Genetic diversity and recombination of bovine enterovirus strains in China.

Bovine enterovirus (BEV) consisting of enterovirus species E (EV-E) and F (EV-F) is the causative agent associated with respiratory and gastrointestinal diseases in cattle. Here, we reported the characterization, genetic diversity, and recombination of novel BEV strains isolated from the major cattle-raising regions in China during 2012-2018. Twenty-seven BEV strains were successfully isolated and characterized. Molecular characterization demonstrated that the majority of these novel BEV strains (24/27) were EV-E, while only few strains (3/27) were EV-F. Sequence analysis revealed the diversity of the circulating BEV strains such as species and subtypes where different species or subtype coinfections were detected in the same regions and even in the same cattle herds. For the EV-E, two novel subtypes, designated as EV-E6 and EV-E7, were revealed in addition to the currently reported EV-E1-EV-E5. Comparative genomic analysis revealed the intraspecies and interspecies genetic exchanges among BEV isolates. The representative strain HeN-B62 was probably from AN12 (EV-F7) and PS-87-Belfast (EV-F3) strains. The interspecies recombination between EV-E and EV-F was also discovered, where the EV-F7-AN12 might be from EV-E5 and EV-F1, and EV-E5-MexKSU/5 may be recombined from EV-F7 and EV-E1. The aforementioned results revealed the genetic diversity and recombination of novel BEV strains and unveiled the different BEV species or subtype infections in the same cattle herd, which will broaden the understanding of enterovirus genetic diversity, recombination, pathogenesis, and prevention of disease outbreaks. IMPORTANCE: Bovine enterovirus (BEV) infection is an emerging disease in China that is characterized by digestive, respiratory, and reproductive disorders. In this study, we first reported two novel EV-E subtypes detected in cattle herds in China, unveiled the coinfection of two enterovirus species (EV-E/EV-F) and different subtypes (EV-E2/EV-E7, EV-E1/EV-E7, and EV-E3/EV-E6) in the same cattle herds, and revealed the enterovirus genetic exchange in intraspecies and interspecies recombination. These results provide an important update of enterovirus prevalence and epidemiological aspects and contribute to a better understanding of enterovirus genetic diversity, evolution, and pathogenesis.

Surrogate Selection for Foot-and-Mouth Disease Virus in Disinfectant Efficacy Tests by Simultaneous Comparison of Bacteriophage MS2 and Bovine Enterovirus Type 1.

In South Korea, testing disinfectants against foot-and-mouth disease virus (FMDV) that are contagious in livestock or that require special attention with respect to public hygiene can be manipulated only in high-level containment laboratories, which are not easily available. This causes difficulties in the approval procedure for disinfectants, such as a prolonged testing period. Additionally, the required biosafety level (BSL) in the case of FMDV has hindered its extensive studies. However, this drawback can be circumvented by using a surrogate virus to improve the performance of the efficacy testing procedure for disinfectants. Therefore, we studied bacteriophage MS2 (MS2) and bovine enterovirus type 1 (ECBO) with respect to disinfectant susceptibility for selecting a surrogate for FMDV according to the Animal and Plant Quarantine Agency (APQA) guidelines for efficacy testing of veterinary disinfectants. Effective concentrations of the active substances in disinfectants (potassium peroxymonosulfate, sodium dichloroisocyanurate, malic acid, citric acid, glutaraldehyde, and benzalkonium chloride) against FMDV, MS2, and ECBO were compared and, efficacies of eight APQA-listed commercial disinfectants used against FMDV were examined. The infectivity of FMDV and ECBO were confirmed by examination of cytopathic effects, and MS2 by plaque assay. The results reveal that the disinfectants are effective against MS2 and ECBO at higher concentrations than in FMDV, confirming their applicability as potential surrogates for FMDV in efficacy testing of veterinary disinfectants.

Antiviral Effect of Bovine Lactoferrin against Enterovirus E.

Enterovirus E (EV-E), a representative of the Picornaviridae family, endemically affects cattle across the world, typically causing subclinical infections. However, under favorable conditions, severe or fatal disorders of the respiratory, digestive, and reproductive systems may develop. There is no specific treatment for enterovirus infections in humans or animals, and only symptomatic treatment is available. The aim of this study was to determine the in vitro antiviral effect of bovine lactoferrin (bLF) against enterovirus E using virucidal, cytopathic effect inhibition, and viral yield reduction assays in MDBK cells. The influence of lactoferrin on the intracellular viral RNA level was also determined. Surprisingly, lactoferrin did not have a protective effect on cells, although it inhibited the replication of the virus during the adsorption and post-adsorption stages (viral titres reduced by 1-1.1 log). Additionally, a decrease in the viral RNA level in cells (by up to 75%) was observed. More detailed studies are needed to determine the mechanism of bovine lactoferrin effect on enterovirus E. However, this highly biocompatible protein ensures some degree of protection against infection by bovine enterovirus, which is particularly important for young animals that receive this protein in their mother's milk.

