Bovine viral diarrhea virus is a suitable viral vector for stable expression of heterologous gene when inserted in between N(pro) and C genes.

Bovine viral diarrhea virus (BVDV) is a group of small enveloped viruses with a single-stranded, positive-oriented RNA genome of approximately 12.3 kb. BVDV genome directs the production of a viral polyprotein that is subsequently cleaved to release the mature viral proteins. To explore the potential of using BVDV as viral vector for stable expression of heterologous genes, eGFP2A was inserted in between N(pro) and C genes of a noncytopathic type-I BVDV strain SD1. eGFP2A was designed with eGFP protein in frame fused to the N terminus of the foot-and-mouth disease virus 2A protease. This strategy promised not only the correct processing of both viral N(pro) and C protein but also releasing of the chimeric protein from the nascent viral polyprotein. The recombinant reporter virus was successfully rescued in MDBK cells. In vitro study showed that eGFP2A protein, as expected, was expressed and processed properly from the nascent viral polyprotein. The reporter virus was similar to wt SD1 in viral RNA replication and protein expression and comparable to wt SD1 in growth kinetics except that this virus had a peak virus titer approximately 0.5 log(10) lower and a maximum yield about 4h later than wt SD1. In summary, these results indicated that BVDV is a suitable viral vector for stable expression of heterologous genes when inserted in between N(pro) and C genes.

Replicon Particle Expressing the E2 Glycoprotein of Bovine Viral Diarrhea Virus Immunization and Evaluation of Antibody Response.

The objective of this study was to develop a new antigen delivery system using an alphavirus replicon particle (RP) to induce humoral antibody responses against bovine viral diarrhea virus (BVDV) recombinant antigen produced from envelope glycoprotein E2. An alphavirus RP expressing the E2 glycoprotein of BVDV was used for immunization of pigs. A fluorescent microsphere immunoassay (FMIA) has been applied to detect BVDV E2 antigen-specific antibody isotype in pig immunized with alphavirus RP. Full-length BVDV E2 (aa 1-375) was cleaved into several pieces, eight E2 DNA fragments, including full-length DNA, were cloned into expression vector pHUE, and the recombinant proteins expressed in BL-21 (DE3) Escherichia coli. After successful conjugation of purified proteins with microsphere beads, a multiplex FMIA platform was constructed, and BVDV E2 alphavirus-based RP-immunized animal serum samples were tested in the presence of bead-bound antigen targets. The results were represented as mean fluorescence intensity (MFI); the MFI values were converted to sample value/positive value (S/P) ratios. BVDV E2 (aa 1-183) showed the highest MFI values of eight recombinant E2 fragments when the specific activity of each fragment was tested. In immunized animals, data for BVDV E2-specific IgA, IgG, and IgM in serum and only IgG and IgA in oral fluids were recorded. The MFI values for the positive serum sample showed a 100-fold increase compared with the negative serum sample. Antibody isotype to BVDV E2 antigens showed that IgG > IgM > IgA in serum, whereas IgG > IgA > IgM in oral fluids. The data presented in this study suggested that boosting with the same doses of alphavirus RP in 3-week intervals may potentially enhance antibody response. The experimental results demonstrate that alphavirus RP-expressing BVDV E2 antigen induces antibody response in pig.

Nylon membrane-immobilized PCR for detection of bovine viruses.

Bridge Technology is an amplification technique in which pairs of primers are immobilized on a solid support, allowing amplification only at the location of the primer pair spot. The technique has diagnostic potential since an array of primer pairs, each specific for a different pathogen, can be used with a diagnostic sample without inter-pair interactions that plague the development of multiplex PCRs. As a result, one assay should be able to determine which of multiple pathogens are present and which are absent in each sample. As test material, we examined the specificity of detection of the RNA-containing bovine viral diarrhea virus (BVDV) and two DNA-containing bovine herpesviruses 1 and 2 (BHV-1 and BHV-2). Nylon membranes with two spots of UV-immobilized primer pairs--one for BVDV and one for BHV--were used in amplification with both corresponding templates, with each template singly and with no template. When amplification was assayed by chemiluminescent detection of incorporated DIG-nucleotides, the expected amplification patterns were obtained.

