Do modified live virus vaccines against bovine viral diarrhea induce fetal cross-protection against HoBi-like Pestivirus?

Bovine Pestivirus heterogeneity is a major challenge for vaccines against bovine viral diarrhea (BVD). In breeding herds, fetal protection is a high priority issue. To some degree, fetal infections in vaccinated heifers have been attributed to the antigenic diversity of bovine Pestiviruses. The purpose of this study was to assess fetal protection against a divergent bovine Pestivirus (Hobi-like Pestivirus, HoBiPeV) with a commercially available modified live vaccine (MLV) claiming fetal protection against BVDV 1 and BVDV 2 up to one year after the first inoculation. Five vaccinated and four unvaccinated heifers were challenged by intranasal inoculation with the HoBiPeV Italy-1/10-1 strain between 82 and 89 days after insemination, i.e. between 4 and 6 months after vaccination. At challenge, neutralizing antibody titers to HoBiPeV in vaccinated heifers were low or even undetectable. Of the four unvaccinated heifers, one control animal aborted (fetus not available) and the remaining three gave birth to HoBiPeV positive calves. Among the heifers of the vaccinated group, one aborted the fetus in the sixth month of pregnancy, which tested Pestivirus negative, while three others gave birth to healthy, HoBiPeV negative calves; the remaining heifer delivered one HoBiPeV positive calf. The results suggest that the BVDV vaccine might be able to elicit a partial fetal protection against HobiPeV, even in absence of a strong specific antibody response.

Serological evidence of Coxiella burnetii, Leptospira interrogans Hardjo, Neospora caninum and bovine pestivirus infections in a dairy cattle herd from the United Arab Emirates.

The serostatus of five abortigenic agents and the association between abortion history and Coxiella burnetii seropositivity were assessed in 350 dairy cattle from Al Ain, UAE. The bovine sera were ELISA-screened for C. burnetii, Leptospira Hardjo, Neospora caninum, and Brucella abortus antibodies, plus bovine pestivirus (BVDV) antigen. The serology data were collated and the level of significance between the proportions of C. burnetii-seropositive cattle with and without abortion history assessed by the Z test of two proportions. Of the 350 cattle, 41.4%, 1.7%, 1.4%, 0.3%, and 0.0% were seropositive to the above pathogens, respectively. Besides, 61.9%, 2.9%, 1.0%, 0.0%, and 0.0% of the 105 cattle with history of abortion and 32.7%, 1.2%, 1.6%, 0.0% and 0.0% of the 245 seropositive cattle with no history of abortion were also seropositive for the above pathogens respectively. Moreover, the proportion of C. burnetii-seropositive cattle with history of abortion were significantly higher than the C. burnetii-seropositive ones without abortion history (p-value < 0.01). Apparent C. burnetii infections were relatively higher than the other four pathogens suggesting this bacterium contributed to abortion in the herd. Additional research on the public and bovine health implications of C. burnetii and Leptospira in the UAE are urgently needed.

Estimation of the within-herd transmission rates of bovine viral diarrhoea virus in extensively grazed beef cattle herds.

Many research groups have developed mathematical models to simulate the dynamics of BVDV infections in cattle herds. However, most models use estimates for within-herd BVDV transmission rates that are either based on expert opinion or adapted from other dairy herd simulation models presented in the literature. There is currently little information on the transmission rates for BVDV in extensively grazed beef herds partly due to the logistical challenges in obtaining longitudinal data of individual animal's seroconversion, and it may not be appropriate to apply the same transmission rates from intensive dairy herds given the significant differences in herd demographics and management. To address this knowledge gap, we measured BVDV antibody levels in 15 replacement heifers in each of 75 New Zealand beef breeding farms after their first calving and again at pregnancy scanning or weaning to check for seroconversion. Among these, data from 9 farms were used to infer the within-herd BVDV transmission rate with an approximate Bayesian computation method. The most probable within-herd BVDV transmission rate was estimated as 0.11 per persistently infected (PI) animal per day with a 95% highest posterior density interval between 0.03 and 0.34. This suggests that BVDV transmission in extensively grazed beef herds is generally slower than in dairy herds where the transmission rate has been estimated at 0.50 per PI animal per day and therefore may not be sufficient to ensure that all susceptible breeding females gain adequate immunity to the virus before the risk period of early pregnancy for generating new PI calves.