Isolation and Identification of Two Clinical Strains of the Novel Genotype Enterovirus E5 in China.

Most enterovirus (EV) infections are subclinical but, occasionally, can cause severe and potentially fatal diseases in humans and animals. Currently, EVs are divided into 12 types (A to L) based on phylogenetic analysis and on their natural hosts. Bovine enterovirus (BEV) is an essential member of the enterovirus belonging to the types E and F that attacks cattle as its natural host and causes clinical disorders in the digestive, respiratory, and reproductive tracts. In 2020, several dairy farms in China experienced cow mortality with acute clinical signs, including fever, and diarrhea. In these cases, GX20-1 and JS20-1 virus strains were isolated and sequenced. Cellular adaptation of these two strains showed efficient replications on Madin-Darby bovine kidney (MDBK) cells and produced a significant cytopathogenic effect (CPE). However, on baby hamster kidney (BHK-21) and Vero cells, viral replication was inefficient and did not produce CPE. As noted in comparative genomics analysis, these two strains showed distant evolutionary relationships with the well-known E1 to E4 and F1 to F4 subtypes of BEV and high sequence identities with the candidate type Enterovirus E5, a novel genotype recently identified based on the genomic data of three strains, including the GX20-1 and JS20-1 strains. This study provides the first evidence of a novel genotype bovine enterovirus infection in Chinese cattle herds, a potential threat to the cattle industry in China. IMPORTANCE Bovine enterovirus (BEV) is a cattle-infecting pathogen. This study is the first report of natural infection of a novel genotype of enterovirus in herds of cattle in China. The homology of the novel enterovirus is far different from the structural protein of other enteroviruses and has different cellular adaptations. This study provides a reference for the biological characteristics and prevalence of the novel enterovirus in Chinese cattle populations.

Isolation and Identification of Type F Bovine Enterovirus from Clinical Cattle with Diarrhoea.

Recently, bovine enterovirus (BEV) has caused several respiratory and gastrointestinal diseases outbreaks in cattle. Monitoring the epidemiological and pathogenic characteristics of this virus is crucial to controlling its spread. We isolated a BEV strain with typical cytopathic effects from the faeces of cows with significant diarrhoeal symptoms in China and observed the viral particles within 20-30 nm through transmission electron microscopy. Then, we designated this strain as HB19-1 in this study. The multistep growth curves showed that the virus propagated well in the MDBK cells. Molecular genetic analysis of VP1 indicated that HB19-1 belonged to the BEV-F1 group. Although the challenged ICR mice did not exhibit typical disease symptoms in animal infection assay, we observed significant pathological damage in the lungs, intestines, and muscle tissues. In summary, we isolated a BEV strain HB19-1 causing severe diarrhoea in cattle and proposed reinforcing the epidemiological surveillance of this virus.

Experimental immunization of mice with a recombinant bovine enterovirus vaccine expressing BVDV E0 protein elicits a long-lasting serologic response.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is a cause of substantial economic loss to the cattle industry worldwide, and there are currently no effective treatment or preventive measures. Bovine enterovirus (BEV) has a broad host range with low virulence and is a good candidate as a viral vaccine vector. In this study, we explored new insertion sites for the expression of exogenous genes in BEV, and developed a recombinant infectious cDNA clone for BEV BJ101 strain expressing BVDV E0 protein. METHODS: A recognition site for the viral proteinase 3Cpro was inserted in the GpBSK-BEV plasmid at the 2C/3A junction by overlapping PCR. Subsequently, the optimized full-length BVDV E0 gene was inserted to obtain the recombinant infectious plasmid GpBSK-BEV-E0. The rescued recombinant virus was obtained by transfection with linearized plasmid. Expression of BVDV E0 in the recombinant virus was confirmed by PCR, western blotting, and immunofluorescence analysis, and the genetic stability was tested in MDBK cells over 10 passages. We further tested the ability of the recombinant virus to induce an antibody response in mice infected with BVDV and immunized them with the recombinant virus and parental strain. RESULTS: The rescued recombinant virus rBEV-E0 was identified and confirmed by western blot and indirect immunofluorescence. The sequencing results showed that the recombinant virus remained stable for 10 passages without genetic changes. There was also no significant difference in growth dynamics and plaque morphology between the recombinant virus and parental virus. Mice infected with both recombinant and parental viruses produced antibodies against BEV VP1, while the recombinant virus also induced antibodies against BVDV E0. CONCLUSION: A new insertion site in the BEV vector can be used for the prevention and control of both BEV and BVDV, providing a useful tool for future research on the development of viral vector vaccines.

