In vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains using the PrimeFlow RNA assay.

Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.

Molecular characterization of bovine viral diarrhea virus type 2 isolate originating from a native Indian sheep (Ovies aries).

The wide spread prevalence of bovine viral diarrhea virus type 1 (BVDV-1) in cattle and recent identification of BVDV-2 in goats in India warranted pestivirus surveillance in sheep. Nested reverse transcription-polymerase chain reaction (RT-PCR) was used to detect BVDV-2 in one of 1561 blood samples collected randomly from 78 sheep flocks in 11 states of India. Antigenic characterization of the isolated pestivirus using polyclonal and monoclonal antibodies typed the isolate as BVDV-2. When analyzed at genetic level in N(pro) (N-terminal autoprotease) and entire gene region coding structural proteins, namely capsid (C) protein and envelope proteins E(rns) (ribonuclease secreted), E1 and E2 genomic organization was the same for all pestiviruses, the nucleic acid and amino acid sequences showed highest similarity with those of BVDV-2. When compared with BVDV-2 isolate 890, the sequence homology was 83.7% for C, 84.6% for E(rns) and E1, and 81.5% for E2. The cleavage site C/E(rns) was found totally conserved while N(pro)/C was conserved only from C-terminus of N(pro), E(rns)/E1 site was conserved only from C-terminus of E(rns) and E1/E2 site was conserved only from C-terminus of E1. Phylogenetic analysis of nucleotide sequences in 5' untranslated regions (UTR), N(pro), E2, NS3 and NS5B regions placed the sheep isolate in a separate clade within BVDV-2 subtype b. This was supported by the presence of unique mutations in the structural protein coding regions beside NS3 and NS5B. To our knowledge this is the first report on the sequence analysis of the entire structural gene coding region of a BVDV-2b isolate. This is also the first occurrence of BVDV-2 subtype b in sheep, providing the evidence that this subtype can also occur in species other than cattle.

Platelet function and association of bovine viral diarrhea virus with platelets of persistently infected cattle.

OBJECTIVE: To determine whether viral involvement with platelets obtained from cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) is associated with altered platelet function or decreased platelet counts. SAMPLE POPULATION: Platelets obtained from 8 cattle PI with BVDV and 6 age-, sex-, and breed-matched uninfected control cattle. PROCEDURE: Manual platelet counts were determined, and platelet function was assessed through optical aggregometry by use of the aggregation agonists ADP and platelet-activating factor. Identification of BVDV in serum and preparations of purified platelets was determined by use of virus isolation tests. RESULTS: No significant difference in platelet counts was detected between cattle PI with BVDV and control cattle. In response to the aggregation agonists, maximum aggregation percentage and slope of the aggregation curve were not significantly different between cattle PI with BVDV and control cattle. We isolated BVDV from serum of all PI cattle and from purified platelets of 6 of 8 PI cattle, but BVDV was not isolated from serum or platelets of control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Isolation of BVDV from platelets in the peripheral circulation of cattle immunotolerant to BVDV does not result in altered platelet function or decreases in platelet counts.