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.

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.

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.

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.

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.

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.

Genetic variations in regions of bovine and bovine-like enteroviral 5'UTR from cattle, Indian bison and goat feces.

BACKGROUND: Bovine enteroviruses (BEV) are members of the genus Enterovirus in the family Picornaviridae. They are predominantly isolated from cattle feces, but also are detected in feces of other animals, including goats and deer. These viruses are found in apparently healthy animals, as well as in animals with clinical signs and several studies reported recently suggest a potential role of BEV in causing disease in animals. In this study, we surveyed the presence of BEV in domestic and wild animals in Thailand, and assessed their genetic variability. METHODS: Viral RNA was extracted from fecal samples of cattle, domestic goats, Indian bison (gaurs), and deer. The 5' untranslated region (5'UTR) was amplified by nested reverse transcription-polymerase chain reaction (RT-PCR) with primers specific to BEV 5'UTR. PCR products were sequenced and analyzed phylogenetically using the neighbor-joining algorithm to observe genetic variations in regions of the bovine and bovine-like enteroviral 5'UTR found in this study. RESULTS: BEV and BEV-like sequences were detected in the fecal samples of cattle (40/60, 67 %), gaurs (3/30, 10 %), and goats (11/46, 24 %). Phylogenetic analyses of the partial 5'UTR sequences indicated that different BEV variants (both EV-E and EV-F species) co-circulated in the domestic cattle, whereas the sequences from gaurs and goats clustered according to the animal species, suggesting that these viruses are host species-specific. CONCLUSIONS: Varieties of BEV and BEV-like 5'UTR sequences were detected in fecal samples from both domestic and wild animals. To our knowledge, this is the first report of the genetic variability of BEV in Thailand.

Isolation and molecular characterization of bovine enteroviruses in Egypt.

Enteroviruses belong to the Picornaviridae family and infect a wide range of mammals including cattle. Bovine enterovirus (BEV) has recently been reclassified into E and F serotypes. BEV was first isolated in Egypt in 1966 although it has been known in other countries since the 1950s. In this study, BEV-F2 was isolated from calves with severe diarrhea and the isolated viruses were subjected to molecular characterization. Illumina sequencing of one of the isolates revealed the presence of a complete BEV-F genome sequence. The phylogenetic analysis revealed nucleotide substitutions along the genome in comparison with other known strains of BEV-F (HQ663846, AY508697 and DQ092795). Two primer sets were designed from the 3D and 5'NTR regions and used for the examination of the remaining isolates, which were confirmed to be of the BEV-F2 serotype. The availability of the complete genome sequence of this virus adds to the sequence database of the members of Picornaviridae and should be useful in future molecular studies of BEV.

[Establishment and Preliminary Application of the SYBR Green I Real-time PCR Assay for Detection of the Bovine Enterovirus].

The bovine enterovirus (BEV) is a pathogen found the digestive tracts of cattle. Recently, the BEV was discovered in cattle in a province in China. A rapid and effective detection method for the BEV is essential. An assay was carried out using two specific primers designed to amplify a highly conserved sequence of the 3D gene. A recombinant plasmid containing the target gene 3D was constructed as a standard control. The limit of detection of the reaction was 7.13 x 10(1) plasmid copies/µL of initial templates, which was tenfold more sensitive than the conventional reverse-transcription-polymerase chain reaction (RT-PCR). Moreover, the assay was highly specific because all negative controls and other viruses of clinical relevance did not develop positive results. Assay performance on field samples was evaluated on 44 (41 diarrhea and 3 aerosol) samples and compared with the conventional RT-PCR assay. Sixteen diarrhea samples were positive (16/41, 39. 02%) and 3 aerosol samples were positive (3/3, 100%). Preliminary results for clinical detection showed that the SYBR Green I real-time PCR assay was highly sensitive, specific and reproducible. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications for epidemics and in BEV research.

Identification of a novel enterovirus E isolates HY12 from cattle with severe respiratory and enteric diseases.