Longitudinal and cross-sectional detection of four bovine enteric viruses by multiplex- reverse transcription polymerase chain reaction: Identification of possible indicator viruses to assess biosecurity level at bovine farms.

It can be judged that if the detection frequency of prevalent pathogenic viruses decreases, biosecurity has been enhanced. To monitor bovine farm biosecurity levels, one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) for the simultaneous detection of group A rotavirus (RVA), bovine torovirus (BToV), bovine enterovirus (BEV), and bovine coronavirus (BCV) was designed, with the aim of configuring candidates for "viral pathogen indicators". A total of 322 bovine fecal samples were collected from calves aged less than three months at 48 bovine farms in Ibaraki and Chiba prefectures. At farm A, 20 calves were selected and sampled weekly for 12 weeks (184 samples); at farm B, 10 calves were selected and sampled for five weeks (50 samples); and at the rest of the 46 farms, 88 calves were sampled once. The screening on the 358 field samples proved positive for 27 RVA, 4 BToV, 55 BEV, and 52 BCV. In the successive sampling, RVA was detected once but not continuously, whereas BEV and BCV were detected in succession for up to five weeks. The results revealed that RVA was the primary agent among the positive samples obtained from calves aged three weeks or less, while BEV was the primary among those from the older than three weeks old. They can be employed as useful viral pathogen indicators for soundly evaluating biosecurity at bovine farms.

A serological investigation of Bovine enterovirus-1, Bovine herpesvirus-1, Bovine viral diarrhea virus, and Parainfluenza-3 infections in camelsin Western Turkey.

Camels (Camelus dromedarius) are bred in Western Turkey, particularly in the province of Aydin, for touristic, social and cultural purposes. Bovine enterovirus­1 (BEV­1), Bovine herpesvirus type­1 (BHV­1), Bovine viral diarrhea virus (BVDV), and Parainfluenza­3 (PI­3) virus infections are significant causes of health and/or economic concerns in several animal species. These agents have not been investigated in the camel population in Turkey. The objective of this study was to serologically investigate the presence and infection rates of these viruses in camels in Aydin province, Western Turkey. Ninety­two serum samples were taken from clinically healthy camels that were kept in private farms or brought to the local slaughterhouses. Serum neutralization test was performed to assess the presence and the titers of specific antibodies against BEV­1, BHV­1, BVDV, and PI­3 virus in camel sera. Of the 92 camels tested, 30 (32.61%), 2 (2.17%), 54 (58.7%), and 20 (21.74%) were seropositive for BEV­1, BHV­1, BVDV, and PI­3, respectively. These results suggest that, except for BHV­1, these viral infections are common among camels in Western Turkey. To our knowledge, this the first comprehensive, large­scale study investigating these viral infections in camels in Turkey.

Structural basis for hijacking of the host ACBD3 protein by bovine and porcine enteroviruses and kobuviruses.

Picornaviruses infect a wide range of mammals including livestock such as cattle and swine. As with other picornavirus genera such as Aphthovirus, there is emerging evidence of a significant economic impact of livestock infections caused by members of the genera Enterovirus and Kobuvirus. While the human-infecting enteroviruses and kobuviruses have been intensively studied during the past decades in great detail, research on livestock-infecting viruses has been mostly limited to the genomic characterization of the viral strains identified worldwide. Here, we extend our previous studies of the structure and function of the complexes composed of the non-structural 3A proteins of human-infecting enteroviruses and kobuviruses and the host ACBD3 protein and present a structural and functional characterization of the complexes of the following livestock-infecting picornaviruses: bovine enteroviruses EV-E and EV-F, porcine enterovirus EV-G, and porcine kobuvirus AiV-C. We present a series of crystal structures of these complexes and demonstrate the role of these complexes in facilitation of viral replication.

