A phylogenetic analysis of Bovine Viral Diarrhoea Virus (BVDV) isolates from six different regions of the UK and links to animal movement data

Bovine Viral Diarrhoea Virus (BVDV) is a pestivirus which infects cattle populations worldwide and is recognised as a significant source of economic loss through its impact on health and productivity. Studies investigating the molecular epidemiology of BVDV can give invaluable information about the diversity of viral strains present in a population and this, in turn, can inform control programs, drive vaccine development and determine likely infection sources. The current study investigated 104 viral isolates from forty farms across the UK. Through phylogenetic and nucleotide sequence analysis of the 5′UTR and N^pro regions of the isolates investigated, it was determined that BVDV 1a was the predominant sub-genotype. However, BVDV 1b, 1e and 1i were also identified and, for the first time in the UK, BVDV 1d. Through analysis of animal movement data alongside the phylogenetic analysis of these BVD isolates, it was possible to link animal movements to the viral isolates present on several premises and, for the first time, begin to elucidate the routes of viral transmission. With further work, this type of analysis would enable accurate determination and quantification of the true biosecurity risk factors associated with BVDV transmission.     

Phylogenetic, antigenic and clinical characterization of type 2 BVDV from North America

Bovine viral diarrhea virus (BVDV) infection continues to have a significant impact upon US cattle producers despite the availability of more than 140 federally licensed vaccines. Detection and control is hampered by viral heterogeneity that results in differences in neutralizing epitopes, cytopathology and virulence. Recently it was found that there are two different genotypes, BVDV1 and BVDV2, among BVDV. BVDV2 isolates make up a significant proportion of the BVDV isolated in North America. Serologically BVDV2 viruses can be distinguished from BVDV1 and border disease viruses. Mab binding also distinguishes between BVDV1, BVDV2 and BDV. Like the BVDV1 viruses, BVDV2 viruses may exist as one of two biotypes, cytopathic or noncytopathic, based on their activity in cultured cells. Cytopathogenic effects on cultured cells does not correlate with virulence in vivo, as BVDV2 associated with hemorrhagic syndrome (HS) are noncytopathic. Variation among BVDV1 and BVDV2 in the 5' UTR is similar. Phylogenetic analysis and differences in virulence suggest that BVDV2 are heterogeneous. Symptoms resulting from BVDV2 infections may range from clinically inapparent to clinically severe. Recently, disease outbreaks associated with acute uncomplicated BVDV infection have been reported in the US and Canada. These outbreaks of clinically severe disease, termed HS, were all associated with viruses from the BVDV2 genotype. Not all BVDV2 isolates cause clinically severe disease. Avirulent BVDV2 isolates do exist and may predominate over virulent BVDV2 in nature. When virulent BVDV2 viruses are inoculated into calves they induce a disease characterized by fever, diarrhea, leukopenia, lymphopenia, neutropenia, thrombocytopenia, and death. Infection with avirulent BVDV2 results in a reduction of luekocytes that may be accompanied by a low-grade fever. These viruses do not cause clinical disease or a clinical leukopenia.     

Onset of protection from experimental infection with type 2 bovine viral diarrhea virus following vaccination with a modified-live vaccine

The onset of protection after the administration of a modified-live bovine viral diarrhea virus (BVDV) vaccine was determined. Protection was determined following experimental infection with a virulent type-2 BVDV (strain 1373) in cattle vaccinated 3, 5, or 7 days before BVDV infection. Protection, as measured by reduced virus shedding, lack of leukopenia, reduction in viremia, and reduced mortality, was present as early as 3 days after vaccination with a single dose of modified-live BVDV vaccine. Complete protection was obtained in cattle vaccinated 5 or 7 days before BVDV experimental infection.     

