Recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing a partial deletion within the Nsp2-encoding region.

Non-structural protein 2 (Nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the most variable region and postulated to play an important role in cell and tissue tropism of PRRSV. To investigate the role of Nsp2 in the viability and growth of PRRSV in cells in vitro, two cDNA clones were constructed containing a deletion of 63 consecutive nucleotides (pWSK-DCBAd63) or 117 nucleotides (pWSK-DCBAd117) within the Nsp2-encoding region of PRRSV (BJ-4). The clone pWSK-DCBAd63 was infectious and produced viable recombinant virus, whereas clone pWSK-DCBAd117 could not be rescued. The rescued virus was able to induce CPE typical of PRRSV on MARC-145 cells and was stably propagated during sequential in vitro cell passages, like the virus recovered from the full-length cDNA clone of PRRSV BJ-4. In comparison to the parental virus (BJ-4) and the virus recovered from the full-length cDNA clone of the BJ-4 strain, the rescued virus from pWSK-DCBAd63 exhibited enhanced growth kinetics, reaching the peak progeny virus titer by 48 h postinfection. These observations suggest that the Nsp2-encoding region is necessary for productive virus infection, and partial deletion does not influence the viability and propagation of PRRSV in cell culture, which may provide a way to insert a foreign gene into the viral genome as a marker for differentiation.

DNA priming and killed virus boosting vaccination strategy can augment the immune response to pseudorabies virus.

The immune efficacy of DNA vaccines containing three plasmids encoding gB, gC, and gD glycoproteins (Mix DNA) of Pseudorabies virus (PRV) or the plasmid for gC only (gC DNA), killed virus (KV) vaccine or combination of gC DNA, Mix DNA and KV vaccines was evaluated in mice using primeboost strategy. The mice vaccinated twice with Mix DNA, and once with KV generated higher levels of gCspecific and virus neutralization (VN) antibodies and a stronger cellular immune response than the mice vaccinated three times with the Mix DNA vaccine only. The highest level of VN antibodies were detected in mice vaccinated twice with KV vaccines alone or with combination of DNA and KV vaccines. The challenge of vaccinated mice with the lethal dose of PRV showed that the complete protection against PRV was achieved in the group of mice immunized with the DNA and KV vaccines combined. The results suggested that DNA priming followed by KV vaccine boosting could enhance the antibody response and cellular immunity against PRV infection in mice.

Genomic analysis of two porcine encephalomyocarditis virus strains isolated in China.

Two strains of encephalomyocarditis virus (EMCV), designated BJC3 and HB1, were isolated from an aborted fetus and the heart tissue of a dead piglet that had pericardial fluid, respectively. The complete genomic sequences of the two viruses were determined and analyzed. The size of the genomes of BJC3 and HB1 were 7746 and 7735 nucleotides, respectively, including poly(A) tails. Comparative analysis with the genomic sequences of other EMCV strains showed that BJC3 and HB1 shared higher identity (92.5-99.6%) with BEL-2887A/91, EMCV-R and PV21, but lower identity (83.3-84.6%) with EMC-B, EMC-D and D variants, and only 81.0% with Mengo virus. Two amino acid mutations in the leader protein of the two viruses and one amino acid substitution in VP1 of BJC3 were found in comparison to other EMCV strains Phylogenetic analysis based on the amino acid sequences of the entire ORF revealed that the two Chinese isolates BJC3 and HB1 clustered together with the strains BEL-2887/91, EMCV-R and PV21, which belong to the same genetic subgroup as EMCV-30. Our results provide genomic information for EMCV isolated in China.