Evidence of circulation of the novel border disease virus genotype 8 in chamois.

Evidence of association between the novel putative border disease virus genotype 8 (BDV-8) and fatal disease in an Alpine chamois (Rupicapra rupicapra) is reported. Diagnostically, we also demonstrated, as already previously reported, the failure of BDV-specific primers (PDB1 and PDB2) to detect BDV-8.

A new genotype of border disease virus with implications for molecular diagnostics.

Border disease virus (BDV) is a (+) single-stranded RNA pestivirus affecting mainly sheep and goats worldwide. Genetic typing of BDV has led to the identification of at least seven major genotypes. This study reports the detection of a BDV strain from a goat in northwestern Italy during routine investigations. Sequence analysis revealed mutations in the 5'-UTR of the virus with implications for BDV molecular diagnostics. Moreover, subsequent phylogenetic analysis based on the combined 5'-UTR and Npro/partial C genes, showed divergence from known BDV genotypes, revealing the detection of a novel pestivirus group, for which we propose the name BDV genotype 8.

Characterization of one sheep border disease virus in China.

BACKGROUND: Border disease virus (BDV) causes border disease (BD) affecting mainly sheep and goats worldwide. BDV in goat herds suffering diarrhea was recently reported in China, however, infection in sheep was undetermined. Here, BDV infections of sheep herds in Jiangsu, China were screened; a BDV strain was isolated and identified from the sheep flocks in China. The genomic characteristics and pathogenesis of this new isolate were studied. RESULTS: In 2012, samples from 160 animals in 5 regions of Jiangsu province of China were screened for the presence of BDV genomic RNA and antibody by RT-PCR and ELISA, respectively. 44.4% of the sera were detected positively, and one slowly grown sheep was analyzed to be pestivirus RNA positive and antibody-negative. The sheep kept virus positive and antibody negative in the next 6 months of whole fattening period, and was defined as persistent infection (PI). The virus was isolated in MDBK cells without cytopathic effect (CPE) and named as JSLS12-01. Near-full-length genome sequenced was 12,227 nucleotides (nt). Phylogenetic analysis based on 5'-UTR and N(pro) fragments showed that the strain belonged to genotype 3, and shared varied homology with the other 3 BDV strains previously isolated from Chinese goats. The genome sequence of JSLS12-01 also had the highest homology with genotype BDV-3 (the strain Gifhorn). Experimental infections of sheep had mild clinical signs as depression and short-period mild fever (5 days). Viremia was detected in 1-7 days post-infection (dpi), and seroconversion began after 14 dpi. CONCLUSIONS: This study reported the genomic and pathogenesis characterizations of one sheep BDV strain, which confirmed the occurrence of BDV infection in Chinese sheep. This sheep derived BDV strain was classified as BDV-3, together with the goat derived strains in China. These results might be helpful for further understanding of BDV infection in China and useful for prevention and control of BDV infections in the future.

Genetic characterization of border disease virus (BDV) isolates from small ruminants in Italy.

Border disease virus (BDV) belongs to the Pestivirus genus in the family Flaviviridae. Genetic analyses of pestiviruses that have been isolated from sheep and goat have led to the proposal that BDV isolates can be phylogenetically segregated into at least seven clusters, subtypes BDV-1 to BDV-7. In order to investigate the genetic heterogeneity of small ruminant pestivirus isolates in Italy, a selection of 5'-UTR sequences from isolates that were collected from clinical specimens between 2002 and 2014 was analysed. Phylogenetic reconstructions indicated that the BDV-positive samples clustered within the BDV-1, BDV-3, BDV-5, and BDV-7 groups. These results suggested high genetic diversity within the Italian BDV field isolates. The phylogenetic analysis indicated the first evidence of BDV-1 and BDV-5 circulation in Italy. The marked diversity of the pestivirus isolates might reflect the sheep trade with foreign countries.

Detection and Genetic Characterization of Border Disease Virus (BDV) Isolated from a Persistently Infected Sheep in a Migratory Flock from Rajasthan State, Northwestern India.