Vaccination of calves with contaminated batches of bovine herpes virus 1 vaccines did not result in infection with bovine virus diarrhea virus

The aim of the experiment was to study whether bovine herpesvirus 1 (BHV1) marker vaccine batches known to be contaminated with bovine virus diarrhoea virus (BVDV) type 1 could cause BVD in cattle. For this purpose, four groups of cattle were used. The first group (n = 4 calves, the positive control group), was vaccinated with vaccine from a batch contaminated with BVDV type 2. The second group (n = 4 calves, the negative control group), was vaccinated with vaccine from a batch that was not contaminated with BVDV. The third group (n = 39 calves), was vaccinated with a vaccine from one of four batches contaminated with BVDV type 1 (seronegative experimental group). The fourth group (n = 6 seropositive heifers), was vaccinated with a vaccine from one of three batches known to be contaminated with BVDV type 1. All cattle were vaccinated with an overdose of the BHV1 marker vaccine. At the start of the experiment, all calves except those from group 4 were seronegative for BVDV and BHV1. The calves from group 4 had antibodies against BVDV, were BVDV-free and seronegative to BHV1. After vaccination, the positive control calves became severely ill, had fever for several days, and BVDV was isolated from nasal swabs and white blood cells. In addition, these calves produced antibodies to BVDV and BHV1. No difference in clinical scores of the other groups was seen, nor were BVDV or BVDV-specific antibody responses detected in these calves; however, they did produce antibodies against BHV1. The remainder of each vaccine vial used was examined for the presence of infectious BVDV in cell culture. From none of the vials was BVDV isolated after three subsequent passages. This indicates that BVDV was either absent from the vials or was present in too low an amount to be isolated. Thus vaccination of calves with vaccines from BHV1 marker vaccine batches contaminated with BVDV type 1 did not result in BVDV infections.     

Genomic analyses of bovine viral diarrhea viruses isolated from cattle imported into Japan between 1991 and 2005

Thirty-one isolates of bovine viral diarrhea virus (BVDV) isolated within the past 15 years from imported cattle by the Japanese Animal Quarantine Service (AQS) were used in this study in which a 5'-untranslated region of each isolate was genetically analyzed. Twenty-six of the 31 isolates were classified as BVDV1 and the remainder as BVDV2. Phylogenetic analysis of the RT-PCR fragments amplified from the isolates showed the presence of viruses belonging to the BVDV1a, BVDV1b, BVDV1c, unclassified BVDV1 genotypes, and BVDV2. From the cattle of Australian origin, 16 of 17 isolates were classified as BVDV1c. This result was in agreement with a report showing that BVDV1c was a predominant subgenotype in Australia. From the cattle of North American origin, BVDV1 and BVDV2 species were both found. BVDV2 from the North American cattle was identified as the same cluster as the BVDV 890 strain, which is the prototype of BVDV2. These results suggest that the BVDVs isolated from exported cattle at the AQS reflect the predominant genotypes of BVDVs found in the exporting countries. The unclassified BVDV1 genotype of Chinese origin was in the same cluster as the ZM-95 strain, which was isolated from pigs in China. In this study, the genomic properties of 31 isolates of BVDV collected in the AQS were investigated. We concluded that isolates are genetically heterogeneous but geographically restricted. The information obtained from this report will be useful when carrying out epidemiological surveys of BVDV isolated in Japan.     

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.     

Attenuation of a virulent type 2 bovine viral diarrhea virus

The purpose of this study was to produce an attenuated bovine viral diarrhea virus (BVDV) type 2 strain as a tool for identifying potential virulence markers in the BVDV2 genome. The attenuation of the virulent strain, BVDV2-24515, was accomplished by in vivo and in vitro passage. The strain was initially used to infect an elk (Cervus elaphus) [J. Wildl. Dis. 35 (1999) 671], re-isolated at 7 days post-inoculation from serum, and then subsequently passaged 56 times in cell culture. Two groups of calves were inoculated intranasally with either BVDV2-24515 or the putative attenuated virus, designated BVDV2-LATT. Calves inoculated with BVDV2-24515 had cumulative clinical scores which ranged from 6 to 53. Clinical signs in these calves consisted of anorexia, depression, dehydration, diarrhea (±bloody), and pneumonia. Several calves developed leukocytopenia, primarily a neutrocytopenia, and presented lesions of enteritis or pneumonia at necropsy. In contrast, cattle inoculated with BVDV2-LATT had cumulative clinical scores which ranged from 0 to 2. This was not significantly different from that of controls which received no virus (range: 0–1). Calves inoculated with BVDV2-LATT produced high neutralizing antibody titers against BVDV2. Thus, in addition to its potential use as a tool for identifying virulence markers, the attenuated virus is also worthy of further study as a candidate virus for inclusion in a modified-live vaccine.     

Comparison of type I and type II bovine viral diarrhea virus infection in swine.

