Evolution Of Porcine Reproductive And Respiratory Syndrome Virus Under Antibody Selective Pressures

Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is a highly contagious disease in pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). Since the first outbreak in the United States 1987,it has spread to the principal pigs cultivation countries around the world currently. In summer of 2006, pigs "high fever" disease characterized by high fever took place in south of China in hot and humid season, the study showes it was caused mainly by porcine reproductive and respiratory syndrome virus as the main pathogen associated with a variety of pathogenic infection and secondary infection of other pathogen at the same time. The "high fever" epidemic in Shandong also occured and caused unprecedented loss since September 2006. In this study, a pig "high fever" of one big pig farm in Shandong was diagnosised and a highly pathogenic porcine reproductive and respiratory syndrome virus was isolated and identified, named for the SD0612. The evolution of it's genetic variation and quasispecies diversity under the antibody selective pressure was studied.1,The isolation and identification of porcine reproductive and respiratory syndrome virus SD0612 and it's pathogenicity testUsing DNA hybridization combined with PCR and RT-PCR, a "high fever" of a pig farms in Shandong was diagnosed and the main pathogen is determined as porcine reproductive and respiratory syndrome virus. Marc-145 cells was used to isolate the virus and a highly pathogenic porcine reproductive and respiratory syndrome virus was isolated and named for the SD0612. its main gene NSP2, ORF3-ORF7 was cloned and sequenced, the results showed that the virus was a deletion mutation with NSP2 missing 90 bp, sequence analysis showed it share highly homologous with other representative strains in the same period. pathogenicity test of the strain for the 1 month old piglets showed that mortality rate up to 100%, indicating that SD0612 is highly pathogenic variant.2,Evolution of porcine reproductive and respiratory syndrome virus SD0612 under antibody selective pressuresTo study effects of antibody immune selective pressures on antigenic evolutions of porcine reproductive and respiratory syndrome virus (PRRSV), the field strain SD0612 was continuously passed in Marc-145 cell cultures without or with anti-SD0612 pig sera in 6 separated lineages for 40 passages. The resistant virus of the lineage F with anti-SD0612 sera at 40th passage was collected as strain F40 and used for the second step of 40 passages again in another 6 lineages with or without anti-F40 sera. The resistant virus of the lineage e at 40th passage was collected to prepare anti-e40 pig sera. Infected cells were collected for extraction of viral RNA every 10 passages. ORF3, 4, 5 genes were amplified, cloned and sequenced. Cross viral neutralization indicated that serological homology between SD0612 and its two resistant mutants F40 or e40 were 62.5%, 50%; homology between F40 and e40 was 80%. Sequence analysis indicated that non-synonymous vs synonymous mutation ratios (NS/S) of ORF3 , 4, 5 in two steps were 11.0 and 3.5, 0.5 and 0.67, 13.0 and 4.0 respectively in passages with anti-PRRSV sera, but only in the range of 0.33-1.4 in passages without anti-PRRSV sera. It indicated that anti-PRRSV sera had strong immune selective pressures on viral evolution in its ORF3 and ORF5 genes. Besides, there were total 14 stable NS mutation sites in ORF5 and 4 in ORF3 in 3 lineages during the 80 passages with anti-PRRSV sera, which were not detected in passages without specific antibodies.3,Comparison of PRRSV quasispecies diversity and it's evolution under the antibody selective pressureTo study the evolution of quasispecies diversity for PRRSV under the antibody selective pressvre, PRRSV wild strain SD0612 was passed in Marc-145 cells for two series, the series ? was cultured with medium without anti-SD0612 serum, including A, B, C three parallel passage lines; while series ?? was cultured with medium added proportional anti-SD0612 serum, including D, E, F three parallel passage. After 40 continuous passages we obtained the derivants A40-F40. the F40 was passed in a second round passage as above, after 40 continuous passages a40-f40 were obtained. 13 strains from three generation(the original of SD0612, the first-generation A40-F40, the second-generation a40-f40) were cultured and it's ORF5 gene was amplified and cloned, All positive clones of each strain were sequenced and analyzed with DNAStar software. The results show that SD0612 is a population composed of different quasispecies which nucleic acid homology ranging from 97.7% -100%, among the 60 total sequence 17 sequences were the same and account for the main advantages of the populations; in the two passage trial, the quasispecies diversity are declining while the rate for advantage quasispecies is on the rise for group ?? with antibody, the result for group ? without antibody is on the contrast; there are more points mutation for group ? without antibody than group ?? with antibody. The advantage quasispecies of group ?? with antibody shared lower and lower identity with the original strain with the continuous passage. The study indicates that antibodies selective pressure can significantly affect the quasispecies diversity of PRRSV,particularly causing the changes of advantage quasispecies .4,The comparision of viremia and the antibody response in pigs between SD0612 original strain and different mutants in the passage experimentsTo compare the dynamic of viremia and antibody response in pigs between SD0612 original strain and it's mutants in the selective pressure passage experiment, two animal experiments were conducted in this study. In the first experiment, 8 healthy pigs were divided into four groups as the SD0612 original strain infection group, the first passage mutant F40 infection group and the second passage mutant e40 infection group and negative control group, antibody was determined by ELISA kit in 1w, 2w, 3w, 4w, 5w after challenge, and virus concentration in blood was tested by fluorescence quantitative PCR. In the 40th days all group was inoculated with virulent PRRSV and temperature was measured and incidence of performance was observed daily. The results showed that: SD0612 original strain infection can induce high antibody response and the antybody can resistance to virulent virus attack. Antibodies of the F40 infection group and e40 infection group is later than the original strain group. The antibody caused by F40 can protect the pig withstand the virulent virus attack, while the e40 infection group all died. Showing e40 antigencity have taken place great changes.The dynamic of viremia and antibody response between F40 and it's derivants a40 in the passage without antibody was compared in the second experiment. 15 pigs were divided into three groups as F40 infection group, a40 infection group and negative control group. Antibody was determined by ELISA kit in 1w, 2w, 3w, 4w, 5w after challenge, and virus concentration in blood was tested with TCID50 in 100ul blood serum. In the 40th days all group was inoculated with virulent PRRSV and temperature was measured and incidence of performance was observed daily. The results showed that the infection of F40 and a40 can induce high antibody titer and protect the pig withstand the virulent virus attack, while the viremia level of the F40 infection group is higher and earlier than the a40 infection group. 3 pigs of the negative control group died at last.