Cellular insertions in the NS2-3 genome region of cytopathic bovine viral diarrhoea virus (BVDV) isolates

When compared to noncytopathic (ncp) bovine viral diarrhoea virus (BVDV), some cytopathic (cp) BVDV contain additional sequences in the NS2-3 genomic region. One of these insertions, which is 270 nucleotides long and of host origin (cINS), was first described for strain NADL. To find out how frequently this type of insertion occurs in other cp BVDV, 32 cp BVDV field isolates and the BVDV reference cp strain Indiana were screened using RT-PCR which detected cINS in NADL. For most cp viruses an RT-PCR product of 402bp indicated the presence of NS2-3 genes without insertions. In addition, one or two DNA fragments, around 600-850bp in size, were amplified from the genomes of 13 cp viruses indicating the presence of insertions. Sequencing of the PCR products, i.e. 402bp DNA fragment (with no insertion) and longer fragments (with insertion) revealed the location of the insertions in the NS2-3 coding region of eight cp BVDV genomes. All of the insertions were confirmed to be of the cINS type and were located in a very similar position to that found previously in the NADL genome. They were in the same reading frame as the viral polypeptide and they encoded 90-140 amino acids. The 5' and 3' ends of the insertions were different in most of the cp isolates studied. Interestingly, a 14-amino-acid stretch at the 5'-end of the insertion in the cp 5569 isolate as well as 15 amino acids at the 3'-end of the insertion in the cp 5.19516 isolate were not homologous to the cINS sequence. No significant matches for these stretches were found in the EMBL and Swissprot databases.     

Effect of the bovine viral diarrhoea virus (BVDV) infection on dairy calf rearing

The aim of this study was to compare the cumulative incidence of mortality, clinical diarrhoea and respiratory disease in calves, during their first six months of age, in herds with different bovine viral diarrhoea virus (BVDV) infection status. Calves’ health indicators were tested by comparing proportions in 101 farms with dissimilar infection condition. The results indicate that there was a significant relationship between the BVDV status (actively infected herd or not) and the cumulative incidence of mortality and respiratory disorders.     

Principles for eradication of bovine viral diarrhoea virus (BVDV) infections in cattle populations

Systematic eradication of BVDV without vaccination started in Scandinavia in 1993. In principle, the schemes include; (1) identification of non-infected and infected herds using different combinations of serological herd tests such as bulk milk tests and spot tests (sample of animals in a certain age), (2) monitoring/certification of non-infected herds by repeated sampling, applying one of the above-mentioned methods and (3) virus clearance in infected herds aimed at removing persistently infected (PI) animals in a cost- and time-efficient manner. In the virus clearance protocol described, an initial test is performed on all animals with subsequent follow-up of calves born as well as of dams seronegative in the initial test. It is generally recommended to perform an initial antibody test on all samples. This should be done not only to screen for seronegative animals on which virus isolation should be attempted (i.e. possible PI animals), but more in order to identify non-immune animals in reproductive age, that is, the key animals in herd-level persistence of infection. In Sweden, a common finding has been self-clearance, where the infection ceases without any other intervention than controlled introduction of new animals. Other epidemiological observations concern the course of events following virus introduction. Important risk factors for spreading BVDV are discussed, where livestock trade is perceived as the most central to control. Live vaccines, imported semen and embryos constitute special hazards, since they may act as vehicles for the introduction of new BVDV strains. The importance of making farmers aware of herd biosecurity and their own responsibility for it is stressed, and in order to maintain a favourable situation after a scheme has been concluded, effort must be put into establishing such a persisting attitude in the farming community.     

Epitopic characterisation of Turkish bovine viral diarrhoea virus (BVDV) isolates using monoclonal antibodies

In this study, 15 bovine viral diarrhoea viruses (BVDV) isolated from the field in Turkey were characterised for their biotype, cloned and eventually analysed for their epitopic composition in terms of glycoprotein E2. Immunoplaque assay, plaque assay, limiting dilution and streptavidin-biotin-peroxidase techniques were used for biotype characterisation, cloning of cytopathic (cp) and noncytopathic (ncp) biotypes and epitope analysis, respectively. While 14 out of 15 BVDV isolates were distinguished as ncp biotype, 1 isolate was found to be containing both biotypes (cp + ncp). According to the reactivity patterns of isolates with 15 monoclonal antibodies, 4 different antigenic groups could be formed. There were no antigenic differences between the isolates derived from the same animal with various time intervals. On the other hand, biotype clones isolated from the same animal exhibited difference in one epitope. This is the first study describing antigenic characterisation of BVDV field isolates in Turkey.     