Some isolates of type II bovine viral diarrhea virus (BVDV) are capable of causing severe clinical disease in cattle. Bovine viral diarrhea virus infection has been reported in pigs, but the ability of these more virulent isolates of type II BVDV to induce severe clinical disease in pigs is unknown. It was our objective to compare clinical, virologic, and pathologic findings between type I and type II BVDV infection in pigs. Noninfected control and BVDV-infected 2-month-old pigs were used. A noncytopathic type I and a noncytopathic type II BVDV isolate were chosen for evaluation in feeder age swine based upon preliminary in vitro and in vivo experiments. A dose titration study was performed using 4 groups of 4 pigs for each viral isolate. The groups were inoculated intranasally with either sham (control), 10(3), 10(5), or 10(7) TCID50 of virus. The pigs were examined daily and clinical findings were recorded. Antemortem and postmortem samples were collected for virus isolation. Neither the type I nor type II BVDV isolates resulted in clinical signs of disease in pigs. Bovine viral diarrhea virus was isolated from antemortem and postmortem samples from groups of pigs receiving the 10(5) and the 10(7) TCID50 dose of the type I BVDV isolate. In contrast, BVDV was only isolated from postmortem samples in the group of pigs receiving the 10(7) TCID50 dose of the type II BVDV isolate. Type I BVDV was able to establish infection in pigs at lower doses by intranasal instillation than type II BVDV. Infection of pigs with a type II isolate of BVDV known to cause severe disease in calves did not result in clinically apparent disease in pigs.     

Sequence-optimised E2 constructs from BVDV-1b and BVDV-2 for DNA immunisation in cattle

We report DNA immunisation experiments in cattle using plasmid constructs that encoded glycoprotein E2 from bovine viral diarrhoea virus (BVDV)-1 (E2.1) and BVDV-2 (E2.2). The coding sequences were optimised for efficient expression in mammalian cells. A modified leader peptide sequence from protein gD of BoHV1 was inserted upstream of the E2 coding sequences for efficient membrane export of the proteins. Recombinant E2 were efficiently expressed in COS7 cells and they presented the native viral epitopes as judged by differential recognition by antisera from cattle infected with BVDV-1 or BVDV-2. Inoculation of pooled plasmid DNA in young cattle elicited antibodies capable of neutralising viral strains representing the major circulating BVDV genotypes.     

Evaluation of the efficacy of a modified-live combination vaccine against bovine viral diarrhea virus types 1 and 2 challenge exposures in a one-year duration-of-immunity fetal protection study

This study demonstrated that the modified-live bovine viral diarrhea virus (BVDV) type 1 and 2 fractions of a multivalent vaccine protected pregnant heifers and their fetuses against virulent BVDV types 1 and 2 challenge exposures at 370 days after vaccination. All BVDV vaccinated heifers inoculated with either BVDV type 1 or 2 at approximately 62 to 94 days of gestation delivered fetuses or calves that were negative for BVDV by ear-notch immunohistochemistry and virus isolation and serum neutralization on a prenursing serum sample. In comparison, eight of nine and 10 of 10 fetuses or calves from non-BVDV-vaccinated heifers were considered persistently infected following exposure to BVDV type 1 and type 2, respectively.     

Serological and molecular diagnosis of bovine viral diarrhoea virus and evidence of other viral infections in dairy calves with respiratory disease in Venezuela

An investigation based on 2 studies was carried out to assess the involvement of bovine virus diarrhoea virus (BVDV), bovine herpesvirus type 1 (BHV-1), and bovine respiratory syncytial virus (BRSV) in calf respiratory disease in dairy farms in Venezuela. In the first study, 8 farms were selected and paired serum samples from 42 calves with respiratory disease were tested by ELISA for antibodies to the 3 viruses. Seroconversion to BVDV, BHV-1, and BRSV was found to 5, 2, and 6 farms out of the 8, respectively. The proportion of calves that showed seroconversion to BVDV, BHV-1, and BRSV were 19%, 14%, and 26%, respectively. In the second study, another farm having previous serological evidence of BVDV infection was selected. The decline of maternal antibodies against BVDV was monitored in 20 calves and the half-life of maternal antibodies was 34 +/- 12 days presumably indicating an early natural infection with BVDV. Furthermore, sera free of BVDV antibodies that were collected in studies 1 and 2 and were assayed for the presence of BVDV by nested RT-PCR. Two BVDV strains were detected and compared to those of ruminant and porcine pestiviruses. Both strains were assigned to subgroup Ib of type I BVDV. This investigation provides information on BVDV genotypes circulating in Venezuela and may contribute to the establishment of official control programmes against the viruses studied.     