Detection of bovine pestiviruses in sera of beef calves by a RT-PCR based on a newly designed set of pan-bovine pestivirus primers.

The pestiviruses bovine viral diarrhea virus 1 and 2 (BVDV-1 and -2, respectively) and HoBi-like pestivirus (HoBiPeV) are important pathogens of cattle, and a number of reverse-transcription PCR (RT-PCR)-based assays have been developed for their detection in clinical specimens. We evaluated a newly designed set of pan-bovine pestivirus primers (BP189-389) in a gel-based RT-PCR screening test for pestiviruses in the sera of beef calves destined for export from southern Brazil. Serum samples positive for BVDV antigens by an antigen ELISA ( n = 135) were submitted to RT-PCR assays using different sets of primers, followed by nucleotide sequencing of the amplicons. RT-PCR with pestivirus primers 324-326 detected 110 positive samples: BVDV-1 ( n = 62), BVDV-2 ( n = 38), and HoBiPeV ( n = 10). A PCR using primers HCV90-368 detected 97 positive samples (64 BVDV-1; 33 BVDV-2). An additional RT-PCR round using BVDV-2-specific primers (2F-2R) detected 45 positive samples (including 38 detected by primers 324-326 and 33 by HCV90-368); whereas a RT-PCR using HoBiPeV-specific primers (N2-R5) detected 26 positive samples (including 10 detected by primers 324-326).The assay using the primers BP189-389 detected all 135 ELISA-positive samples, including the 26 HoBiPeV detected by primers N2-R5. Our results demonstrated that primers BP189-389 compare favorably against other primer sets in the detection of bovine pestiviruses, especially HoBiPeV. This conventional PCR may be useful for efficient detection of pestiviruses in bovine sera and other specimens as well, especially in laboratories without real-time PCR equipment.

Experimental infection of mice with noncytopathic bovine viral diarrhea virus 2 increases the number of megakaryocytes in bone marrow.

BACKGROUND: Bovine viral diarrhea virus (BVDV) causes significant economic losses worldwide in the cattle industry through decrease in productive performance and immunosuppression of animals in herds. Recent studies conducted by our group showed that mice can be infected with BVDV-1 by the oral route. The purpose of this study was to assess the clinical signs, hematological changes, histopathological lesions in lymphoid tissues, and the distribution of the viral antigen after oral inoculation with a Korean noncytopathic (ncp) BVDV-2 field isolate in mice. METHODS: Mice were orally administered a low or high dose of BVDV-2; blood and tissue samples were collected on days 2, 5, and 9 postinfection (pi). We monitored clinical signs, hematological changes, histopathological lesions, and tissue distribution of a viral antigen by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) and then compared these parameters with those in ncp BVDV-1 infections. RESULTS: None of the infected mice developed any clinical signs of the illness. Significant thrombocytopenia was found in both low- and high-dose-inoculated mice on day 2 pi. Leukopenia was apparent only in low-dose-inoculated mice on day 2 pi, whereas lymphopenia was not observed in any ncp BVDV-2-infected animal. Viral RNA was found in the spleen in of low- and high-dose-inoculated mice by RT-PCR. According to the results of IHC, the viral antigen was consistently detected in lymphocytes of bone marrow and spleen and less frequently in bronchus-associated lymphoid tissue (BALT), mesenteric lymph nodes, and Peyer's patches. Despite the antigen detection in BALT and mesenteric lymph nodes, histopathological lesions were not observed in these tissues. Lympholysis, infiltration by inflammatory cells, and increased numbers of megakaryocytes were seen in Peyer's patches, spleens, and bone marrow, respectively. In contrast to ncp BVDV-1 infection, lympholysis was found in the spleen of ncp BVDV-2-infected mice. These histopathological lesions were more severe in high-dose-inoculated mice than in low-dose-inoculated mice. CONCLUSIONS: Our results provide insight into the pathogenesis of ncp BVDV-2 infection in mice. Collectively, these results highlight significant differences in pathogenesis between ncp BVDV-1 and ncp BVDV-2 infections in a murine model.