A study regarding bovine enterovirus type 1 infection in domestic animals and humans: An evaluation from the zoonotic aspect.

Bovine enteroviruses (BEV) are members of Enterovirus genus of the family Picornaviridae. BEV1 has a broad host spectrum, including humans. The virus usually causes subclinical infection, but fatal/severe cases have also been reported in different animal species. There is quite limited data regarding BEV1 in humans. The purpose of this study is to investigate human infection and to identify possible risk factors for viral exposure. For this purpose, blood serum samples (n=1,526) were collected from a city center and nearby villagers simultaneously from humans and farm animals in Elazig province in Eastern Anatolia. As a result of serum neutralisation test, BEV1 specific antibody presence detected in cattle was 85.3% (163/191), 73.5% in donkeys (64/87), 71.8% in goats (115/160), 46.5% in sheep (93/200), 43.9% in horses (40/91), 41.3% in dogs (19/46) and 33% in humans (248/751). Although a high contamination potential was mentioned for people living in rural areas, it was determined that infection rates in rural areas (31.6%) and urban centers (32.2%) were very close. There was no difference according to sex. Viral exposure is higher in the 40 to 70 age range. In addition, the serological evidence of the infection in donkeys was identified for the first time with this study.

Detection and first molecular characterisation of three picornaviruses from diarrhoeic calves in Turkey.

The involvement of picornaviruses in calf diarrhoea was evaluated by the analysis of 127 faecal samples collected from diarrhoeic calves during 2014-2016. Virus detections were carried out by PCR using generic or specific primer pairs. One-third of the faecal samples (33.86%) were found to be positive for one or more of the studied viruses. Bovine kobuvirus was detected in 22.83%, bovine hungarovirus in 11.02%, while bovine enterovirus 1 in 5.51% of the samples. The sequences of the PCR products indicated the existence of novel variants in all the three virus species. When comparing the partial sequences, the nucleotide sequence identities between our newly detected viruses and those previously deposited to the GenBank ranged between 76 and 99%. Phylogenetic analyses revealed a novel lineage within the species Hunnivirus A. Our findings suggest that these viruses should be regarded as possible aetiological agents of calf diarrhoea. Based on the newly determined sequences, we designed and tested a new generic PCR primer set for the more reliable detection of bovine hungaroviruses. This is the first report on the molecular detection of the presence of bovine hungarovirus, bovine kobuvirus and bovine enterovirus 1 in the faecal samples of diarrhoeic calves in Turkey.

Identification of three linear B cell epitopes using monoclonal antibodies against bovine enterovirus VP2 protein.

Bovine enterovirus (BEV) VP2 protein is a structural protein that plays an important role in inducing protective immunity in the host. The function of VP2 has been characterized, but there is little information on its B cell epitopes. Three monoclonal antibodies (mAbs) directed against BEV VP2 were generated and characterized from mice immunized with the recombinant VP2 protein. Three minimal linear epitopes 152FQEAFWLEDG161, 168LIYPHQ173, and 46DATSVD51 reactive to the three mAbs were identified using western blotting analysis. Three-dimensional model of the BEV-E virion and the VP2 monomer showed that epitope 152FQEAFWLEDG161 is exposed on surface of the virion and epitopes 46DATSVD51 and 168LIYPHQ173 are located inside the virion. Alignment of the amino acid sequences corresponding to the regions containing the three minimal linear epitopes in the VP2 proteins and their cross-reactivity with the three mAbs showed that epitope 168LIYPHQ173 is completely conserved in all BEV strains. Epitope 46DATSVD51 is highly conserved among BEV-E strains and partly conserved among BEV-F strains. However, epitope 152FQEAFWLEDG161 is not conserved among BEV-F strains. Using the mAbs of 3H4 and 1E10, we found that VP2 localized in the cytoplasm during viral replication and could be used to monitor the viral antigen in infected tissues using immunohistochemistry. A preliminary 3H4-epitope-based indirect ELISA allowed us to detect anti-BEV-strain-HY12 antibodies in mice. This study indicates that the three mAbs could be useful tools for investigating the structure and function of the viral VP2 protein and the development of serological diagnostic techniques for BEV infection.

Detection and molecular characterisation of bovine Enterovirus in Brazil: four decades since the first report.

