CaCV strains provides significant evidence to support the formation of a new CaCV species in the Vesivirus genus.

Characterization of a Vesivirus Associated with an Outbreak of Acute Hemorrhagic Gastroenteritis in Domestic Dogs.

Four of eleven affected dogs died despite aggressive treatment during a 2015 focal outbreak of hemorrhagic gastroenteritis following a stay in a pet housing facility. Routine diagnostic investigations failed to identify a specific cause. Virus isolation from fresh necropsy tissues yielded a calicivirus with sequence homology to a vesivirus within the group colloquially known as the vesivirus 2117 strains that were originally identified as contaminants in CHO cell bioreactors. In situ hybridization and reverse transcription-PCR assays of tissues from the four deceased dogs confirmed the presence of canine vesivirus (CaVV) nucleic acids that localized to endothelial cells of arterial and capillary blood vessels. CaVV nucleic acid corresponded to areas of necrosis and hemorrhage primarily in the intestinal tract, but also in the brain of one dog with nonsuppurative meningoencephalitis. This is the first report of an atypical disease association with a putative hypervirulent vesivirus strain in dogs, as all other known strains of CaVV appear to cause nonclinical infections or relatively mild disease. After identification of the CU-296 vesivirus strain from this outbreak, four additional CaVV strains were amplified from unrelated fecal specimens and archived stocks provided by other laboratories. Broader questions include the origins, reservoir(s), and potential for reemergence and spread of these related CaVVs.

Serological and molecular investigation of 2117-like vesiviruses in cats.

Vesivirus 2117 was first discovered as a contaminant in Chinese hamster ovary (CHO) cell cultures used for human drug production. Similar vesiviruses (VeVs) have been detected recently in dogs. In order to address the hypothesis that cats may also be exposed to 2117-like VeVs, in this study, we screened 236 feline sera using an enzyme-linked immunosorbent assay (ELISA) based on a recombinant VP1 protein from the canine VeV Bari/212/07/ITA. IgG antibodies against the 2117-like VeV were detected in 37.3% of the sera tested. Also, by screening cat faecal specimens, the RNA of a 2117-like VeV was detected in a clinically healthy cat.

Vesivirus 2117 capsids more closely resemble sapovirus and lagovirus particles than other known vesivirus structures.

Vesivirus 2117 is an adventitious agent that, in 2009, was identified as a contaminant of Chinese hamster ovary cells propagated in bioreactors at a pharmaceutical manufacturing plant belonging to Genzyme. The consequent interruption in supply of Fabrazyme and Cerezyme (drugs used to treat Fabry and Gaucher diseases, respectively) caused significant economic losses. Vesivirus 2117 is a member of the Caliciviridae, a family of small icosahedral viruses encoding a positive-sense RNA genome. We have used cryo-electron microscopy and three-dimensional image reconstruction to calculate a structure of vesivirus 2117 virus-like particles as well as feline calicivirus and a chimeric sapovirus. We present a structural comparison of several members of the Caliciviridae, showing that the distal P domain of vesivirus 2117 is morphologically distinct from that seen in other known vesivirus structures. Furthermore, at intermediate resolutions, we found a high level of structural similarity between vesivirus 2117 and Caliciviridae from other genera: sapovirus and rabbit hemorrhagic disease virus. Phylogenetic analysis confirms vesivirus 2117 as a vesivirus closely related to canine vesiviruses. We postulate that morphological differences in virion structure seen between vesivirus clades may reflect differences in receptor usage.

Vesivirus 2117: Cell line infectivity range and effectiveness of amplification of a potential adventitious agent in cell culture used for biological production.

Vesivirus 2117 (VV-2117) has been recently described as a contaminant of cell culture operations in several biologics manufacturing facilities. VV-2117 has been poorly studied and little information exists about its biology, pathogenicity and infectivity range in cell culture settings. In this study we evaluated the potential in vitro viral infectivity of VV-2117 using a range of established mammalian cell lines from various species, the effectiveness of virus amplification in CHO-K1 cells at differing infection levels, and the relative sensitivity of two test methods (cytopathic effect [CPE] and polymerase chain reaction [PCR]) to detect infection and viral amplification. Of eight cell culture systems studied, two originating from hamster (CHO-K1 and BHK-21) and one from canine (MDCK) were positive for CPE and also showed a marked increase of viral RNA in a reverse transcriptase quantitative PCR (RT-qPCR) test. CHO-K1 cell cultures inoculated at 10, 1 and 0.1 genome copies per cell (gc/cell) showed both CPE and amplification of VV-2117 RNA, indicating that infection had occurred in these cultures. CHO-K1 cultures inoculated at 0.01, 0.001, 0.0001 and 0.00001 gc per cell showed neither CPE nor VV-2117, indicating that infection had not occurred. Therefore, the minimum dose necessary for infection of CHO-K1 cells was approximately 0.1 genome copies per cell. At any infection level where VV-2117 amplification was observed by RT-qPCR, the cultures also showed CPE. There was no low-level infection that could be detected by RT-qPCR without developing signs of CPE. However, the RT-qPCR assay appeared more sensitive in that it detected VV-2117 infection earlier than the onset of observable CPE.

