Porcine circovirus type 2 (PCV2) is the primary causative agent of post-weaning multisystemic wasting syndrome in pigs. To generate a genetic marker strain of PCV2, the full-length genome of the virus was amplified us...
详细信息
Porcine circovirus type 2 (PCV2) is the primary causative agent of post-weaning multisystemic wasting syndrome in pigs. To generate a genetic marker strain of PCV2, the full-length genome of the virus was amplified using PCR, and two copies of the genome were ligated in tandem to construct an infectious molecular clone. A Sal I restriction enzyme site was inserted into the clone as a genetic marker, and the recombinant plasmid was transfected into porcine kidney cells to generate mutant virus. The antigenicity of the recovered virus was confirmed by immunoperoxidase monolayer assay. The viral antigen was visualized in the nucleus and cytoplasm of the virus-infected cells. The viral genome could be differentiated from the wild-type parent by PCR and restriction fragment length polymorphism (PCR-RFLP). The mutant virus was stable on multiplication through 60 passages in cell culture;the highest titer reached was 106.6 TCID50/mL. Four 35-day-old unvaccinated piglets were inoculated with the virus by the intranasal and intravenous routes. Two of the virus-infected pigs developed high temperatures, progressive weight loss, and swollen lymph nodes. The viral antigen and nucleic acid were detected in numerous tissues of the pigs. The results indicate that the genetic marker strain should be a useful tool in studies on pathogenesis, vaccination, and molecular diagnosis of PCV2.
Six porcine epidemic diarrhea viruses (PEDVs) were isolated from the fecal samples of piglets infected with PEDV in 2006 in China. The membrane (M) protein genes of six PEDV isolates were amplified by reverse transcri...
详细信息
Six porcine epidemic diarrhea viruses (PEDVs) were isolated from the fecal samples of piglets infected with PEDV in 2006 in China. The membrane (M) protein genes of six PEDV isolates were amplified by reverse transcriptase-poly- merase chain reaction (RT-PCR), then cloned, sequenced, and compared with each other as well as those ten PEDV reference strains. The M protein genes of six Chinese PEDV isolates consisted of 692 nucleotides containing a single open reading frame (ORF) of 681 nucleotides, which encoded a 226aa-long peptide. The conserved intergenic motif (ATAAAC), as previously rec- ognized in Br1/87, was found in the 5 nucleotides upstream of the initiator ATG of M protein genes of six Chinese PEDV iso- lates. The hexamer motif was also found in CV777, JMe2, LZC, and QH. The M protein of six isolates had three main trans- membrane domains (aa20～38, aa43～65, aa75～97). The M protein of one isolate, CH/IMT/06, had one potential glycosylation site, but those of the other five isolates had two. The glycosylation sequence Asn-Phe-Thr was highly conserved in the M pro- teins of six PEDV isolates. The six PEDV isolates showed nucleotide sequence homology between 98.8 and 100 % and deduced amino acid sequence homology between 98.2 and 100 % with each other. The nucleotide and amino acid identity of M protein genes between the six PEDV isolates and ten reference PEDV strains varied from 97.2 to 99.4 % and 96.9 to 100 %, respec- tively. On the basis of the phylogenetic relationship of M protein genes, six Chinese PEDV isolates composed of a separate clus- ter including one Chinese strain JS-2004-02, however, not including the Chinese strain LJB/03. These results demonstrated that there was a new genotype of PEDV prevailing in China.
暂无评论