Identification and molecular characterization of border disease virus (BDV) from sheep in India

Pestiviruses isolated from sheep and goats in India thus far have been bovine viral diarrhoea virus 1 (BVDV-1) or BVDV-2. During routine genetic typing of pestiviruses in the years 2009-10, border disease virus (BDV) was detected in eight Indian sheep of a flock showing clinical signs of BD by real time RT-PCR. All the samples yielded positive virus isolates in cell culture but were found negative by a BVDV antigen ELISA. A representative BDV isolate was characterized at genetic and antigenic level. Phylogenetic analysis carried out in 5′-UTR, N^pro and E2 regions of genome typed the Indian BDV isolate as BDV-3. A more detailed analysis in N^pro and entire region coding structural proteins showed that the N^pro (168), C (100 aa), E^rns (227 aa), E1 (195 aa) and E2 (373 aa) proteins were of size characteristic for BDV reference strain X818. Antigenic differences were evident between the BDV-3 isolate and previously reported BDV-1, BDV-5 and BDV-7 strains. Although origin of BDV-3 in India is not clear, the results reflect probable introduction through trade in sheep between India and other countries or BDV-3 may be more widely distributed. Additionally, this study suggests that for diagnosis of BDV infection, the commercial BVDV Ag-ELISA should be used with caution. This is the first identification of BDV in sheep in India which highlights the need for continued pestivirus surveillance and assessing its impact on sheep and goat production.     

Identification of bovine viral diarrhea virus type 1 in yaks (Bos poephagus grunniens) in the Himalayan region

Since cattle are widely infected by bovine viral diarrhea virus (BVDV) in India, we searched for pestivirus infection in yaks. Of 71 pure and crossbred yaks from Himalayan region, pestivirus antigen was detected by Ag-ELISA in three animals. Pestivirus in leukocyte and cell culture isolated virus samples originating from positive yaks was also confirmed by RT-PCR using panpestivirus specific primers selected from 5'-untranslated region (5' UTR). The 5' UTR, N(pro) and E2 regions were sequenced and used for genetic typing. Phylogenetic analysis revealed that pestiviruses detected in three Himalayan yaks were similar genetically, belonging to BVDV-1. Antigenic characterisation of yak pestivirus also confirmed the typing as BVDV-1. This is the first report on the identification of BVDV type 1 in yaks.     

Pestiviruses in wild animals

Pestiviruses are not strictly host-species specific and can infect not only domestic but also wild animals. The most important pestivirus, CSFV, infects domestic pigs and wild boars, which may cause a major problem for successful CSFV eradication programmes. Mainly BVDV specific antibodies have been reported in captive and free-living animals. Virus has been isolated from some of these animal species, but since BVDV can contaminate cell cultures and foetal calf serum, early reports of BVDV isolation have to be considered with caution. Genetic typing of early pestivirus isolates from wild species revealed that the majority were BVDV-1. Of the pestiviruses identified so far three species (CSFV, BVDV-1, giraffe pestivirus) and three genotypes (BDV-2, BDV-4, pronghorn) appear to circulate in wildlife animal populations. The potential for pestiviruses to spread between farm animals and free-living animals is discussed as are epidemiological and technical problems, and the future direction of research.     

Serological survey for antibodies against pestiviruses in sheep in Austria

The prevalence of antibodies to pestiviruses was investigated in 4931 sheep, in 377 flocks, in four federal states of Austria, by means of an indirect elisa that detected antibodies to Border disease virus (BDV) and bovine viral diarrhoea virus (BVDV). The mean flock prevalence was 62.9 per cent and the mean individual prevalence was 29.4 per cent. Comparative neutralisation studies on the elisa-positive samples with BVDV type 1 (BVDV-1), BVDV type 2 (BVDV-2) and BDV recorded 336 samples with higher titres (more than four times average) to BVDV-1, three samples with higher titres to BVDV-2 and 55 samples with higher titres to BDV. The other samples did not show clear differences in antibody titres against the strains of pestivirus tested because of cross-reactions. The seroprevalence of pestiviruses in sheep was significantly higher on farms with cattle. There were significant regional differences between the prevalences in flocks and individual sheep, the highest prevalences being in the region of Austria where communal alpine pasturing of sheep, goats and cattle is an important part of farming.     