Detection and molecular characterization of caliciviruses (vesivirus and norovirus) in an outbreak of acute diarrhea in kittens from Brazil.

Feline caliciviruses (FCVs) have occasionally been described in cats in association with enteric disease, but an etiological role for these viruses in acute gastroenteritis is still unclear. In this study, molecular characterization of FCV and feline norovirus (FNoV) was undertaken using real-time PCR (RT-PCR) and sequence analysis of the ORF1 region in fecal specimens from 29 diarrheic cats. The specimens were also screened for parvovirus, coronavirus, astrovirus and group A rotavirus. A quantitative one step RT-PCR was also performed to detect and quantitate NoV genogroup IV and the role of these animal caliciviruses in feline gastroenteritis was investigated. This is the first description of enteric FCV and FNoV in South America.

Detection and Full-Length Genome Characterization of Novel Canine Vesiviruses.

Vesiviruses have been detected in several animal species and as accidental contaminants of cells. We detected vesiviruses in asymptomatic kennel dogs (64.8%) and symptomatic (1.1%) and asymptomatic (3.5%) household dogs in Italy. The full-length genome of 1 strain, Bari/212/07/ITA, shared 89%-90% nt identity with vesiviruses previously detected in contaminated cells.

Genetic characterization of a reptilian calicivirus (Cro1).

BACKGROUND: Vesiviruses in the family Caliciviridae infect a broad range of animal hosts including mammals, birds, fish, amphibians and reptiles. The vesivirus Cro1 strains were isolated from diseased snakes in the San Diego zoo in 1978 and reported as the first caliciviruses found in reptiles. The goal of this study was to characterize the Cro1 strain 780032I that was isolated in cell culture from a rock rattlesnake (Crotalus lepidus) in the original outbreak. RESULTS: We re-amplified the original virus stock in Vero cells, and determined its full-length genome sequence. The Cro1 genome is 8296 nucleotides (nt) in length and has a typical vesivirus organization, with three open reading frames (ORF), ORF1 (5643 nt), ORF2 (2121 nt), and ORF3 (348 nt) encoding a nonstructural polyprotein, the major capsid protein precursor, and a minor structural protein, respectively. Phylogenetic analysis of the full-length genome sequence revealed that the Cro1 virus clustered most closely with the VESV species of the genus Vesivirus, but was genetically distinct (82-83% identities with closest strains). CONCLUSIONS: This is the first description of a full-length genome sequence from a reptile calicivirus (Cro1). The availability of the Cro1 genome sequence should facilitate investigation of the molecular mechanisms involved in Cro1 virus evolution and host range.

Characterization of San Miguel sea lion virus populations using pyrosequencing-based methods.

San Miguel Sea Lion Virus (SMSV) is a small RNA virus in the genus Vesivirus with an unusually broad host range. Three populations of SMSV were examined by PCR amplification of the capsid precursor and putative helicase genes, followed by pyrosequencing. The populations were nasal swabs from two SMSV infected California sea lions (Zalophus californianus) from two different years, and a virus isolate from the earlier swab that was passaged in cell culture five times. In the capsid precursor, extensive deletions were prevalent in the passaged virus but uncommon in the clinical samples. A greater prevalence of point mutations was seen in the capsid precursor gene than in the putative helicase gene. In culture, the minority sequence in the capsid precursor at nucleotide position 5826 rapidly shifted after five passages to become the majority sequence. Levels of diversity at individual sites showed much more similarity between the two clinical samples than between the earlier clinical sample and the passaged culture from the same sample. SMSV appears to behave as a quasispecies. Assessment of original patient samples is preferable for understanding clinical SMSV populations.