In this study, a virus strain designated as HY12 was isolated from cattle with a disease of high morbidity and mortality in Jilin province. Biological and physiochemical properties showed that HY12 isolates is cytopathic with an extremely high infectivity. HY12 is resistant to treatment of organic solvent and acid, and unstable at 60°C for 1 h. Electron microscopy observation revealed the virus is an approximately 22-28 nm in diameter. The complete genome sequence of HY12 consists of 7416 nucleotides, with a typical picornavirus genome organization including a 5'-untranslated region (UTR), a large single ORF encoding a polyprotein of 2176 amino acids, and a 3'-UTR. Phylogenetic analysis clustered HY12 isolates to a new serotype/genotype within the clade of enterovirus E (formerly BEV-A). Alignment analysis revealed a unique insertion of 2 amino acid residues (NF) at the C-terminal of VP1 protein between aa 825 and 826, and several rare mutations in VP1 and VP4 of HY12 isolates in relation to known bovine enterovirus (BEV) strains. This is the first report of an enterovirus E in China, which is potentially associated with an outbreak in cattle with severe respiratory and enteric diseases.

The mechanism of translation initiation on Type 1 picornavirus IRESs.

Picornavirus Type 1 IRESs comprise five principal domains (dII-dVI). Whereas dV binds eIF4G, a conserved AUG in dVI was suggested to stimulate attachment of 43S ribosomal preinitiation complexes, which then scan to the initiation codon. Initiation on Type 1 IRESs also requires IRES trans-acting factors (ITAFs), and several candidates have been proposed. Here, we report the in vitro reconstitution of initiation on three Type 1 IRESs: poliovirus (PV), enterovirus 71 (EV71), and bovine enterovirus (BEV). All of them require eIF2, eIF3, eIF4A, eIF4G, eIF4B, eIF1A, and a single ITAF, poly(C) binding protein 2 (PCBP2). In each instance, initiation starts with binding of eIF4G/eIF4A. Subsequent recruitment of 43S complexes strictly requires direct interaction of their eIF3 constituent with eIF4G. The following events can differ between IRESs, depending on the stability of dVI. If it is unstructured (BEV), all ribosomes scan through dVI to the initiation codon, requiring eIF1 to bypass its AUG. If it is structured (PV, EV71), most initiation events occur without inspection of dVI, implying that its AUG does not determine ribosomal attachment.

VP1 B-C and D-E loops of bovine enterovirus cluster B can effectively display foot-and-mouth disease virus type O-conserved neutralizing epitope.

On the basis of generation of an infectious cDNA clone for the BHM26 strain of bovine enterovirus cluster B (BEV-B), 22 sites on different loops of the BHM26 capsid were selected according to an alignment of its sequence with the structural motifs of BEV-A strain VG-5-27 for insertion of the foot-and-mouth disease virus (FMDV) type O-conserved neutralizing epitope 8E8. Two recombinant viruses, rBEV-A1 and rBEV-DE, in which the FMDV epitope was inserted into the VP1 B-C or D-E loops, were rescued by transfection of BHK-21 cells with the in vitro-transcribed RNA of the recombinant BHM26 genome-length cDNA constructs. The two epitope-inserted viruses were genetically stable and exhibited growth properties similar to those of their parental virus in BHK-21 and IBRS-2 cells, which are susceptible to both BEV and FMDV. However, the two recombinant BEVs (rBEVs) had a significantly lower growth titre than those of the parental virus BHM26 in MDBK and Marc145 cells, which are susceptible to BEV but not to FMDV. These results indicated that insertion of the FMDV epitope into the VP1 B-C or D-E loops of the BEV particle altered the replication properties of BEV. In addition, the two rBEVs were sensitive to neutralization by the FMDV type O-specific mAb 8E8, and anti-FMDV IgG antibodies were induced in mice by intramuscular inoculation with the rBEV-A1 and rBEV-DE viruses. Our results demonstrate that the VP1 B-C and D-E loops of the BEV-B particle can effectively display a foreign epitope, making this an attractive approach for the design of BEV-vectored and epitope-based vaccines.

Discovery of a bovine enterovirus in alpaca.