Genetic and antigenic characterization of bovine viral diarrhoea virus type 2 isolated from cattle in India

Previous studies have shown that bovine viral diarrhoea virus type 1 (BVDV-1) subtype b is predominantly circulating in Indian cattle. During testing for exotic pestiviruses between 2007 and 2010, BVDV-2 was identified by real time RT-PCR in two of 1446 cattle blood samples originating from thirteen states of India. The genetic analysis of the isolated virus in 5′ UTR, N^pro, entire structural genes (C, E^rns, E1 and E2), nonstructural genes NS2-3 besides 3′ UTR demonstrated that the nucleotide and amino acid sequences showed highest similarity with BVDV-2. The entire 5′ and 3′ UTR consisted of 387 and 204 nucleotides, respectively, and an eight nucleotide repeat motif was found twice within the variable part of 3′ UTR that may be considered as a characteristic of BVDV-2. The phylogenetic analysis revealed that the cattle isolate and earlier reported goat BVDV-2 isolate fall into separate clades within BVDV-2a subtype. Antigenic typing with monoclonal antibodies verified the cattle isolate also as BVDV-2. In addition, cross-neutralization tests using antisera raised against Indian BVDV strains circulating in ruminants (cattle, sheep, goat and yak) displayed significant antigenic differences only between BVDV-1 and BVDV-2 strains. This is the first identification of BVDV-2 in Indian cattle that may have important implications for immunization strategies and molecular epidemiology of BVD.     

Genetic diversity of ruminant <Emphasis Type="Italic">Pestivirus</Emphasis> strains collected in Northern Ireland between 1999 and 2011 and the role of live ruminant imports

Background

The genus pestivirus within the family Flaviviridae includes bovine viral diarrhoea virus (BVDV) types 1 and 2, border disease virus (BDV) and classical swine fever virus. The two recognised genotypes of BVDV are divided into subtypes based on phylogenetic analysis, namely a-p for BVDV-1 and a-c for BVDV-2.

Methods

Three studies were conducted to investigate the phylogenetic diversity of pestiviruses present in Northern Ireland. Firstly, pestiviruses in 152 serum samples that had previously tested positive for BVDV between 1999 and 2008 were genotyped with a RT-PCR assay. Secondly, the genetic heterogeneity of pestiviruses from 91 serum samples collected between 2008 and 2011 was investigated by phylogenetic analysis of a 288 base pair portion of the 5’ untranslated region (UTR). Finally, blood samples from 839 bovine and 4,437 ovine animals imported in 2010 and 2011 were tested for pestiviral RNA. Analysis of animal movement data alongside the phylogenetic analysis of the strains was carried out to identify any links between isolates and animal movement.

Results

No BVDV-2 strains were detected. All of the 152 samples in the first study were genotyped as BVDV-1. Phylogenetic analysis indicated that the predominant subtype circulating was BVDV-1a (86 samples out of 91). The remaining five samples clustered close to reference strains in subtype BVDV-1b. Out of the imported animals, 18 bovine samples tested positive and 8 inconclusive (Ct ≥36), while all ovine samples were negative. Eight sequences were obtained and were defined as BVDV-1b. Analysis of movement data between herds failed to find links between herds where BVDV-1b was detected.

Conclusion

Given that only BVDV-1a was detected in samples collected between 1968 and 1999, this study suggests that at least one new subtype has been introduced to Northern Ireland between 1999 and 2011 and highlights the potential for importation of cattle to introduce new strains.