Border disease virus (BDV) causes significant economic losses in sheep farming worldwide. In India, BDV has not yet been studied in sheep migrating for summer pasturing. This study aimed to determine the extent of BDV infection in migratory sheep and provide genetic characteristics of BDV. Blood and serum samples from 90 lambs of a migratory sheep flock (600) in Central India were collected and subjected to molecular detection, phylogenetic analysis and virus neutralization test (VNT). We detected BDV in two lambs through real-time RT-PCR, while 64.4% (58/90) of in-contact lambs had BDV neutralizing antibodies. One apparently healthy lamb was found to be persistently infected with BDV. Phylogenetic analysis of 5'-UTR and Npro genes and the concatenated datasets typed the BDV isolate from PI sheep as BDV-3 genotype. However, it showed a closer relationship with BDV-3 strains from China than the previously reported Indian BDV-3 strains. This is the first report on the detection of BDV persistently infected migratory sheep in India. Additionally, we provided evidence of genetic variability among BDV-3 strains in India. The findings improve our understanding of epidemiology and genetic characteristics of BDV in India and highlight the potential risks associated with the traditional practice of sheep migration for summer pasturing.

Detection of border disease virus (BDV) in goat herds suffering diarrhea in eastern China.

BACKGROUND: Border disease virus (BDV) is an important pathogen in sheep and goat production. Neither epidemiological investigation nor any reports of BDV infection was available in China. During Jan to Apr, 2012, several herd goats in Anhui and Jiangsu provinces in eastern China suffered unremitting diarrhea, with morbidity and mortality of about 28-37% and 10-15%, respectively. In the present study, sera and tissue samples from diseased goats of four farms were taken for BDV detection, isolation and identification. RESULTS: Panpesti generic primers and border disease virus (BDV)-specific primers targeting the 5'-UTR region produced RT-PCR positive bands for sera (24/28) and tissue samples (7/30). Twenty positive sera and tissue samples were inoculated onto Madin-Darby bovine kidney (MDBK) cells for virus isolation. Finally, three different strains of BDV, named AH12-01, AH12-02 and JS12/04, were successfully isolated as identified by RT-PCR using 5'-UTR and N(pro) gene primers, sequencing and electron microscopy. Sequences of 5'-UTR and N(pro) genes of them were used for phylogenetic analysis and comparison to other reference sequences available in GenBank. The results indicated AH12-01, AH12-02 and JS12/04 possess high relationship with the BDV 3 group viruses and differed with each other. CONCLUSION: This is the first detection of BDV from goats with diarrhea and confirmation of BDV infection in China.

Genetic characterization of a border disease virus isolate originating from Slovakia.

In this study, a major part of genome of the pestivirus isolate 297 from Slovakia, comprising the 7195 nt-long 5΄-UTR-NS3 region was sequenced and analyzed. Conserved cleavage sites between individual viral proteins of this region were determined and the number of amino acids of respective proteins was estimated as follows: 168 for Npro, 100 for C, 227 for Erns, 195 for E1, 373 for E2, 70 for p7, 453 for NS2, and 683 for NS3. Based on sequence and phylogenetic analysis of 5΄-UTR, Npro, and E2 the isolate 297 was characterized as a border disease virus of genotype 3. It was found to be distinct from other BDV-3 strains analyzed so far, consequently forming a distinct branch within the phylogenetic clade. All these data expand a relatively limited knowledge of genetic properties of individual BDV genotypes and strains circulating in the Central Europe.

Global Distribution and Genetic Heterogeneity of Border Disease Virus.

Border disease virus (BDV) belongs to the genus Pestivirus of the family Flaviviridae. Interspecies transmission of BDV between sheep, cattle, and pigs occurs regularly, sometimes making diagnosis a challenge. BDV can yield substantial economic losses, including prenatal and postnatal infections in lambs, which are the primary source of infection and maintenance of the virus in the population. Since BDV is antigenically and genetically related to bovine viral diarrhea virus (BVDV), it might pose a significant risk to cattle, influencing BVDV eradication campaigns. Similarly, the presence of BDV in swine herds due to pestivirus spillover between small ruminants and pigs might cause uncertainty in classical swine fever virus (CSFV) diagnostics. Therefore, knowledge of BDV epidemiology in different geographical regions will help prevent its spread and optimize control measures. Previous epidemiological studies have shown that various BDV genotypes are predominant in different countries. This review provides an overview of the spread of BDV world-wide in different host species.