Expression and self-assembly of virus-like particles from two genotypes of marine vesiviruses and development of an ELISA for the detection of antibodies.

Sequences encoding the major and minor capsid proteins (VP1 and VP2) from two marine vesivirus isolates (Steller sea lion viruses V810 and V1415) were engineered for expression of virus-like particles (VLPs) in the baculovirus system. The resulting VLPs were morphologically similar to native vesivirus virions. Purified VLPs were probed in immunoblots with pooled antisera specific for nine San Miguel sea lion virus (SMSV) types, and a predominant protein of approximately 60kDa was detected. An enzyme linked immunosorbent assay (ELISA) for the detection of antibodies was developed in which the VLPs served as antigen. The VLPs were adsorbed to the wells of a microplate, and the specificity of the ELISA was established with hyperimmune sera raised against 24 serotypes of the genus Vesivirus. The ELISA was used to screen for the presence of vesivirus specific antibodies in the sera of free-ranging Steller sea lions. The ELISA results demonstrated that Steller sea lions that inhabit the Pacific Ocean waters of southeast Alaska are widely exposed to antigenically related marine vesiviruses, while no previous exposure could be demonstrated using VLP antigens in 17 Steller sea lions from the Aleutian Islands. The broad reactivity of these VLPs and their non-infectious nature will facilitate global sero-epidemiological studies aimed at determining the incidence and prevalence of marine vesiviruses in mammals that inhabit the Pacific and Atlantic oceans as well as susceptible terrestrial animals.

A capsid gene-based real-time reverse transcription polymerase chain reaction assay for the detection of marine vesiviruses in the Caliciviridae.

A real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was developed for the identification of marine vesiviruses. The primers were designed to target a 176-nucleotide fragment within a highly conserved region of the San Miguel sea lion viruses (SMSVs) capsid gene. The assay detected viral RNA from nine marine vesivirus serotypes described previously, including two serotypes (SMSV-8 and -12) not identified with presently available molecular assays, a highly related bovine vesivirus strain (Bos-1), a mink vesivirus strain (MCV), and two novel genotypes isolated recently from Steller sea lions (SSL V810 and V1415). The real-time assay did not amplify sequences from the corresponding genomic regions of feline calicivirus (also in the genus Vesivirus) and representative members of the genus Norovirus. The rtRT-PCR assay described below may prove useful as a diagnostic tool for the detection of currently circulating, emerging and previously described marine vesiviruses in clinical samples, especially when large numbers are screened in surveillance studies of these restricted viruses.

A new generic real-time reverse transcription polymerase chain reaction assay for vesiviruses; vesiviruses were not detected in human samples.

Different viruses belonging to the genus Vesivirus infect a broad range of animals, and cause gastroenteritis, vesicular skin lesions, hemorrhagic disease, respiratory diseases and other conditions. A recent report on Vesivirus viremia, as detected by PCR, in samples from patients with hepatitis of unknown etiology in the USA suggested a zoonotic potential for vesiviruses. These results have not been confirmed by another laboratory. In order to do so, a generic PCR assay on the RNA polymerase region was developed, and validated with RNA from 69 different Vesivirus species. Except SMSV serotype-8, all species tested were detected, including the ones that were suggested to be involved in zoonotic transmission in the USA (SMSV serotype-5). The generic Vesivirus assay was used on RNA extracted from serum samples from patients with hepatitis, stool samples from patients with gastroenteritis, throat-swab specimens of patients with rash illnesses, throat-swab and nose-swabs of patients with acute respiratory diseases, and cell cultures with cytopathologic effect from enterovirus surveillance in which no pathogen was found. None were found positive. In this study a generic Vesivirus assay was developed and it was concluded that vesiviruses are an unlikely cause of common illnesses in humans in the Netherlands.

Genomic characterization of novel marine vesiviruses from Steller sea lions (Eumetopias jubatus) from Alaska.

Marine vesiviruses were isolated in cell culture from oral and rectal swabs and vesicular fluid from Alaskan Steller sea lions (SSL; Eumetopias jubatus). Further characterization by RT-PCR, complete genomic sequencing, and phylogenetic analyses indicated that these viruses are most closely related to the marine vesiviruses, but are distinct viruses and represent two novel genotypes. The complete genome of these two SSL isolates was sequenced after cloning their viral cDNA. The genomes were found to be 8302 and 8305 nucleotides in length, organized in three open reading frames and contained 5' and 3' untranslated regions (UTR) of 19 and 180 nucleotides, respectively. The complete genomes of both SSL viruses were most closely related to each other and shared 83.0% nucleotide identity. Using the very limited number of complete genomic vesivirus sequences available in the NCBI database, these novel SSL vesiviruses seem most closely related to vesicular exanthema of swine virus-A48 and least related to rabbit vesivirus and walrus calicivirus. Specific antiserum against some evolutionary closer marine vesiviruses did not neutralize these isolates supporting the novel nature of these SSL viruses.