A systematic worldwide review of the direct monetary losses in cattle due to bovine viral diarrhoea virus infection.

Bovine viral diarrhoea virus (BVDV) is an important infectious agent of cattle worldwide that affects herd productivity and reproduction. In this systematic review of the impact of BVDV, studies were analysed with a particular focus on the monetary implications and types of direct losses, the initial infection status of herds, production systems, time periods of assessment, calculation level, study types and whether or not country-specific assessments were published. A linear mixed model was applied to analyse factors that influence the level of monetary direct losses due to BVDV infection. The 44 studies included in this review covered 15 countries and assessed direct monetary losses due to BVDV incurred over the past 30 years. Direct losses between and within countries were largely heterogeneous with respect to the monetary level and types of direct losses, ranging from 0.50 to 687.80 US dollars (USD) per animal.1 Average direct losses per naïve dairy cow were USD24.85 higher than per beef cow. Country-specific assessments of direct losses due to BVDV were provided in 38/44 (86.4%) studies. Mortality, morbidity, premature culling, stillbirths, abortion, reinfection, country and study type had a significant influence on the monetary level of direct losses (r2 = 0.69). Countries recording direct losses were more likely to carry out voluntary or compulsory control and eradication programmes (odds ratio = 10.2; 95% confidence interval 1.7-81.9; P = 0.004).

Molecular Characterization of a Novel Bovine Viral Diarrhea Virus Isolate SD-15.

As one of the major pathogens, bovine viral diarrhea virus caused a significant economic loss to the livestock industry worldwide. Although BVDV infections have increasingly been reported in China in recent years, the molecular aspects of those BVDV strains were barely characterized. In this study, we reported the identification and characterization of a novel BVDV isolate designated as SD-15 from cattle, which is associated with an outbreak characterized by severe hemorrhagic and mucous diarrhea with high morbidity and mortality in Shandong, China. SD-15 was revealed to be a noncytopathic BVDV, and has a complete genomic sequence of 12,285 nucleotides that contains a large open reading frame encoding 3900 amino acids. Alignment analysis showed that SD-15 has 93.8% nucleotide sequence identity with BVDV ZM-95 isolate, a previous BVDV strain isolated from pigs manifesting clinical signs and lesions resembling to classical swine fever. Phylogenetic analysis clustered SD-15 to a BVDV-1m subgenotype. Analysis of the deduced amino acid sequence of glycoproteins revealed that E2 has several highly conserved and variable regions within BVDV-1 genotypes. An additional N-glycosylation site (240NTT) was revealed exclusively in SD-15-encoded E2 in addition to four potential glycosylation sites (Asn-X-Ser/Thr) shared by all BVDV-1 genotypes. Furthermore, unique amino acid and linear epitope mutations were revealed in SD-15-encoded Erns glycoprotein compared with known BVDV-1 genotype. In conclusion, we have isolated a noncytopathic BVDV-1m strain that is associated with a disease characterized by high morbidity and mortality, revealed the complete genome sequence of the first BVDV-1m virus originated from cattle, and found a unique glycosylation site in E2 and a linear epitope mutation in Erns encoded by SD-15 strain. Those results will broaden the current understanding of BVDV infection and lay a basis for future investigation on SD-15-related pathogenesis.

[PESTIVIRUSES IN RUMINANTS].

The genus Pestivirus includes four species: bovine viral diarrhea virus 1, bovine viral diarrhea virus 2, classical swine fever disease virus, and ovine border disease virus. Pestiviruses infect many species of domestic and wild animals. Bovine viral diarrhea virus is a prototypical representative of the pestiviruses of ruminant animals. Recently, new candidates appeared for including in this genus: two viruses of the wild ruminant animals that have not been officially classified and one HoBi-like virus discovered for the first time in the bovine fetal serum. The circulation of the ruminant animal pestiviruses within population of domestic and wild animals, the presence of these viruses in bioproducts stimulates studies of the infection reservoirs and their influence on the effect of the bovine viral diarrhea control programs.

Cross-priming amplification for detection of bovine viral diarrhoea virus species 1 and 2.