Selection of BVDV genotype II isolates using a monoclonal antibody and FACS analysis]

The infection of cattle with the bovine viral diarrhea virus (BVDV) in Germany is gaining attention and guidelines for the "protection of cattle farms against BVDV infections" were passed in 1997. New investigations about the damages induced by BVDV infections as well as the new occurrence of so-called BVDV genotypes (BVDV I and II) made the problems to become aware. The newly described BVDV genotype considerably differs both genetically and antigenetically from the up to now known BVD-viruses (BVDV I). The subdivision in BVDV genotypes I and II is based on genomic differences, which are determined by sequence analyses of different parts of the viral genome. Here, we describe the classification of BVDV in genotypes using a monoclonal antibody and indirect immunofluorescence with flow cytometry (FACS) based analysis. The suitability of the mab WB160 (Central Veterinary Laboratory, Weybridge; UK) for the classification of both BVDV-genotypes was first checked using genetically defined BVDV isolates. While all BVDV I isolates (n = 20) reacted with high fluorescence signals, the mab WB160 could not detect any of the defined BVDV II isolates (n = 20). Subsequently, 505 BVDV field strains isolated between 1993-1997 were screened for both genotypes using the mab WB160 and FACS analysis. 33 (6.5%) of the BVDV isolates were classified as BVDV II.     

Experimental persistent infection with bovine viral diarrhea virus in white-tailed deer

Bovine viral diarrhea virus (BVDV) infections cause substantial economic losses to the cattle industries. Persistently infected (PI) cattle are the most important reservoir for BVDV. White-tailed deer (Odocoileus virginianus) are the most abundant species of wild ruminants in the United States and contact between cattle and deer is common. If the outcome of fetal infection of white-tailed deer is similar to cattle, PI white-tailed deer may pose a threat to BVDV control programs. The objective of this study was to determine if experimental infection of pregnant white-tailed deer with BVDV would result in the birth of PI offspring. Nine female and one male white-tailed deer were captured and housed at a captive deer isolation facility. After natural mating had occurred, all does were inoculated intranasally at approximately 50 days of pregnancy with 10(6) CCID(50) each of a BVDV 1 (BJ) and BVDV 2 (PA131) strain. Although no clinical signs of BVDV infection were observed or abortions detected, only one pregnancy advanced to term. On day 167 post-inoculation, one doe delivered a live fawn and a mummified fetus. The fawn was translocated to an isolation facility to be hand-raised. The fawn was determined to be PI with BVDV 2 by serial virus isolation from serum and white blood cells, immunohistochemistry on skin biopsy, and RT-PCR. This is the first report of persistent infection of white-tailed deer with BVDV. Further research is needed to assess the impact of PI white-tailed deer on BVDV control programs in cattle.     

Bovine herpes virus expressing envelope protein (E2) of bovine viral diarrhea virus as a vaccine candidate.

The gene encoding the envelope protein (E2) of bovine viral diarrhea virus (BVDV) was expressed under the thymidine kinase (TK) promoter of Korean bovine herpesvirus 1 (BHV-1) isolate. Thymidine kinase negative (TK-) BHV-1 recombinants expressing E2 of BVDV were constructed and the expression of E2 was identified by immunofluorescence and Western blotting. Compared to wild type BHV-1, the recombinant BHV-1 had a delayed cytopathogenic effect in cells. The immunogenicity of the recombinant BHV-1 was examined in guinea pigs and cattle. Although an increase in body temperature was detected for a few days, the inoculated cattle returned to normal temperature with the development of neutralizing antibodies to BVDV.     