Border disease virus shedding and detection in naturally infected Pyrenean chamois (Rupicapra pyrenaica).

Pyrenean chamois (Rupicapra pyrenaica) populations of the central and eastern Pyrenees have been affected by severe outbreaks associated with Border disease virus (BDV) since 2001. Eight Pyrenean chamois (7 males and 1 female) from 1 to 8 years of age with clinical signs consistent with BDV infection were studied. At necropsy, whole blood, tissue samples (skin, brain, prescapular lymph node, thyroid gland, lung, liver, spleen, kidney, small intestine, bone marrow, and testicle), urine, and nasal, oral, and rectal swabs were obtained. The fetus from a pregnant female was also studied. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the virus in all samples, and virus isolation was performed. Sera and tissue samples were positive to RT-PCR, and the virus was isolated from all chamois. The nasal, oral, and rectal swabs and urine samples were RT-PCR positive in 100%, 85.71%, 71.43%, and 100% of chamois, respectively, confirming the excretion of the virus via these 4 routes. In addition, sera were tested for BDV antibodies using enzyme-linked immunosorbent assay and seroneutralization techniques, with negative results. Sequence analysis of the 5' untranslated region in 7 of the chamois confirmed that the virus is grouped into the BDV-4 genotype, the same BDV previously described in Pyrenean chamois. To the authors' knowledge, this is the first study of naturally infected Pyrenean chamois, providing evidence that infected animals shed BDV through nasal, oral, fecal, and urinary excretion routes.

Characterization of internal ribosome entry sites according to secondary structure analysis to classify border disease virus strains.

Applying palindromic nucleotide substitutions (PNS) method, variable loci of the internal ribosome entry site (IRES) secondary structure in the 5' untranslated region (UTR) of Border disease virus sequences were analysed allowing their allocation into ten IRES classes within the species. Sequence characteristics of Turkish and Chinese strains were highly divergent from other genogroups, indicating geographic segregation and micro-evolutive steps within the species. Observed heterogeneity in the BDV species has to be considered for potential implications on diagnostic tests, control and preventive measures.

Border disease virus among chamois, Spain.

Approximately 3,000 Pyrenean chamois (Rupicapra pyrenaica pyrenaica) died in northeastern Spain during 2005-2007. Border disease virus infection was identified by reverse transcription-PCR and sequencing analysis. These results implicate this virus as the primary cause of death, similar to findings in the previous epizootic in 2001.

Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses.

This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5' biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.

Border disease virus (BDV) infections of small ruminants in Turkey: a new BDV subgroup?

Blood samples from sheep and/or goats from eight small ruminant flocks in the Turkish provinces of Aydin and Burdur were tested for the presence of Pestiviruses using an antigen-capture ELISA. From clinically affected animals, pathological and immunohistochemical findings were recorded. Post mortem examination of a virus-positive lamb showing abnormal fleece and paralysis of the hind legs revealed nonsuppurative meningoencephalomyelitis with hypomyelinogenesis. By immunohistochemistry Pestivirus antigen was detected in all parts of the brain including cerebellum, cerebral hemispheres and midbrain. Two Pestivirus isolates from a sheep and a goat kid, respectively, were isolated from samples that were positive in the antigen-capture ELISA. Genetic typing using the 5'-NTR (288bp) and N(pro) (738bp) showed that both were Border disease virus (BDV) isolates. By phylogenetic analysis, they formed a cluster clearly separated from the known clusters BDV-1 to BDV-6 and might therefore represent a new subgroup (BDV-7?). This is the first report confirming the occurrence and partial characterisation of BDV infection in small ruminants in Turkey.