AIMS: The aim of the study was the development of cross-priming amplification for ubiquitous detection of bovine viral diarrhoea virus (BVDV) species 1 and 2. METHODS AND RESULTS: Three and five specific primers, respectively, for the detection of BVDV-1 and BVDV-2, were designed on the basis of the sequences of the 5'UTR region. Incubation temperature and reaction time were determined. The optimal incubation conditions using water bath were 63°C for 75 min. Reverse transcription step (RT) was not required. The results were visualized under UV-light as a bright yellow fluorescence in positive samples. Additional method for results interpretation was agarose gel electrophoresis. Positive samples showed the presence of ladder-like banding patterns, formed by harpin-like cross-priming amplification (CPA) products. Sensitivity of CPA was compared with conventional RT-PCR and real-time RT-PCR. The CPA detection limit was 3500 copies for BVDV-1 and 80000 copies for BVDV-2 per reaction. For RT-PCR it was 350 and 80 copies for BVDV-1 and BVDV-2, respectively, and for real-time RT-PCR it was 35 copies for BVDV-1 and 80 copies for BVDV-2. The sensitivity of the developed method is sufficient to detect persistently infected (PI) animals. Positive results were found in 24 of 25 BVDV isolates belonging to species 1 and 2. Additionally, one false-negative result for BVDV-2 was detected. There were no false-positive results in negative samples and in the negative control. Both sets of primers used for the detection of BVDV-1 and BVDV-2 were not able to detect atypical pestiviruses. CPA positive results were confirmed by RT-PCR and real-time RT-PCR. CONCLUSIONS: CPA is a rapid method for the detection of BVDV-1 and BVDV-2 in field samples from PI animals. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report on the application of the CPA method for the detection of BVDV.

First report of bovine viral diarrhoea virus-2 infection in cattle in Poland.

This report describes the first identification in Poland of bovine viral diarrhoea virus (BVDV)-2 in a dairy herd where severe clinical disease with losses of young animals was observed. The virus was readily cultivated in cell culture and a phylogenetic analysis of the nucleotide sequences and secondary structures of the viral genomic 5' untranslated region confirmed virus identity. The economic impact of the infection was significant compared to the previously prevalent BVDV-1 infections confirming that this genotype of BVDV can cause severe sickness in affected herds. The use of BVDV-1 vaccine did not prevent the infection with the BVDV-2 genotype.

Insertion and stable expression of Gaussia luciferase gene by the genome of bovine viral diarrhea virus.

As a tool to address selected issues of virus biology, we constructed a recombinant cDNA clone of bovine viral diarrhea virus (BVDV) expressing Gaussia luciferase (Gluc) reporter gene. A full-length genomic cDNA clone of a non-cytopathic BVDV isolate was assembled by recombination in yeast Saccharomyces cerevisiae. The Gluc gene was inserted between the N(pro) and Core protein coding regions by recombination. The cDNA transcribed in vitro was infectious upon transfection of MDBK cells, resulting in reporter gene expression and productive virus replication. The rescued viruses were stable for 15 passages in cell culture, maintaining the replication kinetics, focus size and morphology similar to those of the parental virus. Expression and correct processing of the reporter protein were also maintained, as demonstrated by Gluc activity. These results demonstrate that genes up to 555 bp are simply assembled by a single step in yeast recombination and are stably expressed by this cDNA clone.

Genetic diversity of bovine viral diarrhea viruses in commercial bovine serum batches of Chinese origin.

Bovine viral diarrhea virus (BVDV) is often detected in commercial bovine serum. BVDV genetic diversity was investigated in commercial bovine serum of Chinese origin. Twenty-two batches of bovine serum were obtained from 10 suppliers with different geographic origins in China, and 20 batches of bovine serum were positive by reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. Phylogenetic reconstructions of partial 5'UTR sequences indicated that the samples examined in this work clustered within the BVDV type 1 and BVDV type 2 genotypes. Interestingly, 3 sample sequences clustered into CSFV. These results suggest a high genetic diversity in Chinese BVDV field isolates. This study will benefit epidemiological surveys of BVDV detected in China.

Effects of in utero pestivirus infection on bovine fetal bone geometry, biomechanical properties and composition.