A cytopathic virus was isolated using Madin-Darby bovine kidney (MDBK) cells from lung tissue of alpaca that died of a severe respiratory infection. To identify the virus, the infected cell culture supernatant was enriched for virus particles and a generic, PCR-based method was used to amplify potential viral sequences. Genomic sequence data of the alpaca isolate was obtained and compared with sequences of known viruses. The new alpaca virus sequence was most similar to recently designated Enterovirus species F, previously bovine enterovirus (BEVs), viruses that are globally prevalent in cattle, although they appear not to cause significant disease. Because bovine enteroviruses have not been previously reported in U.S. alpaca, we suspect that this type of infection is fairly rare, and in this case appeared not to spread beyond the original outbreak. The capsid sequence of the detected virus had greatest homology to Enterovirus F type 1 (indicating that the virus should be considered a member of serotype 1), but the virus had greater homology in 2A protease sequence to type 3, suggesting that it may have been a recombinant. Identifying pathogens that infect a new host species for the first time can be challenging. As the disease in a new host species may be quite different from that in the original or natural host, the pathogen may not be suspected based on the clinical presentation, delaying diagnosis. Although this virus replicated in MDBK cells, existing standard culture and molecular methods could not identify it. In this case, a highly sensitive generic PCR-based pathogen-detection method was used to identify this pathogen.

Construction of a bovine enterovirus-based vector expressing a foot-and-mouth disease virus epitope.

A recombinant infectious bovine enterovirus (BEV) vector was constructed to express a foot-and-mouth disease virus (FMDV) capsid protein (VP1) epitope. Sequences encoding the VP1 epitope (amino acid residues 141-160) of FMDV (vaccine strain O1/Manisa/Turkey/69) were inserted into pBLUBEV at the VP1/2A junction. The growth characteristics of the parental virus and viruses derived from recombinant plasmids (pBLUBEV, pBLUBEV-Manisa-epi) were determined by plaque assay and one-step growth curve analysis. There were no significant differences in the growth kinetics and plaque morphologies between transfectant viruses and their parental virus. The expressed VP1 epitope was detected successfully by using indirect immunofluorescence assay with a polyclonal antibody against the FMDV VP1 epitope from Madin Darby bovine kidney (MDBK) cells infected with BEV-Manisa-epi transfectant virus. This study demonstrated a novel alternative live viral vector that may be utilized as a candidate vaccine vector for veterinary applications.

Isolation of two Chinese bovine enteroviruses and sequence analysis of their complete genomes.

In this study, RNA corresponding to bovine enterovirus (BEV) was detected in 24.6 % of faecal samples (17/69) from diarrheic and healthy cattle in six different areas in China by an RT-PCR screening method. Furthermore, two cytopathic agents, designated as BHM26 and BJ50, were isolated from the bovine diarrheic fecal samples. During passage in MA104 cells, ultrathin sections of virus-infected monolayers were examined using a transmission electron microscope, and a large number of symmetrical virus crystals were seen in the cytoplasm, with monomorphic small viral particles of 27-30 nm in diameter. The full-length RNA genomes were 7433 and 7416 nucleotides long, respectively, with a genome organization analogous to that of picornaviruses. Phylogenetic analysis of the VP1 and VP3 capsid protein coding sequences suggested that the viruses BHM26 and BJ50 belong to genotype 2 of the BEV cluster B (BEV-B). In addition, sequence comparisons of the 5' and 3' UTRs and P1, P2 and P3 subgenomic regions of the two isolates suggested that there were intergenotypic recombination events occurring during evolution of the BHM26 and BJ50 isolates.

Natural interspecies recombinant bovine/porcine enterovirus in sheep.

Members of the genus Enterovirus (family Picornaviridae) are believed to be common and widespread among humans and different animal species, although only a few enteroviruses have been identified from animal sources. Intraspecies recombination among human enteroviruses is a well-known phenomenon, but only a few interspecies examples have been reported and, to our current knowledge, none of these have involved non-primate enteroviruses. In this study, we report the detection and complete genome characterization (using RT-PCR and long-range PCR) of a natural interspecies recombinant bovine/porcine enterovirus (ovine enterovirus type 1; OEV-1) in seven (44 %) of 16 faecal samples from 3-week-old domestic sheep (Ovis aries) collected in two consecutive years. Phylogenetic analysis of the complete coding region revealed that OEV-1 (ovine/TB4-OEV/2009/HUN; GenBank accession no. JQ277724) was a novel member of the species Porcine enterovirus B (PEV-B), implying the endemic presence of PEV-B viruses among sheep. However, the 5' UTR of OEV-1 showed a high degree of sequence and structural identity to bovine enteroviruses. The presumed recombination breakpoint was mapped to the end of the 5' UTR at nucleotide position 814 using sequence and SimPlot analyses. The interspecies-recombinant nature of OEV-1 suggests a closer relationship among bovine and porcine enteroviruses, enabling the exchange of at least some modular genetic elements that may evolve independently.