Vesivirus viremia and seroprevalence in humans.

Pathogenic caliciviruses of the genus Vesivirus circulate in oceanic ecosystems and spread to and among terrestrial mammals. Isolation of Vesivirus from natural and laboratory infections in humans led to this investigation of Vesivirus seroprevalence and viremia. Sera from four groups were tested for antibodies to Vesivirus as follows: blood donors whose units were cleared for donation, blood donors whose units were not accepted for donation solely because of elevated blood liver alanine aminotransferase (ALT) concentrations, patients with clinical hepatitis of unknown but suspected infectious cause, and patients with clinical hepatitis of unknown cause but associated with blood transfusion or dialysis. Additionally, sera were tested for Vesivirus genome by three methods: dot-blot and two reverse transcription-polymerase chain reaction (RT-PCR) methods. The calculated seroprevalence against Vesivirus virions within these groups (N = 765) was 12%, 21%, 29%, and 47%, respectively (P < 0.001 for group differences). Additionally, 11 (9.8%) of 112 sera tested yielded RT-PCR amplicons that by nucleotide sequence were distinct from each other and related to known Vesivirus. These data indicate that some blood donors in the population tested have serologic evidence of previous Vesivirus infection and some also have Vesivirus viremia. These results justify further investigation of an association between Vesivirus infection and illness in humans.

Isolation and characterization of a new Vesivirus from rabbits.

This report describes the isolation, cDNA cloning, complete genome nucleotide sequence, and partial characterization of a new cultivable calicivirus isolated from juvenile feeder European rabbits (Oryctolagus cuniculus) showing symptoms of diarrhea. Absence of neutralization by type-specific neutralizing antibodies for 40 caliciviruses and phylogenetic sequence comparisons of the open reading frame 1-encoded polyprotein with those of other caliciviruses demonstrate that this new calicivirus is a putative novel member of the Vesivirus genus which is closely related to the marine calicivirus subgroup. According to its putative classification, this new virus has been named rabbit vesivirus.

Inter- and intragenus structural variations in caliciviruses and their functional implications.

The family Caliciviridae is divided into four genera and consists of single-stranded RNA viruses with hosts ranging from humans to a wide variety of animals. Human caliciviruses are the major cause of outbreaks of acute nonbacterial gastroenteritis, whereas animal caliciviruses cause various host-dependent illnesses with a documented potential for zoonoses. To investigate inter- and intragenus structural variations and to provide a better understanding of the structural basis of host specificity and strain diversity, we performed structural studies of the recombinant capsid of Grimsby virus, the recombinant capsid of Parkville virus, and San Miguel sea lion virus serotype 4 (SMSV4), which are representative of the genera Norovirus (genogroup 2), Sapovirus, and Vesivirus, respectively. A comparative analysis of these structures was performed with that of the recombinant capsid of Norwalk virus, a prototype member of Norovirus genogroup 1. Although these capsids share a common architectural framework of 90 dimers of the capsid protein arranged on a T=3 icosahedral lattice with a modular domain organization of the subunit consisting of a shell (S) domain and a protrusion (P) domain, they exhibit distinct differences. The distally located P2 subdomain of P shows the most prominent differences both in shape and in size, in accordance with the observed sequence variability. Another major difference is in the relative orientation between the S and P domains, particularly between those of noroviruses and other caliciviruses. Despite being a human pathogen, the Parkville virus capsid shows more structural similarity to SMSV4, an animal calicivirus, suggesting a closer relationship between sapoviruses and animal caliciviruses. These comparative structural studies of caliciviruses provide a functional rationale for the unique modular domain organization of the capsid protein with an embedded flexibility reminiscent of an antibody structure. The highly conserved S domain functions to provide an icosahedral scaffold; the hypervariable P2 subdomain may function as a replaceable module to confer host specificity and strain diversity; and the P1 subdomain, located between S and P2, provides additional fine-tuning to position the P2 subdomain.