Transplacental viral infection of the fetus can result in abnormal trabecular and cortical bone modeling in long bones through impaired bone resorption and formation. Although such infections are frequently associated with neonatal fractures in humans and animals, their effect on the biomechanical properties of the developing skeleton remain poorly understood. The goal of this study was to determine the effects of transplacental bovine viral diarrhea virus (BVDV) infection on the biomechanical properties of fetal femora. Pregnant heifers were inoculated intranasally with non-cytopathic BVDV or media alone on day 75 of gestation to produce persistently infected (PI) and control fetuses, respectively, which were then removed on days 192 and 245 of gestation. Histomorphometry, compositional analysis and 'four-point bending until failure' were performed on fetal femora. Altered cortical geometry largely accounted for differences in calculated elastic modulus (PI vs. control, and day 192 vs. day 245) and ultimate stress (day 192 vs. day 245). Fetal infection with BVDV did not significantly impair inherent biomechanical properties of bone but rather resulted in decreased periosteal apposition rates, manifested as smaller femoral mid-diaphyseal diameters. There were no differences between PI and control fetuses in cortical thickness ratio, ash density or calcium/phosphorous content; however, cortical thickness ratio decreased with fetal age. Thus even when cortical thickness ratios are similar, differences in mid-diaphyseal diameter affect the error associated with the calculation of stress and strain by classical beam theory equations.

A study of the effectiveness of a needle-free injection device compared with a needle and syringe used to vaccinate calves against bovine viral diarrhea and infectious bovine rhinotracheitis viruses.

The aim of this study was to compare the effectiveness of a needle-free injection device (NF) with a needle and syringe (NS) when used to vaccinate calves against bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV). The study was conducted in two independent phases. Ninety-six crossbred beef calves were vaccinated in the spring and 98 beef calves in the autumn. The calves were vaccinated using a NF or NS at 2 months of age (day 0) and again on day 119, with a modified-live virus vaccine containing IBRV, BVDV (types 1 and 2), parainfluenza-3 virus, and bovine respiratory syncytial virus. In each herd 10 calves were left unvaccinated to determine whether exposure to either BVDV or IBRV occurred. Visible vaccine residue at the surface of the skin/hair was apparent immediately following vaccination with NF in 30% of the spring-born calves following both the primary and booster vaccination. In the autumn, visible vaccine residues occurred in 19% and 8% of NF-vaccinated calves following the primary and booster vaccination. Post-vaccination skin reactions recorded on days 21, 42, 119 and 140 occurred with greater frequency in NF-vaccinated calves than NS-vaccinated ones. Blood samples were collected on days 0, 21, 42, 119, and 140 and tested for antibodies to BVDV and IBRV. Vaccination technique had no significant effect on BVDV or IBRV antibody concentrations at any time point. NF was as effective as NS vaccination in eliciting BVDV and IBRV antibody responses.

An evaluation of circulating bovine viral diarrhea virus type 2 maternal antibody level and response to vaccination in Angus calves.

Vaccination against viruses has been shown to help prevent bovine respiratory disease in cattle. However, both passively acquired maternal antibody concentration and calf age have been shown to impact the ability of the immune system of a calf to respond to vaccination. The objectives of this study were to identify and evaluate environmental and management factors that affect 1) passively acquired bovine viral diarrhea virus (BVDV) type 2 antibody level, 2) decay rate of passively acquired BVDV type 2 antibody level, and 3) responses to BVDV type 2 vaccinations. A 2-shot modified live vaccine was administered to 1,004 Angus calves that were weaned at either the initial vaccination (n = 508) or the booster vaccination (n = 496). Calves weaned at the initial vaccination averaged 139 d whereas calves weaned at booster vaccination averaged 128 d of age. Bovine viral diarrhea virus type 2 antibodies were measured in 3 approximately 21-d intervals, serially collected serum samples to quantify antibody levels at initiation and end of vaccination protocol in addition to responses to initial, booster, and overall vaccination protocol. Amount of passively transferred antibody in the calf increased as dam age increased from 2 to 6 yr (P < 0.05) with no differences after dams reached 6 yr (P > 0.05). Calf age nested within birth year-season and dam age affected both initial and final antibody level, initial response, booster response, and overall antibody response to vaccination. The level of circulating, passively acquired maternal antibodies present at the time of vaccination had a significant (P < 0.05) negative effect on antibody responses to vaccination (initial response, booster response, and overall response). Calves that were weaned at the time of initial vaccination had significantly (P < 0.05) greater final antibody level, initial response, and overall response to vaccination than animals weaned at booster vaccination. In order for a calf to mount an overall antibody response to vaccination, maternal antibodies in circulation need to be less than 3.12 titers. However, the age at which a calf reached this antibody threshold was dependent on dam age. This information will help cattle managers and consultants design vaccination protocols to successfully mount an antibody response to vaccination.