     

Genetic comparison of ovine and bovine pestiviruses

Viral RNA oligonucleotide fingerprinting was used to compare genetic relationship among pestiviruses originating from ovine or bovine host species. Ovine pestiviruses, including reference border disease virus and 2 border disease isolates originating from natural pestivirus infections of sheep, appeared to have a more distant genetic relationship among themselves than with certain bovine pestiviruses. A closer genetic relatedness was evident between border disease virus and 3 noncytopathic bovine pestiviruses, including Draper bovine viral diarrhea virus (BVDV), a BVDV isolate that originated from aborted bovine fetuses, and a virus that was isolated from the serum of a calf that had a chronic BVDV infection. Four noncytopathic bovine viruses, including Draper BVDV and 3 field isolates, were closely related. Reference Oregon C24V BVDV, a cytopathic virus, was closely related to only 1 of the 7 noncytopathic viruses in this study.     

Variation in pestivirus growth in testicle primary cell culture is more dependent on the individual cell donor than cattle breed

The causes of bovine respiratory disease complex (BRDC) are multifactorial and include infection with both viral and bacterial pathogens. Host factors are also involved as different breeds of cattle appear to have different susceptibilities to BRDC. Infection with bovine pestiviruses, including bovine viral diarrhea virus 1 (BVDV1), BVDV2 and ‘HoBi’-like viruses, is linked to the development of BRDC. The aim of the present study was to compare the growth of different bovine pestiviruses in primary testicle cell cultures obtained from taurine, indicine and mixed taurine and indicine cattle breeds. Primary cells strains, derived from testicular tissue, were generated from three animals from each breed. Bovine pestivirus strains used were from BVDV-1a, BVDV-1b, BVDV-2a and ‘HoBi’-like virus. Growth was compared by determining virus titers after one passage in primary cells. All tests were run in triplicate. Virus titers were determined by endpoint dilution and RT-qPCR. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by the Tukey’s Multiple Comparison Test (P?0.05). Significant differences in virus growth did not correlate with cattle breed. However, significant differences were observed between cells derived from different individuals regardless of breed. Variation in the replication of virus in primary cell strains may reflect a genetic predisposition that favors virus replication.     

Prevalence of antibodies to pestiviruses in goats in Austria

Serological investigations were carried out to determine the prevalence of pestiviral infections in goats in Austria, and to investigate the possible relations to herd management practices. The prevalence of antibodies to pestiviruses was investigated in 549 goats in 80 flocks from four regions of Austria. The examination for antibodies was performed using an indirect enzyme-linked immunosorbent assay detecting antibodies to the border disease virus (BDV) and bovine viral diarrhoea virus (BVDV). The observed individual prevalence was 11.5% and the flock prevalence was 31.3%. Comparative neutralization studies on the 63 seropositive samples with BVDV-1, BVDV-2 and the BDV yielded in 32 samples higher titres (> or =4-fold) to BVDV-1 and in two samples to BDV. The remaining samples did not show distinct differences in antibody titres against the pestivirus strains tested because of the cross-reactions. There was a significant (P < 0.05) association between the prevalence of antibodies to pestiviruses and the presence of cattle on the farm. Significant (P < 0.05) geographical variations in individual prevalence were obtained, ranging from 3.5% in lower Austria to 20.2% in Vorarlberg.     

Kinetics of single and dual infection of calves with an Asian atypical bovine pestivirus and a highly virulent strain of bovine viral diarrhoea virus 1

Atypical bovine pestiviruses related to bovine viral diarrhoea virus (BVDV) have recently been detected in cattle from South America, Asia and Europe. The purpose of this study was to compare the clinical and virological aspects of dual infection with BVDV-1 (Horton 916) and an Asian atypical bovine pestivirus (Th/04_KhonKaen) in na?ve calves, in comparison to single infections. Milder clinical signs were observed in the animals infected with single Th/04_KhonKaen strain. Leukocytopenia and lymphocytopenia were observed in all infected groups at a similar level which correlated with the onset of viraemia. Co-infection with both viruses led to prolonged fever in comparison to single strain inoculated groups and simultaneous replication of concurrent viruses in blood and in the upper respiratory tract. Following the infections all the calves seroconverted against homologous strains. Atypical pestiviruses pose a serious threat to livestock health and BVDV eradication, since they may have the potential to be widely spread in cattle populations without being detected and differentiated from other BVDV infections.     