Increased phylogenetic diversity of bovine viral diarrhoea virus type 1 isolates in England and Wales since 2001.

Currently, there are two recognised genotypes of Bovine viral diarrhoea virus (BVDV), type 1 and type 2. These genotypes are divided into subtypes based on phylogenetic analysis, namely a-p for BVDV-1 and a-c for BVDV-2. Within this study, the genetic heterogeneity of BVDV-1 in England and Wales was investigated and compared to the situation in 1996/1997. Viral RNA was extracted from 316 blood samples collected between 2004 and 2009 that were previously identified as BVDV-1 positive. A region of the 5' untranslated region (UTR) was amplified by RT-PCR and the PCR products were sequenced. Phylogenetic analysis of the 5'UTR demonstrated the existence of five subtypes of BVDV-1 circulating in England and Wales, namely BVDV-1a (244 samples), BVDV-1b (50), BVDV-1e (3), BVDV-1f (1) and BVDV-1i (18). Phylogenetic analysis of the nucleotide sequence for the N(pro) region of the viral genome supported the classification obtained with the 5'UTR. Given the fact that only three subtypes were detected in 1999 this report supports the notion that the restocking of cattle from continental Europe, after the mass culling during the Foot-and-Mouth outbreak in 2001 and slaughter of cattle due to bovine tuberculosis infection, has increased the genetic diversity of BVDV-1 subtypes in England and Wales in the past 10 years.

Acute bovine viral diarrhea associated with extensive mucosal lesions, high morbidity, and mortality in a commercial feedlot.

In 2008, a northwest Texas feedlot underwent an outbreak of Bovine viral diarrhea virus (BVDV) causing high morbidity and mortality involving 2 lots of calves (lots A and B). Severe mucosal surface lesions were observed grossly in the oral cavity, larynx, and esophagus. Mucosal lesions varied from small (1-3 mm) infrequent mucosal ulcerations to large (5 mm to 1 cm) and coalescing ulcerations. Necrotic debris was present in ulcerations of some mortalities with some having plaque-like debris, but other mortalities presented more proliferative lesions. A calf persistently infected with BVDV arrived with one lot and the isolated virus was genotyped as BVDV-1b. Identical BVDV-1b strains were isolated from 2 other mortalities. A BVDV-2a genotype was also isolated in this outbreak. This genotype was identical to all BVDV-2a strains isolated in both lots. Serum samples were collected from exposed and unexposed animals and tested for antibodies for multiple viral pathogens. Seropositivity ranged from zero percent for calicivirus to 100% positive to Pseudocowpox virusx. At the end of the feeding period, the morbidity and mortality for the 2 lots involved was 76.2% and 30.8%, respectively, for lot A, and 49.0% and 5.6%, respectively, for lot B. Differential diagnoses included vesicular stomatitis viruses, Bovine papular stomatitis virus, and Foot-and-mouth disease virus. Based on the present case, acute BVDV should be considered when mucosal lesions are observed grossly.

Vaccination of cattle with a DNA plasmid encoding the bovine viral diarrhoea virus major glycoprotein E2.

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle that is ubiquitously distributed worldwide. In this study, cattle were immunized by intramuscular injections with plasmid DNA expressing the BVDV type 1 major glycoprotein E2. Animals either received injections of naked DNA (N-DNA) or DNA in cationic liposomes (L-DNA). Both DNA preparations induced virus-specific neutralizing antibodies in vaccinates, although the response was much lower in N-DNA-immunized animals. N-DNA-vaccinated animals also showed virus-specific lymphocyte proliferation responses to type 1, live BVDV in vitro, whereas L-DNA vaccination induced no such responses. After 16 weeks, DNA-vaccinated and mock-vaccinated animals were challenged with a USDA-certified BVDV type 1 strain. Four significant observations were made: (1) N-DNA-vaccinated calves showed limited protection from virus challenge, (2) L-DNA-vaccinated animals did not show any signs of protection, (3) the challenge induced strong memory responses in the production of serum neutralizing antibodies to both genotypes (type 1 and 2 of BVDV), and (4) the challenge induced a mucosal memory response in nasal secretions of both L- and N-DNA-vaccinated animals.