Epidemiological Investigation And Mutants Pathogenicity Of Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2), a member of the genus Circovirus of the Circoviridae family, is a small DNA virus with negative-sense circular genome (1767 or 1768bp in length). Two major open reading frames (ORFs) have been identified for PCV2, ORF1, named the rep gene, encodes a protein with a molecular weight of 35.7 kDa involved in virus replication, and ORF2, named the cap gene, encodes a immunogenic capsid protein with a molecular weight of 27.8 kDa. There are still nine potential ORFs besides ORF1 and ORF2 in the genome. PCV2 is the primary cause of postweaning multisystemic wasting syndrome (PMWS) in the pig. PCV2, resulting in immunosuppression due to lymphocyte depletion. The objects of study were to undertake systemic serological and epidemiological investigations of PCV2 infections in pig herds, human and other spicies, and to investigate the pathogenicity of potential ORFs (except ORF1 and 2) in genome of PCV2.Using PCV-free PK-15 cell line, ten isolates of porcine circovirus type 2 were isolated from 11 districts in China Zhejiang province. The isolated virus is a 17nm in diameter and icosahedral virion by transmission electron microscope. The complete genome of ten isolates is composed of 1767 nucleotides.A total of 4307 serum samples were collected from 104 pig farms in Zhejiang province from 2000 to 2004 years. Serum samples were tested for the presence of antibodies against porcine circovirus 2 (PCV2) using indirect immunefluorescent sassay (IFA). Antibodies against PCV2 were detected in all 104 pig farms. Overall seroprevalences were 68.10% for all, 66.88% for sows, 82.10% for post-weaning piglets, 44.83% for Landrace sows and 64.28% for Landrace piglets. The seroprevalences of Landrace sows was higher than Yorkshire and Duroc sows. In the same pig herd, the higher seroprevalences of antibodies against PCV2 were; the lower seroprevalences of antibodies against PRRSV and PrV were. On the contrary, there were positive relation between rate of PCV2 infection and PRRSV infection in the pig farms without PRRSV vaccine. These results revealed that the infection of PCV2 was prevalent in the pig herds, the susceptibility of the various pig species was diverse to PCV2, the PCV2 infection reduced the immune responses to vaccine, and PCV2 infection enhanced PRRSV infection.No antibody against PCV2 were detected in 227 duck serum samples, 204 chicken serum samples, 493 cattle serum samples and 38 goat serum samples in Zhejiang province. By screening 2,352 human serum samples, using IFA and IMPA, the antibody against PCV2-like virus was detected in 689 human serum samples collected from 2004 to 2005 years. The seroprevalence of PCV2 was 29.3%. Statistical analysis showed that PCV2 seroprevalence in 2005 increased significantly, compared with that in 2004 (p<0.01). Logistic regression analysis indicated that positive ratios of PCV2 antibody in women was higher than that in men, and the percent of anti-PCV2 like virus antibody in 40 to 49 years old humans was higher than that in other human populations. The seroprevalence of PCV2 like virus in human populations was not associated with elevated ALT level and HBV infection significantly.The genomes of PCV2-HZ0201 or with mutated ORFs (ORF3/4, ORF5 to 11) were amplified by the special primers and cloned into pMD18T vector. The circular DNA of PCV2 normal infectious clones (IC), PCV2-MF3/4 (ORF3 and ORF4 mutation), PCV2-MF3-4 (ORF3-4 single mutation, kept in our laboratory) and PCV2-MF5 to 11 (ORF5-11 single mutation) were generated by ligating the genome with normal or mutational sequence from recombinant plasmids. These circular DNA clones were transfected into PK-15 cells, respectively. Transfected cells were passaged 6 generations. Transfected and passaged cells were detected by IFA and PCR. The results indicated that IC, PCV2-MF3, PCV2-MF4, PCV2-MF5, PCV2-MF9, PCV2-MF10 and PCV2-MF11 could produce progeny; whereas PCV2-MF3/4, PCV2-MF6, PCV2-MF7 and PCV2-MF8 could not produce PCV2 virions. The TCID50 of rescued virus was the same with wild PCV2. Sequenceing showed the mutated site in PCV2-MF5, PCV2-MF9, PCV2-MF10 and PCV2-MF11 still existed in the progeny virus. The results suggested that ORFs5, 9, 10 and 11 were not necessary for virus replication, whereas ORF3-ORF4, ORFs 6, 7 and 8 were essential in virus repllication.To evaluate the roles of ORFs 3, 4, 5, 10 and 11 in viral pathogenicity in vivo, 8-week-old BABL/c mice inoculated intraperitoneally and intranasally with PCV2-HZ0201 virions, infectious clones (IC) and mutants (PCV2-MF3, PCV2-MF4, PCV2-MF5, PCV2-MF9, PCV2-MF10 and PCV2-MF11). Mice inoculated with HZ0201, IC, PCV2-MF3 and PCV2-MF4 had seroconversion to PCV2 at 14 days postinoculation (dpi) and reached a peak at 28 dpi. Mice inoculated with PCV2-MF5, MF9, MF10, and MF11 had seroconversion to PCV2 at 21dpi and reached a peak at 35 dpi. At 21 dpi, mice infected with the PCV2-HZ0201, normal infectious clones, PCV2-MF9 and PCV2-MF10 showed significant down-regulation of CD3~+CD4~+, CD4~+CD8~+ and CD3~+CD8~+ T cell subsets, and significant up-regulation of apoptotic cells in spleen compared to negative control mice (p<0.05). The apoptosis was induced by Caspase-3 and Caspase-8 pathway in mice infected with the PCV2-HZ0201, normal infectious clones, PCV2-MF9 and PCV2-MF10. At 42 dpi, the above-mentioned lesions discovered. There were not significant down-regulation of CD3~+CD4~+, CD4~+CD8~+ and CD3~+CD8~+ T cell subsets in spleen, and apoptotic cell up-regulation in mouse spleen inoculated with PCV2-MF3 and PCV3-MF5 at 21 and 42 dpi (p>0.05). There were significant down-regulation of CD3~+CD4~+, CD4~+CD8~+ and CD3~+CD8~+ T cell subsets, and significant up-regulation of apoptotic cells in mouse spleen infected with PCV2-MF11 at 42 dpi but not 21 dpi (p>0.05). These data suggest that ORF3, ORF5 and ORF11 play an important role in viral pathogenesis, ORF9, ORF10 are non-necessary to viral pathogenesis, and ORF4 should be identified in further study.