Virus recovery and full-length sequence analysis of atypical bovine pestivirus Th/04_KhonKaen

Phylogenetic analysis of recently identified “atypical” bovine pestiviruses, performed based on different gene regions, has revealed unclear relationships with other established species, therefore, their phylogenetic position could not be determined so far. In this study, the atypical pestivirus Th/04_KhonKaen was recovered from serum of a naturally infected calf and the complete genome sequence was determined and analysed, as means to define its position. The viral genome is 12,337 nucleotides (nt) long, and comprises a 5′-UTR of 383 nt, a 3′-UTR of 254 nt and an open reading frame of 11,700 nt, without duplication of viral sequences or insertions of cellular sequences. The phylogenetic analyses of the full-length sequence, performed by Neighbor-joining, Maximum likelihood, and the Bayesian approach, unanimously placed Th/04_KhonKaen in a single lineage, distinct from the established pestivirus species, and close to bovine viral diarrhea virus types 1 and 2. Furthermore, Th/04_KhonKaen and two previously reported atypical pestiviruses D32/00_‘HoBi’ and CH-KaHo/cont formed a well-supported monophyletic clade in trees based on the complete N^pro and E2 gene regions. The finding provides conclusive classification of the Th/04_KhonKaen virus and confirms the standing of the “atypical” bovine pestiviruses as a novel pestivirus species.     

Heterogeneity of ruminant pestiviruses: academic interest or important basis for the development of vaccines and diagnostics?

Pestiviruses cause economically important diseases of farm animals. Members of the Pestiviruses are bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, classical swine fever virus (CSFV) and border disease virus (BDV). Phylogenetic analyses based on the entire nucleic acid sequence encoding the Npro allow a statistically significant segregation of established species and of subgroups within the species. BVDV-1 strains isolated in Germany can be associated with at least five different subgroups. In contrast all BVDV-2 isolates detected in Germany so far are closely related, belonging to one subgroup. A group of virus isolates from sheep and zoo animals is clearly different from established pestivirus species and can be designated as BDV-2. Antigenetic relatedness of pestiviruses was studied using defined virus isolates and antisera in cross-neutralization assays. Six antigenic groups were distinguished corresponding to the genetic clusters BVDV-1, BVDV-2, CSFV, BDV-1, BDV-2 and Giraffe-1. A significant antigenic difference was also observed between members of subgroups 1a and 1b of BVDV-1. Studies on the genetic and antigenic heterogeneity of pestiviruses are important for the development of new vaccines, diagnostic tests and for eradication programs.     

Genetic typing of pestiviruses from New Zealand

AIM: To investigate the genetic type of 20 pestiviruses collected from New Zealand over the period 1967-97. METHODS: The pestiviruses were genetically typed by the sequencing of polymerase chain reaction (PCR) products. The primers selected were from the 5'-untranslated region (5'-UTR) of the pestivirus genome and consistently amplified a 288 bp fragment from all samples tested. RESULTS: Sequencing and phylogenetic analysis of PCR products revealed that all samples obtained from cattle represented bovine viral diarrhoea (BVDV) type I. Two sheep isolates were characterised as border disease virus (BDV). A pestivirus isolated from foetal calf serum of USA origin was typed as BVDV type II. CONCLUSIONS: The findings show that the evolution of pestiviruses in New Zealand has been similar to Europe and North America, indicating the occurrence of a conservative phylogenetic branch of BVDV type I in cattle and the presence of BDV in the sheep population.     

Reactivity and prevalence of neutralizing antibodies against Japanese strains of bovine viral diarrhea virus subgenotypes

Bovine viral diarrhea virus (BVDV) field isolates show genetic and antigenic diversity. At least 14 subgenotypes of BVDV-1 and 4 of BVDV-2 have been identified in Artiodactyla worldwide. Of these, 6 subgenotypes of BVDV-1 and 1 of BVDV-2 have been isolated in Japan. Previously, we reported that each subgenotype virus expresses different antigenic characteristics. Here we investigated the reactivity of neutralizing antibodies against representative strains of Japanese BVDV subgenotypes using sera from 266 beef cattle to estimate the prevalence of this epidemic virus among cattle in Japan. Antibody titers at concentrations at least 4-fold higher than antibodies against other subgenotype viruses were considered subgenotype specific. Subgenotype-specific antibodies were detected from 117 (80.7%) of 145 sera samples (69.7% against BVDV-1a, 1.4% against BVDV-1b, 8.3% against BVDV-1c, and 1.4% against BVDV-2a). The results suggest that neutralization tests are useful in estimating currently epidemic subgenotypes of BVDV in the field.     