Genetic change in the open reading frame of bovine viral diarrhea virus is introduced more rapidly during the establishment of a single persistent infection than from multiple acute infections.

Bovine viral diarrhea viruses (BVDV) are ubiquitous viral pathogens of cattle with a high degree of sequence diversity amongst strains circulating in livestock herds. The driving force behind change in sequence is not well established but the inaccurate replication of the genomic RNA by a viral RNA polymerase without proof-reading capabilities as well as immune pressure on immunodominant proteins are thought to play major roles. Additionally, it is not clear when the majority of changes are introduced, whether during acute infections with exposure to innate and adaptive immune responses or in establishment of persistent infections (PI) in utero. To examine which generates greater sequence diversity, two groups of viruses were compared. The first was six isolates of a single strain of BVDV-2 that were isolated over greater than a year's time. These viruses caused a series of severe acute (SA) BVD outbreaks over a large geographic area. Changes in nucleotide sequence were determined by comparison of the sequence of each strain to the six virus consensus sequence. The second group was composed of six BVDV strains isolated from PI calves whose dams were exposed to PI cattle. Changes were identified by comparison of the sequence of the progenitor PI virus to that of the progeny viruses from the single in vivo 'passage'. The open reading frames (ORF) of the six SA isolates were >99% identical at the nucleotide level with 30% of the changes being nonsynonymous changes. The amount of genetic change increased with time and distance from the original outbreak. Similarly, the PI viruses isolated from single passage PI calves had >99% identity with the progenitor virus. The number of nucleotide changes in these viruses was equal to or greater than that observed in the SA viruses. The majority of the nonsynonymous changes were found in the structural proteins, with 65% of these occurring in the immunodominant E2 protein. Antigenic mapping studies using a monoclonal antibody panel specific for the BVDV E2 protein showed no antigenic differences amongst the six SA viruses, nor between the progenitor and progeny type 1a and type 2 persistent viruses. However, antigenic differences were observed in the two type 1b progeny viruses that possessed the greatest number of amino acid changes. Two antibodies were found to have altered staining patterns. These results suggest that the establishment of a single persistent infection results in more rapid generation of genetic diversity in BVDV strains than a series of acute infections and may contribute to antigenic change in the absence of an immune response.

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.

Characterisation of a new infectious full-length cDNA clone of BVDV genotype 2 and generation of virus mutants.

Based on their genomic sequences, two genotypes of Bovine viral diarrhea virus (BVDV) can be differentiated, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). The complete genomic sequence of the highly virulent BVDV-2 strain 890 was cloned as cDNA to establish the infectious cDNA clone p890FL. In vitro-synthesised full-length RNA of p890FL was transfected into bovine cells and infectious virus could be recovered (v890FL). In vitro, recombinant v890FL showed similar growth characteristics as wild type virus v890WT. However, infection experiments in calves revealed an attenuation of recombinant v890FL in comparison to the parental isolate. Both leukocytopenia and fever were less pronounced in v890FL-infected calves. Nevertheless, viremia and virus shedding were comparable between recombinant and parental BVDV 890. Furthermore, mutants with partial deletions of the genomic region encoding for the autoprotease N(pro) (p890DeltaN(pro)) or the capsid protein (p890DeltaC) were constructed and characterised. In order to generate pseudovirions, replicon p890DeltaC was efficiently trans-complemented on a helper cell line. In summary, the newly developed construct p890FL represents the first infectious full-length cDNA clone for the BVDV-2 strain 890 and offers a useful tool for further studies on the pathogenesis of BVDV-2 and the development of novel recombinant BVDV-2 specific vaccine candidates.

In vitro amplification of BVDV field strains isolated in Argentina: effect of cell line and culture conditions.

The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD-420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.