Natural changes in the spread of bovine viral diarrhoea virus (BVDV) among Estonian cattle

The results of a survey conducted during 1993-2000 to study the spread of bovine viral diarrhoeal virus (BVDV) among Estonian cattle are presented. The BVDV infection status of a representative random sample of cattle herds housing 20 or more dairy cows was established to estimate the prevalence of herds with active BVDV infection [potentially having persistently infected (PI) cattle--suspect PI herds]. The herds investigated comprised approximately 70% of all Estonian dairy cows. The BVDV infection status was established in 315-350 herds (making the sampling fraction about 20%) during three sampling periods: 1993-95, 1997-98, 1999-2000. BVDV antibodies were detected in herd bulk milk samples and/or sera from young stock by a liquid-phase-blocking enzyme-linked immunosorbent assay developed in the Danish Veterinary Institute for Virus Research. The results of the survey demonstrate the reduction in the prevalence of herds with active BVDV infection in the studied fraction of the Estonian cattle population. During the first sampling period (1993-95) a prevalence of 46% (+/- 5%) for suspect PI herds was observed, during the second sampling period this prevalence was 16% (+/- 3%) and in the third period it was 18% (+/- 3%). As there is no control programme for BVDV in Estonia, the observed changes reflect the natural course of the infection in the study population. A possible cause for these changes is the decreased trade in breeding animals as a result of the economic difficulties present in cattle farming during the study period. The farming practices (most large herds are managed as closed herds) and the low density of cattle farms have obviously facilitated the self-clearance of herds from the BVDV infection, diminishing the new introduction of infection into the herds.     

Evaluation of long-term antibody responses to two inactivated bovine viral diarrhoea virus (BVDV) vaccines

The aim of the present study was to determine the serological response of heifers after vaccination with two inactivated bovine viral diarrhoea virus (BVDV) vaccines by means of various ELISA tests. Three dairy farms were selected from the Galicia region of Spain. In each herd, a batch of heifers to be vaccinated for the first time was selected and followed for 15 months. Heifers from farm 1 (n = 25) were vaccinated with Vaccine A, whereas heifers from farm 2 (n = 16) were vaccinated with Vaccine B. Heifers from farm 3 (n = 17), where no BVDV vaccines were used, acted as controls. Blood samples were analyzed periodically for BVDV antibodies, using five commercial ELISAs, based on BVDV p80 antigen or whole virus.At the end of the study, none of the animals vaccinated with Vaccine A seroconverted according to p80 antibody status, whereas up to 80% tested positive by ELISA against whole virus antigen. For the animals vaccinated with Vaccine B, 2/16 animals seroconverted according to p80 antibody ELISAs, whereas all had seroconverted according to the ELISA against whole virus antigen. In most cases, based on the use of ELISAs to detect specific antibodies against the p80 protein, at 15 months post-vaccination with inactivated BVDV vaccines the responses did not seem to interfere with detection of antibody to BVDV infection. However, the finding of a small proportion of vaccinated animals seropositive against BVDV p80 antigen suggests that antibodies that interfere with diagnosis of BVDV infection within the herd could exist, even when using p80 ELISAs.     

Survivorship of animals persistently infected with bovine virus diarrhoea virus (BVDV)

The survivorship of animals persistently infected with bovine virus diarrhoea virus was studied in ten Danish dairy herds. The ages of 34 persistently infected animals were compared with the ages of non-persistently infected animals in the herds in a cross-sectional study in which the risk rate of removal of animals in the two groups within 1 year was estimated from an exponential probability function of the age. In a cohort study, the 34 persistently infected animals were followed from the date of the initial blood test and onwards and the risk rates of dying or being slaughtered due to unthriftiness or either of these events were calculated.

The attributable risk of leaving the herds within 1 year was 0.35 among persistently infected animals. The risk rate of dying or being slaughtered due to unthriftiness within 1 year was 0.28 and 0.31, respectively; the risk rate of either of these events was 0.5.     

