Microparticle-mediated RNA immunization against bovine viral diarrhea virus.

Infectious transcripts from the full-length infectious clone of the NADL strain of bovine viral diarrhea virus (BVDV) were used to vaccinate cattle and sheep against BVDV. In vitro synthesized RNA delivered by microparticle bombardment with a Helios Gene Gun initiated replication of BVDV and consequently induced humoral immunity against type I BVDV (serum neutralization titers, SNT > 2(12)) and type II BVDV (SNT > 2(7)). The quality and long-term stability of the RNA-carrier complexes was assessed by microparticle bombardment of tissue culture monolayers. The RNA cartridges were found to be stable for at least 8 months upon storage. This is the first report on successful RNA vaccination of large ruminants.

Bovine viral diarrhea virus induced apoptosis correlates with increased intracellular viral RNA accumulation.

Non-cytopathic (NCP) and cytopathic (CP) parent-daughter pairs are often isolated from cattle with bovine viral diarrhea virus (BVDV) induced mucosal disease. Alignment of these pair genomes revealed that genetic changes in CP BVDV involve the NS2-3 coding region and correlate with expression of NS3. However, additional mutations are present elsewhere in the genomes of these natural pairs, precluding unambiguous mapping of this function to the NS2-3 region. To evaluate this phenomenon in identical genetic backgrounds, we have constructed an NCP isogenic pair of the NADL by deletion of the cIns from NS2 region. The levels of viral protein synthesis in infected cells revealed no marked difference between the CP and the isogenic NCP BVDV mutant. In contrast, RNA accumulation in cells infected with CP virus was up to 25 times higher than that in cells infected with NCP BVDV. No significant difference in growth kinetics and viral yields were observed between the CP BVDV and the isogenic NCP pair. Analyses of additional NCP/CP parent-daughter field BVDV isolates revealed a similar pattern of macromolecular synthesis, suggesting the generality of this phenomenon. These results implicate increased levels of RNA accumulation in CP BVDV infected cells, along with the production of NS3 as potential contributors to viral cytopathogenicity.

Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts.

Bovine viral diarrhea virus (BVDV) is the most insidious and devastating viral pathogen of cattle in the United States. Disease control approaches must be based on detailed knowledge of virus biology. To develop reverse-genetic systems to study the molecular biology of the virus, we first constructed a plasmid containing the entire genome of BVDV cloned as cDNA. Subsequently, we showed that infectious BVDV was produced by cells transfected with uncapped RNA transcribed in vitro from the cDNA clone. This result defined functional 5' and 3' termini in viral genomic RNA and established the biological importance of the proposed internal ribosome entry site element in the 5' untranslated region of the viral genome. BVDV rescued from the infectious cDNA clone has an in vitro phenotype similar to that of the wild-type parent, the National Animal Disease Laboratory strain of BVDV. A deletion of a single codon in the full-length genomic BVDV cDNA clone, encoding glutamic acid at position 1600, gave rise to sequence-tagged virus easily identified by restriction fragment length polymorphism analysis of reverse transcription-PCR amplicons. Suitability of the molecular clone of BVDV for genomic manipulations was shown by substitution of the major envelope glycoprotein E2/gp53 with that of the Singer strain, giving rise to a chimeric virus. The predicted change in antigenic structure of the chimeric virus could be readily identified with strain-specific monoclonal antibodies by neutralization and immunofluorescence assays. Immediate applications of this system include development of safe and effective live vaccine strains possessing predetermined defined attenuating mutations.