Molecular and phylogenetic analysis of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2)
MOLECULAR AND PHYLOGENETIC ANALYSIS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV) AND PORCINE CIRCOVIRUS TYPE 2 (PCV2)
Submitted by Yick Yeung LI
for the Degree of Master of Philosophy (MPhil) at The University of Hong Kong
in November 2003
Porcine reproductive and respiratory syndrome VIruS (PRRSV) and postweaning multisystemic wasting syndrome (PMWS) have caused a huge economical loss in the global swine industry. The aim of this study is to characterize the prevalence and phylogenetic relationship of local PRRSV and porcine circovirus type 2 (PCV2, causative agent of PMWS), and to develop a DNA vaccine against PRRSV In this study, PRRSV (26% of tested pigs) and PCV2 (15% of tested pigs) were identified in local swineherds.
Phylogenetic analysis on eighteen local PRRSV s from different pig farms demonstrated the existence of both European (45%) and North American (55%) genotypes. Also, the local European typed virus and North American typed virus showed that the amino acid identity of nucleocapsid protein ranged from 89.1 % to 95.3% and 94.4% to 98.4% when compared to their respective prototypes. However, the average pairwise genetic distances of the European genotype and North American genotype were found to be 0.047 and 0.065, respectively, suggesting that the PRRSVs with North American genotype was more diverse than those with European genotype. In addition, two sub-branches of European typed PRRSVs and at least four sub-branches of North American typed PRRSVs were identified in local pig farms.
U sing the phylogenetic tree analysis on the basis of complete nucleotide sequence of local PCV2 viruses and PCV2 isolates around the world, the nucleotide
homology of local viruses ranged from 96.1% to 99.9% whereas the nucleotide homology of that with other PCV2 strains ranged from 94.2% to 99.9%. Also, the amino acid identity of the capsid protein of the 10 local PCV2 ranged from 93.2% to 99.6%. Besides, eight PCV2 infected pigs were found to be co-infected with PRRSV Among them, six were co-infected with North American PRRSV genotypes, one with European genotype and one with both genotypes. To summarize, at least three different PCV2 sub-branches were identified and co-infection with both PRRSV and PCV2 were found in Hong Kong.
Apart from the phylogenetic analysis of the two viruses, four mutants with truncated putative signal sequence (L1 5.4, L1 5.5, L1 5.6 and L1 5.7) were constructed, based on the hydropathy profile of the PRRSV (strain AV), for DNA vaccine development against PRRSV Among all mutants, only mutants L15.6 and
L15.7 demonstrated a high level of expression in E. coli. when compared to the full-length (ORP5) construct and were expressed in transfected COS-7 cell lines. Both mutants triggered humoral responses in immunized mice and pigs. In mice, the antibody titer peaked at week 3 and was sustained for at least another three weeks while serum neutralizing antibody was detected in pigs at week 4 and maintained until week 8 at variable levels. Therefore, these two deletion mutants are potential candidates for DNA vaccine development.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:swine virus diseases genetic aspects dna vaccines
Date of Publication:01/01/2004