Mucosal lesions in a sheep infected with the Border Disease Virus (BDV).

A 28-week-old sheep was presented at the animal hospital because of chronic emaciation, anemia and slight diarrhea. Due to poor general condition and bad prognosis the animal was euthanized and submitted for postmortem investigation. Multiple erosions and ulcerations were found in the dorsal region of the tongue, the pharynx, the hard palate, in the esophagus and the ruminal pillars. Histologically, these lesions consisted of necrosuppurative inflammation. The animal was tested positive for pestivirus antigen both by immunohistochemical and by virological examination (cell culture, antigen capture ELISA and RT-PCR). A non-cytopathic Border Disease Virus was identified, and sequencing revealed a virus belonging to the BDV-3 cluster. Based on the macroscopical, histological, immunohistological and virological results this case was diagnosed as Border Disease with mucosal lesions. This is the first report of such a case in Switzerland.

Border disease in cattle.

Within the family Flaviviridae, viruses within the genus Pestivirus, such as Border disease virus (BDV) of sheep, can cause great economic losses in farm animals. Originally, the taxonomic classification of pestiviruses was based on the host species they were isolated from, but today, it is known that many pestiviruses exhibit a broad species tropism. This review provides an overview of BDV infection in cattle. The clinical, hematological and pathological-anatomical findings in bovines that were transiently or persistently infected with BDV largely resemble those in cattle infected with the closely related pestivirus bovine viral diarrhoea virus (BVDV). Accordingly, the diagnosis of BDV infection can be challenging, as it must be differentiated from various pestiviruses in cattle. The latter is very relevant in countries with control programs to eradicate BVDV in Bovidae, as in most circumstances, pestivirus infections in sheep, which act as reservoir for BDV, are not included in the eradication scheme. Interspecies transmission of BDV between sheep and cattle occurs regularly, but BDV in cattle appears to be of minor general importance. Nevertheless, BDV outbreaks at farm or local level can be very costly.

Epidemiological study of border disease virus infection in Southern chamois (Rupicapra pyrenaica) after an outbreak of disease in the Pyrenees (NE Spain).

In 2001 and 2002, an outbreak of a previously unreported disease, associated with a border disease virus (BDV), caused high mortality in the Southern chamois (Rupicapra pyrenaica) population in the Alt Pallars-Aran National Hunting Reserve in the Catalan Pyrenees (NE Spain). Between 2002 and 2006, sera and/or tissue samples taken from 116 healthy chamois shot during the hunting season, plus 42 from chamois affected by different diseases, were studied. A blocking enzyme-immunosorbent assay (ELISA) was used to study pestivirus seroprevalence in 114 healthy hunted and 31 diseased chamois, yielding positive results in 73.7 and 22.6% of the chamois, respectively. Comparative virus neutralization tests (VNT) performed on 42 seropositive samples with 6 pestivirus strains yielded statistically higher titres to BDV Spain 97, followed by BDV chamois, BDV 137/4, BDV Moredun, Bovine Diarrhoea virus-1 (BVDV-1) NADL and BVDV-2 atypical. Virological investigations for pestivirus detection were performed using an antigen ELISA test in 82 healthy and 18 diseased chamois, RT-PCR in 16 healthy and in all diseased chamois, and virus isolation in 14 diseased chamois. No viral antigen was detected in any of the healthy animals. A pestivirus, characterized as BDV by monoclonal antibodies, was detected in the 10 chamois showing clinical signs consistent with BDV infection. Sequence analysis in the 5' untranslated region (5'-UTR) revealed that they were grouped into the BDV-4 genotype. In the remaining chamois, infectious keratoconjunctivitis, pneumonia, trauma and contagious ecthyma were diagnosed. The cause of death was unknown in five chamois. The results suggest that the infection has become endemic in the population and that it could have a significant impact on chamois population dynamics.

Prevalence of border disease virus in Spanish lambs.

