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.

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.

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.

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.

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.

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.

[Epidemiology, etiology and clinical picture of enteroviral meningitis in children in South Moravia].

OBJECTIVES: Enteroviruses (EVs) are the most common cause of aseptic viral meningitis. In some cases, they can cause severe meningoencephalitis and acute flaccid paralysis - an association with some virulent serotypes. The objectives were to describe the epidemiological situation of EV meningitis in children in South Moravia, to elucidate the etiology including the incidence of virulent serotypes and to evaluate the clinical presentation. MATERIAL AND METHODS: A total of 88 children with EV meningitis were prospectively evaluated. In case of aseptic inflammation in the cerebrospinal fluid, EV was detected using real-time PCR. Genotyping was performed in 56 samples using repeated one-step PCR and partial sequencing on a genetic analyzer in the National Reference Laboratory for Enteroviruses in Prague. RESULTS: The patients' age range was 3-17 years; there were more boys than girls. Two epidemics occurred, one involving 17 Roma children and the other involving 8 swimming pool visitors. The most common symptoms were headache, fever and stiff neck. The most frequently (59%) detected agent was Echovirus 30 identified as the cause of the epidemics. In one boy, EV 71 (virulent serotype) was found. The clinical course did not vary from that in other serotypes. All 88 children recovered without complications. CONCLUSIONS: EVs are an important part of the differential diagnosis of neuroinfections, although most infections are benign aseptic meningitis. The clinical presentation did not vary between infections with various serotypes. Higher incidence rates of virulent serotypes were not reported. Echovirus 30 was detected most frequently and was repeatedly identified as the cause of epidemics throughout the Czech Republic.

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.

Simultaneous Concentration of Bovine Viruses and Agricultural Zoonotic Bacteria from Water Using Sodocalcic Glass Wool Filters.

Infiltration and runoff from manured agricultural fields can result in livestock pathogens reaching groundwater and surface waters. Here, we measured the effectiveness of glass wool filters to simultaneously concentrate enteric viruses and bacteria of bovine origin from water. The recovery efficiencies were determined for bovine viral diarrhea virus types 1 and 2, bovine rotavirus group A, bovine coronavirus, poliovirus Sabin III, toxigenic Escherichia coli ,and Campylobacter jejuni seeded into water with three different turbidity levels (0.5, 215, and 447 NTU). Twenty liters of dechlorinated tap water (pH 7) were seeded with the test organisms, and then passed through a glass wool filter using a peristaltic pump (flow rate = 1 liter min(-1)). Retained organisms were eluted from the filters by passing beef extract-glycine buffer (pH 9.5) in the direction opposite of sample flow. Recovered organisms were enumerated by qPCR except for C. jejuni, which was quantified by culture. Mean recovery efficiencies ranged from 55 to 33% for the bacteria and 58 to 16% for the viruses. Using bootstrapping techniques combined with Analysis of Variance, recovery efficiencies were found to differ among the pathogen types tested at the two lowest turbidity levels; however, for a given pathogen type turbidity did not affect recovery except for C. jejuni. Glass wool filtration is a cost-effective method for concentrating several waterborne pathogens of bovine origin simultaneously, although recovery may be low for some specific taxa such as bovine viral diarrhea virus 1.

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.

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.

Molecular identification and characterization of a new type of bovine enterovirus.

Bovine enteroviruses belong to the family Picornaviridae. Little is known about their pathogenic potential; however, they cause asymptomatic infections in cattle and are excreted in feces. In the present study, viruses isolated from environmental samples were sequenced. According to phylogenetic analyses and standard picornavirus nomenclature, these isolates constitute a new type of bovine enterovirus serogroup A.

Grazing management effects on sediment, phosphorus, and pathogen loading of streams in cool-season grass pastures.