Cytopathogenicity markers in the genome of Hungarian cytopathic isolates of bovine viral diarrhoea virus

Since genetic recombination is a major factor in the evolution of the cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) biotypes, in this study the cytopathogenicity markers were investigated in the genomes of two cp BVDV strains recently isolated from mucosal disease (MD) cases in Hungary. In the genome of strain H4956, a Jiv-like insertion was found similar to those described in reference strain NADL and in other BVDV 1, BVDV 2 and border disease virus (BDV) strains. The 133 amino acid Jiv-like sequence is inserted at nucleotide position 4984 (amino acid position 1533), 9 nucleotides upstream of that of strain NADL. The insertion showed 96% amino acid sequence identity with the cellular Jiv protein. In the genome of cp BVDV strain H115/PCR, an ubiquitin-containing duplication was found. The duplicated sequence started at nucleotide position 7978 (amino acid 2531) in the NS4B gene. The duplication contained a complete ubiquitin monomer of 76 amino acids and the complete NS3 gene starting at nucleotide position 5153 (amino acid 1589), which corresponds to the first N-terminal amino acid of NS3. The duplication was located further downstream of the known ubiquitin-containing genomic regions of cp BVDV strains, and it consisted of the shortest inserted nucleotide sequence. The insertions and duplication of strains H4956 and H115/PCR further confirmed that recombinations occurring at positions A and B are the most common mechanisms leading to the development of BVDV cytopathogenicity.     

Fatal congenital anaplasmosis associated with bovine viral diarrhoea virus (BVDV) infection in a crossbred calf

Clinical disease resulting from the vertical transmission of Anaplasma marginale has only been reported on 5 occasions despite studies demonstrating successful in utero transmission. During the reported experimental induction of congenital anaplasmosis in calves, the outcome was variable but mostly led to inapparent or mild infection. There are previous case reports of fatal congenital anaplasmosis following natural infection. The clinical findings in a 2-day-old calf presented to the Onderstepoort Veterinary Academic Hospital with clinical signs of congenital anaplasmosis, which was unresponsive to treatment, are described. Subsequent post mortem diagnostic tests revealed that this calf was co-infected with bovine viral diarrhoea virus (BVDV). It is postulated that immunosuppression resulting from BVDV infection predisposed to severe, fatal anaplasmosis in this calf.     

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.     

Bovine viral diarrhoea virus (BVDV) diversity and vaccination. A review

BVDV is associated with a range of economically important clinical diseases including reproductive disorders and acute fatal haemorrhagic disease in cattle industry. Vaccination is still the most important control strategy for controlling BVDV infections in many countries of the world. The existence of great genetic and antigenic diversity of BVDV isolates is very important concern for BVDV vaccine development and protective efficacy of current vaccines. In this review, the protective efficacies of the selected examples of BVDV vaccines with regard to BVDV diversity and the novel marker vaccine development studies are discussed.     

Expression of the E2 envelope glycoprotein of bovine viral diarrhoea virus (BVDV) elicits virus-type specific neutralising antibodies

A region of genome from the NADL strain of BVDV corresponding to the coding sequence for the E2 glycoprotein has been molecularly cloned using RT-PCR. The viral cDNA sequence was used to construct vaccinia virus recombinants that expressed either the entire E2 coding sequence or fragments of it. These recombinants were used to immunise mice of three H-2 haplotypes to investigate their ability to elicit a neutralising antibody response against BVDV. Sera from mice immunised with the recombinant expressing full length E2 contained high levels of virus neutralising antibodies that in addition to giving neutralisation of the homologous NADL strain were also able to neutralise the Oregon C24V reference strain. These sera failed to give any neutralisation of the Osloss reference strain providing evidence for the division of BVDV isolates into at least two distinct E2 serotypes. These results were confirmed in gnotobiotic lambs. Expression of E2 fragments revealed the presence of at least two distinct neutralising epitopes, one of which was localised within the carboxy terminal 90 amino acids of the protein.     

Detection of bovine viral diarrhea virus (BVDV) in seropositive cattle

Detection of bovine virus diarrhoea virus (BVDV) in one vaccinated beef cattle and three non-vaccinated dairy herds was investigated on peripheral blood leukocytes (PBL) with or without previous treatment followed by a capture ELISA (cELISA). Using the combination of PHA and polycation treatment, PBL from 229 seropositive cattle were studied and could be classified in four different states of BVDV infection. Lysed PBL from four animals were directly positive in cELISA (Category I), PBL of 17 animals were positive after PHA stimulation (Category II), 15 animals were positive only after PHA stimulation plus polycation treatment (Category III), while virus could not be detected in 193 seropositive cattle. Wild-type BVDV strains were isolated by co-culture on polycation-treated MDBK cells from 11 of these seropositive animals. BVDV antibodies of these same animals were able to neutralize their own virus, indicating that virus persists in PBL in spite of strain-specific antibodies. No apparent change of leukocyte subpopulations could be detected in any category of virus-positive animals. Thus, BVDV may be present in the PBL of some cattle, even in the presence of a specific active immune response.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle sera

A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population.     

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappavalue=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.