The prevalence of border disease virus (BDV) viraemia in Spanish lambs was determined from 2089 sera randomly collected at two slaughterhouses in 2001 and 2003, as well as in 126 sera obtained in 2004 from a fattening unit with an acute disease problem. BDV was detected with an indirect peroxidase monolayer assay (IPMA), and for the fattening unit sera also by an antigen ELISA. A subset of sera was additionally tested for BDV antibodies. The BDV prevalence in the slaughterhouse sera was 0.24%, whereas 7.1% of randomly selected and 38.6% of sera from clinically affected lambs in the fattening unit were virus positive. Pestivirus antibodies were found in 17.6% of the slaughterhouse sera and 28.6% of those from randomly selected lambs in the fattening unit. In total, 33 virus isolates and 3 antigen positive samples were identified. Genetic typing of 5'-UTR sequences classified all 36 pestiviruses as of BDV type 4. This shows that from a low BDV prevalence in apparently healthy lambs in the entire sheep population, clinical problems associated with BDV can develop when viraemic sheep are brought into intense rearing units.

Numerical taxonomy of the genus Pestivirus based on palindromic nucleotide substitutions in the 5' untranslated region.

The palindromic nucleotide substitutions (PNS) at the three variable loci (V1, V2 and V3) in the 5' untranslated region (UTR) of Pestivirus RNA have been considered for taxonomical segregation of species, through the evaluation of 430 genomic sequences. On the basis of qualitative and quantitative secondary structure characteristics, six species have been identified: Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Classical swine fever virus (CSFV), Border disease virus (BDV), the tentative species Giraffe and a new proposed taxon named Pronghorn. The first step was qualitative and consisted in the characterization of the different positions of the three stems and loops in the 5' UTR sequences of all the strains under consideration belonging to the genus. Secondary structure sequences showing divergent base-pair combinations have been aligned for comparison. Palindromic positions have been characterized according to changes in nucleotide base-pairs identifying low-variable positions (LVP) including base-pairs present in less than 80% of the genus. The second step was quantitative, allowing the identification of genomic groups by clustering the base-pair combinations according to LVP. Relatedness among types was evaluated to identify homogeneous groups. Cross comparisons between types within the genus have been evaluated by computing the divergence percentage thus clarifying borderline and multirelated sequences.

Genetic and antigenic typing of border disease virus isolates in sheep from the Iberian Peninsula.

A selection of 10 pestiviruses isolated from sheep from the Iberian Peninsula from 2001 to 2004 was characterised at the molecular level. The 5' untranslated region (5'-UTR) and N(pro)-coding gene were amplified by the reverse transcription-polymerase chain reaction (RT-PCR) and sequenced directly from purified products. All isolates were also typed antigenically with a panel of monoclonal antibodies (mAbs) raised against representative isolates of the four recognised pestivirus species. The genetic typing placed all the isolates in a new tentative type 4 of border disease virus (BDV), which was closely related to a pestivirus recently found in Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Overall, the genotyping indicated a relatively wide diversity of the BDV type 4, which was best defined on the basis of N(pro) sequences. Antigenically, the isolates were recognised by two pan-pestivirus specific anti-NS3 mAbs, but only by some of the anti-glycoprotein specific mAbs raised against BDV, indicating partial antigenic overlap with other BDV isolates.

Development of a real time RT-PCR to detect and type ovine pestiviruses.

A real time one-step RT-PCR was designed to detect and type border disease virus (BDV), bovine viral diarrhea virus (BVDV) type 1 and BVDV type 2 in ovine samples. The real time RT-PCR was shown to behave in a linear manner and had limits of detection of 100-1000 copies of viral RNA as judged by in vitro transcribed RNA. The real time RT-PCR was validated on 50 clinical samples from UK flocks and was more sensitive than a virus isolation and a classical nested RT-PCR (nRT-PCR). The results of real time RT-PCR virus typing agreed completely with sequencing. The majority of ovine isolates were BDV; a small proportion were BVDV type 1. BVDV type 2 was not detected in any sample. This test appears reliable and can be used for the typing of ovine pestiviruses in the UK.