It is suggested that bovine enteroviruses (BEV) are involved in the aetiology of enteric infections, respiratory disease, reproductive disorders and infertility. In this study, bovine faecal samples collected in different Brazilian states were subjected to RNA extraction, reverse transcription-polymerase chain reaction analysis and partial sequencing of the 5'-terminal portion of BEV. One hundred and three samples were tested with an overall positivity of 14.5%. Phylogenetic analysis clustered these BEV Brazilian samples into the Enterovirus F clade. Our results bring an important update of the virus presence in Brazil and contribute to a better understanding of the distribution and characterisation of BEV in cattle.

Rescue and characterization of a recombinant HY12 bovine enterovirus carrying a foreign HA epitope in the 3A nonstructural protein.

Full-length infectious cDNA clones for recombinant HY12 bovine enteroviruses designated as rHY12-3A-2-HA, rHY12-3A-3-HA, and rHY12-3A-9-HA were constructed by the insertion of an epitope from influenza virus hemagglutinin (HA) at the N-terminus of the HY12-encoded 3A protein at amino acid positions 2, 3, and 9. The recombinant HY12 viruses expressing the HA epitope were rescued and characterized using immunoperoxidase monolayer assay, western blotting, and electron microscopy. The three rescued recombinant marker viruses showed similar characteristics, such as TCID50 titer, plaque size, and growth properties, to those of parental rHY12 virus. Comparative analysis of the nucleotide sequences demonstrated the three recombinant marker viruses remained stable for 15 passages with no genetic changes. The recombinant viruses remained viable in various permissive cell lines, including BHK-21, Vero, and PK15 cells, suggesting that the insertion of the HA epitope tag had no effect on virus infectivity. Mice infected with the recombinant marker viruses and the parental virus produced anti-HY12-virus antibodies, while the recombinant marker viruses also produced anti-HA-epitope-tag antibodies. Taken together, these results demonstrate that HY12 viruses containing genetic markers may be useful tools for future investigations of the mechanisms of viral pathogenesis and virus replication, as well as for vaccine development.

Effects of bovine enterovirus and type 1 diabetes on liver and kidney pyruvate kinase activity in an animal model

Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of pancreatic beta cells. In addition to genetic factors, enteroviruses have been considered the main environmental factor involved in this pathology. Therefore, the objective of this study was to evaluate the effects of streptozotocin-induced diabetes and bovine enterovirus (BEV) on liver and kidney pyruvate kinase activity in rats. Fourteen male Wistar rats were divided in three groups: control, diabetes and a third group, which was fed with water experimentally contaminated by BEV. Increased blood glucose levels were found in both diabetes and enterovirus groups, whereas there were no alterations in the lipid profile. A reduced pyruvate kinase activity was observed in the liver and kidney of animals from diabetes and enterovirus groups. Under our experimental conditions, the ingestion of water experimentally contaminated by BEV induced alterations in glycaemia, and also interfered in the pyruvate kinase activity in liver and kidney of the rats, which might be one of the possible mechanisms involved in the T1D development.

Photodynamic inactivation of non-enveloped RNA viruses.

We recently reported the photodynamic inactivation (PDI) of bacteriophage MS2 with a photosensitiser- 5, 10, 15, 20-tetrakis (1-methyl-4-pyridinio) porphyrin- tetra- p-toluene sulfonate (TMPyP) in solution and concluded that the A-protein of the virus is the main target of inactivation. Here, we have extended these studies and carried out PDI of bacteriophage Qß, bovine enterovirus 2 (BEV-2) and type 1 murine norovirus (MNV-1). The rate of inactivation observed was in the order MS2 > Qß > MNV-1 > BEV-2. Data suggested that TMPyP-treatment could also target the viral genome as well as result in disintegration/disassembly of viral particles. Although emergence of viral drug resistance is a well-documented phenomenon, it was not possible to generate PDI-resistant MS2. However, emergence of a mutation in the lysis protein was detected after serial exposure to PDI.

Identification and genetic characterization of bovine enterovirus by combination of two next generation sequencing platforms.

Prompt and accurate diagnosis is warranted for infectious diseases of domestic animals which may have a significant impact on animal production or clinical practice. In this study, the identification and genetic characterization of a bovine enterovirus (BEV) strain isolated from a calf with diarrhea, are described. Two different next generation sequencing platforms were employed. Shotgun metagenomic accomplished by MinION sequencing (Oxford Nanopore Technologies) allowed the identification of BEV RNA from a cell-culture isolate. BEV was then confirmed by a specific real time RT-PCR assay. To achieve the whole genome of this isolate, sequence reads obtained by MinION were coupled with those originating from NextSeq500 (Illumina). Genomic relatedness and phylogeny with extant BEV strains is also reported. Overall, this manuscript highlights the use of the portable MinION sequence technology as a tool for support diagnostics in veterinary practice.