Prevention of persistent infection in calves by vaccination of dams with noncytopathic type-1 modified-live bovine viral diarrhea virus prior to breeding

OBJECTIVE: To determine the ability of a modified-live virus (MLV) bovine viral diarrhea virus (BVDV) type 1 (BVDV1) vaccine administered to heifers prior to breeding to stimulate protective immunity that would block transmission of virulent heterologous BVDV during gestation, thus preventing persistent infection of a fetus. ANIMAL: 40 crossbred Angus heifers that were 15 to 18 months old and seronegative for BVDV and 36 calves born to those heifers. PROCEDURE: Heifers were randomly assigned to control (n = 13) or vaccinated (27) groups. The control group was administered a multivalent vaccine where-in the BVDV component had been omitted. The vaccinated heifers were administered a single dose of vaccine (IM or SC) containing MLV BVDV1 (WRL strain). All vaccinated and control heifers were maintained in pastures and exposed to BVDV-negative bulls 21 days later. Thirty-five heifers were confirmed pregnant and were challenge exposed at 55 to 100 days of gestation by IV administration of virulent BVDV1 (7443 strain). RESULTS: All control heifers were viremic following challenge exposure, and calves born to control heifers were persistently infected with BVDV. Viremia was not detected in the vaccinated heifers, and 92% of calves born to vaccinated heifers were not persistently infected with BVDV. CONCLUSIONS AND CLINICAL RELEVANCE: These results document that vaccination with BVDV1 strain WRL protects fetuses from infection with heterologous virulent BVDV1.     

Epidemiology and genetic characterization of BVDV,BHV-1, BHV-4, BHV-5 and Brucella spp. infections in cattle in Turkey

The aim of the study was to determine the epidemiological data of bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV-1), bovine herpesvirus-4 (BHV-4), bovine herpesvirus-5 (BHV-5) and Brucella–associated cattle that were previously reported to have abortion and infertility problems in Ankara, Corum, Kirikkale and Yozgat provinces, Turkey. Whole blood and sera samples were obtained from 656 cattle, and antibodies against Brucella spp. were detected in 45 (6.86%) and 41 (6.25%) animals by Rose Bengal plate and serum tube agglutination tests, respectively. The seropositivity rates against BVDV, BHV-1 and BHV-4 were 70.89%, 41.3% and 28.78%, respectively. RT-PCR and PCR were performed to detect RNA and DNA viruses in blood samples, respectively. The BVDV 5′-untranslated region and BHV-1 gB gene detected in this study were phylogenetically analyzed. The BVDV strains analyzed in this study were closely related to those previously reported from Turkey. The nucleotide sequence from the BHV-1 strain detected in this study is the first nucleotide sequence of BHV-1 circulating in this area of Turkey deposited in the GenBank. The presence of Brucella spp. and prevalence of BHV-1, BHV-4 and BVDV in cattle should be further investigated throughout these regions.     

Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections

Infections with bovine virus diarrhoea virus (BVDV) are widespread throughout the world. Although the prevalence of infection varies among surveys, the infection tends to be endemic in many populations, reaching a maximum level of 1-2% of the cattle being persistently infected (PI) and 60-85% of the cattle being antibody positive. Persistently infected cattle are the main source for transmission of the virus. However, acutely infected cattle as well as other ruminants, either acutely or persistently infected, may transmit the virus. Transmission is most efficient by direct contact. However, as infections have been observed in closed, non-pasturing herds, other transmission routes seem likely to have some practical importance. Differences in BVDV prevalence among regions or introduction of virus in herds previously free of BVDV are often associated with particular epidemiological determinants such as cattle population density, animal trade and pasturing practices. However, on a few occasions there have been no obvious explanations for infection of individual herds. Estimates of economic losses due to BVDV infection vary depending on the immune status of the population and the pathogenicity of the infecting virus strains. Introduction of the infection into a totally susceptible population invariably causes extensive losses until a state of equilibrium is reached. Infection with highly virulent BVDV strains causing severe clinical signs and death after acute infection gives rise to substantial economical losses. At an estimated annual incidence of acute infections of 34%, the total annual losses were estimated as US$ 20 million per million calvings when modeling the losses due to a low-virulent BVDV strain. At the same incidence of infection, the losses due to a high-virulent BVDV strain were estimated as US$ 57 million per million calvings. Low-virulent BVDV infections caused maximum losses at an incidence of 45%, whereas high-virulent BVDV infections caused maximum losses at an incidence of 65%. Thus, cost-benefit analyses of control programs are highly dependent on the risks of new infections under different circumstances and on the strains of the virus involved.