Erosion and runoff from pastures may lead to degradation of surface water. A 2-yr grazing study was conducted to quantify the effects of grazing management on sediment, phosphorus (P), and pathogen loading of streams in cool-season grass pastures. Six adjoining 12.1-ha pastures bisected by a stream in central Iowa were divided into three treatments: continuous stocking with unrestricted stream access (CSU), continuous stocking with restricted stream access (CSR), and rotational stocking (RS). Rainfall simulations on stream banks resulted in greater ( < 0.10) proportions of applied precipitation and amounts of sediment and P transported in runoff from bare sites than from vegetated sites across grazing treatments. Similar differences were observed comparing vegetated sites in CSU and RS pastures with vegetated sites in CSR pastures. Bovine enterovirus was shed by an average of 24.3% of cows during the study period and was collected in the runoff of 8.3 and 16.7% of runoff simulations on bare sites in CSU pastures in June and October of 2008, respectively, and from 8.3% of runoff simulations on vegetated sites in CSU pastures in April 2009. Fecal pathogens (bovine coronavirus [BCV], bovine rotavirus group A, and O157:H7) shed or detected in runoff were almost nonexistent; only BCV was detected in feces of one cow in August of 2008. Erosion of cut-banks was the greatest contributor of sediment and P loading to the stream; contributions from surface runoff and grazing animals were considerably less and were minimized by grazing management practices that reduced congregation of cattle by pasture streams.

Pathogenesis of a bovine enterovirus-1 isolate in experimentally infected calves.

The pathogenesis and virulence of Bovine enterovirus-1 (BEV-1) in cattle is largely unknown. Reports concerning its virulence suggest that there might be an association between BEV-1 infections and a range of diseases in cattle that vary from respiratory to enteric to reproductive disease and infertility. In the current study, the pathogenesis associated with acute infection of BEV-1 in calves experimentally inoculated with the Oklahoma isolate of BEV-1 was described. Although interpretation of the study was limited by lack of an effective control group, results suggest that an association between inoculation of BEV-1, virus localization, and the potential development of lesions in the brain and heart probably exists. In the experiment, BEV-1 virus localized to the terminal ileum, ileocecal and cecocolonic junctions, spiral colon, and ileocecal lymph nodes; BEV-1 virus was detected in the cytoplasm of enterocytes, lamina propria macrophages, endothelium, neurons of the submucosal and myenteric plexi, and lymphocytes of the submucosal lymphoid tissue. Although no clinical signs were noted following acute infection, BEV-1 was localized in the cerebellar white matter of a calf with encephalitis and in the heart of another calf with coronary arteritis. The current study suggests that the BEV-1 isolate is infectious to young calves and that BEV-1 potentially can have a similar pathogenesis to that observed in natural or experimental enterovirus infections in other species.

Use of filter carrier technique to measure the persistence of avian influenza viruses in wet environmental conditions.

A germ carrier technique was adapted for the determination of the persistence of influenza viruses in moist environments. The technique was employed with 3 low pathogenic avian influenza viruses (H4N6, H5N1, and H6N8), one human influenza virus (H1N1), and two model viruses (NDV and ECBO) in lake water at five different temperatures (30, 20, 10, 0, and -10°C). Viral quantitation was carried out at regular intervals on cell culture for a maximum duration of 16 weeks. Serial data were analyzed by linear regression model to calculate T-90 values (time required for one log reduction in the virus titer). Persistence of all of the viruses was highest at -10°C followed by 0, 10, 20, and 30°C. At -10°C, the single freeze-thaw cycle resulted in an abrupt decline in the virus titer, followed by long term persistence. Generally, influenza viruses persisted shorter than model viruses while ECBO has the highest survival time in lake water. Individual influenza viruses differed in their persistence at all temperatures. The findings of the present study suggest that AIV can remain infectious in lake water for extended periods of time at low temperatures.

New recognition of Enterovirus infections in bottlenose dolphins (Tursiops truncatus).

An enterovirus was cultured from an erosive tongue lesion of a bottlenose dolphin (Tursiops truncatus). The morphology of virions on negative staining electron microscopy was consistent with those of enteroviruses. Analysis of 2613 bp of the polyprotein gene identified the isolate as a novel enterovirus strain, tentatively named bottlenose dolphin enterovirus (BDEV), that nests within the species Bovine enterovirus. Serologic evidence of exposure to enteroviruses was common in both free-ranging and managed collection dolphins. Managed collection dolphins were more likely to have high antibody levels, although the highest levels were reported in free-ranging populations. Associations between enterovirus antibody levels, and age, sex, complete blood counts, and clinical serum biochemistries were explored. Dolphins with higher antibody levels were more likely to be hyperproteinemic and hyperglobulinemic.