Dembo polymerase chain reaction technique for detection of bovine abortion, diarrhea, and respiratory disease complex infectious agents in potential vectors and reservoirs

Bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds, and rodents. However, experimental data supporting this possibility are scarce. We collected 117 samples and screened them for 44 bovine abortive, diarrheal, and respiratory disease complex pathogens by using Dembo polymerase chain reaction (PCR), which is based on TaqMan real-time PCR. Fifty-seven samples were positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, Salmonella enterica ser. Dublin, Salmonella enterica ser. Typhimurium, and Neospora caninum; some samples were positive for multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus were the most frequently detected pathogens, especially in flies, suggesting an important role of flies in the transmission of these viruses. Additionally, we detected the N. caninum genome from a cockroach sample for the first time. Our data suggest that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of abortion, diarrhea, and respiratory infectious agents, and that they may transmit more than one pathogen at the same time.

Host microRNA-203a Is antagonistic to the progression of foot-and-mouth disease virus infection.

Sam68 was previously shown to be a critical host factor for foot-and-mouth disease virus (FMDV) replication. MicroRNA (miR) miR-203a is reportedly a negative regulator of Sam68 expression both in vitro and in vivo. Here, transfection of miR-203a-3p and miR-203a-5p mimics separately and in combination in a porcine cell line followed by FMDV infection resulted in diminished viral protein synthesis and a 4 and 6log reduction in virus titers relative to negative controls, respectively. Unexpectedly, Sam68 expression was increased by miR-203a-5p transfection, but not miR-203a-3p. miR-203a-5p also down-regulated Survivin expression, which was predicted to play a role in FMDV infection. Moreover, miR-203a-5p but not miR-203a-3p affected a reduction in FMDV viral RNA. These effects were not replicated with a related Picornavirus, suggesting FMDV specificity. Importantly, miR-203a-3p and miR-203a-5p impaired FMDV infection across multiple FMDV serotypes. We concluded that miR-203a-3p and miR-203a-5p represent attractive potential naturally occurring bio-therapeutics against FMDV.

Identification of a novel bovine enterovirus possessing highly divergent amino acid sequences in capsid protein.

BACKGROUND: Bovine enterovirus (BEV) belongs to the species Enterovirus E or F, genus Enterovirus and family Picornaviridae. Although numerous studies have identified BEVs in the feces of cattle with diarrhea, the pathogenicity of BEVs remains unclear. Previously, we reported the detection of novel kobu-like virus in calf feces, by metagenomics analysis. In the present study, we identified a novel BEV in diarrheal feces collected for that survey. Complete genome sequences were determined by deep sequencing in feces. Secondary RNA structure analysis of the 5' untranslated region (UTR), phylogenetic tree construction and pairwise identity analysis were conducted. RESULTS: The complete genome sequences of BEV were genetically distant from other EVs and the VP1 coding region contained novel and unique amino acid sequences. We named this strain as BEV AN12/Bos taurus/JPN/2014 (referred to as BEV-AN12). According to genome analysis, the genome length of this virus is 7414 nucleotides excluding the poly (A) tail and its genome consists of a 5'UTR, open reading frame encoding a single polyprotein, and 3'UTR. The results of secondary RNA structure analysis showed that in the 5'UTR, BEV-AN12 had an additional clover leaf structure and small stem loop structure, similarly to other BEVs. In pairwise identity analysis, BEV-AN12 showed high amino acid (aa) identities to Enterovirus F in the polyprotein, P2 and P3 regions (aa identity ≥82.4%). Therefore, BEV-AN12 is closely related to Enterovirus F. However, aa sequences in the capsid protein regions, particularly the VP1 encoding region, showed significantly low aa identity to other viruses in genus Enterovirus (VP1 aa identity ≤58.6%). In addition, BEV-AN12 branched separately from Enterovirus E and F in phylogenetic trees based on the aa sequences of P1 and VP1, although it clustered with Enterovirus F in trees based on sequences in the P2 and P3 genome region. CONCLUSIONS: We identified novel BEV possessing highly divergent aa sequences in the VP1 coding region in Japan. According to species definition, we proposed naming this strain as "Enterovirus K", which is a novel species within genus Enterovirus. Further genomic studies are needed to understand the pathogenicity of BEVs.