Multiple diagnostic tests to identify cattle with Bovine viral diarrhea virus and duration of positive test results in persistently infected cattle

Several tests for Bovine viral diarrhea virus (BVDV) were applied to samples collected monthly from December 20, 2005, through November 27, 2006 (day 0 to day 342) from 12 persistently infected (PI) cattle with BVDV subtypes found in US cattle: BVDV-1a, BVDV-1b, and BVDV-2a. The samples included clotted blood for serum, nasal swabs, and fresh and formalin-fixed ear notches. The tests were as follows: titration of infectious virus in serum and nasal swabs; antigen-capture (AC) enzyme-linked immunosorbent assay (ELISA), or ACE, on serum, nasal swabs, and fresh ear notches; gel-based polymerase chain reaction (PCR) testing of serum, nasal swabs, and fresh ear notches; immunohistochemical (IHC) testing of formalin-fixed ear notches; and serologic testing for BVDV antibodies in serum. Of the 12 animals starting the study, 3 died with mucosal disease. The ACE and IHC tests on ear notches had positive results throughout the study, as did the ACE and PCR tests on serum. There was detectable virus in nasal swabs from all the cattle throughout the study except for a few samples that were toxic to cell cultures. The serum had a virus titer ≥ log₁₀ 1.60 in all samples from all the cattle except for 3 collections from 1 animal. Although there were several equivocal results, the PCR test most often had positive results. The BVDV antibodies were due to vaccination or exposure to heterologous strains and did not appear to interfere with any BVDV test. These findings illustrate that PI cattle may be identified by several tests, but differentiation of PI cattle from cattle with acute BVDV infection requires additional testing, especially of blood samples and nasal swabs positive on initial testing. Also, calves PI with BVDV are continual shedders of infectious virus, as shown by the infectivity of nasal swabs over the 11-mo study.     

Seroprevalence of antibodies to ruminant pestiviruses in sheep and goats in Tyrol (Austria)

In this study 2058 blood samples from sheep of 150 flocks from the province of Tyrol were tested by ELISA and serum neutralisation tests for antibodies to ruminant pestiviruses. In the ELISA, positive results were obtained with 34.9% of individual sheep sera and in 89.3% of the sheep flocks. The prevalence in sheep and sheep flocks varied according to areas. Seroprevalence of pestiviruses was significantly (p < 0.05) higher in small ruminants pastured during summertime on the Alps. Comparative neutralisation studies were carried out on all positive blood samples with BVDV-1, BVDV-2 and BDV. 443 seropositive sheep samples exhibited clearly the highest titre against one of the pestivirus strains tested. 413 revealed the highest titres (2 or more fold) to BVDV-1, 6 to BVDV-2 and 24 to BDV. In some areas a very high rate of pestivirus seroprevalence could be found. This fact could be harmful to the BVDV-Elimination and Controlling Program in cattle in Austria.     

Bovine viral diarrhoea virus is internalized by clathrin-dependent receptor-mediated endocytosis

Bovine viral diarrhoea virus (BVDV) is a pestivirus within the family Flaviviridae. In contrast to the members of the genus flavivirus, nothing is known about the viral entry route for pestiviruses. In this study, the process of BVDV infection following attachment to the cell surface was examined. BVDV clearly co-localizes with clathrin, with early endosome antigen-1 (EEA-1), an early endosome marker, and also with lysosomal-associated membrane protein-2 (LAMP-2), a lysosomal marker. BVDV internalization is inhibited by compounds that block clathrin- but not caveolae-dependent endocytosis. These findings demonstrate that BVDV enters the cells via the clathrin-coated pit pathway.