Molecular Evolution And Antigenic Drift Analysis Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),a member of Arterivirus Group,represents the causative agent of a new porcine disease which characterized the symptoms of fever,anorexia,abortion or premature delivery,mummied fetuses and stillbirths in the herds of the productive sows,high mortality and dyspnoea of piglets,and stunt of grower-finisher pigs.Since the disease brokeout first in America in 1987, great damages have took place in pig industry.This disease has been first reported in Taiwan in 1991,and then brokeout in Beijing in 1996,followed by the widely eruption of PRRS and then spread throughout our country.Especially since 2006,the genome of PRRSV has taken some mutation,resulting in the death of about 30 million pigs.The item covered with some pig farms distributed in the sourth of Hebei district.To carry out the epidemiological of PRRS, three consecutive years of randomness and targeted investigations has taken place.Then the Inactivated vaccine has been made.Finally some molecular biological characteristics of the PRRSV and bioinformatics of the Nsp2 and every main structural proteins were analysed.The contents included and the issues revealed in this paper were as follows.1.For the prevention and control of porcine reproductive and respiratory syndrome, reduce the enormous economic losses,more than 16 counties in Hebei province have been carried out the PRRS epidemiological investigation to determine the disease incidence, prevalence and control measures since 2006;On this basis,study on the inactivated oil emulsion vaccine--safety and immune efficacy test has been accomplished.2.From PRRS samples,cDNA fragments of the dieffrent genes of Nsp2 and ORF2-ORF7 were acquired espectively by RT-PCR after designing eight pairs of primers which were based on the ATCC VR-2332(U87392) sequences published from GenBank and optimizing the ampliyfing conditions.Eight gene fragments of 1200bp,1300bp,800bp, 780bp,600bp,600bp,550bp and 400bp were amplified via RT-PCR and eight recombinant plasmid pUC-2.1,pUC-2.2,pUC-2,pUC-3,pUC-4,pUC-5,pUC-6 and pUC-7 were constructed,respectively.3.In order to clarify the source and genetic background of HPPRRSV from different parts of China and from abroad,the sequences of the those genes of PRRSV were analyzed and compared with those of VR-2332,LV,CH-1a,BJ-4,HB-1,HB-2 et al.,and several Vaccine strains by MEGA and PHYLIP.All the phylograms of the main ORFs exhibited well bootstrap-supported topologies.The base composition was basically the same among different HPPRRSV strains in China,all isolates belong to American strain.Homology analysis showed that all HPPRRSV strains in China are closely related to the strain HB-1,Speculating that HPPRRSV was mutated from a domestic wild strain.The results of phylogenetic analysis of HPPRRSV can be seperated into three groups,and Jiangxi isolates distributed in various evolutionary branches,prove that the HPPRRSV originated in Jiangxi from molecular level.4.Through software such as DNaman,amino acid mutations were found to take place in 30 out of 36 antigen clusters,and two more epitopes disappeared;Using DNAstar Protean software process,the amino acid sequence prediction of antigenic analysis,compared with the ATCC VR-2332(U87392) and severay vaccine strains,showed that a great deal of antigenic drift phenomenon occurred to HP PRRSV,and explained the reasons of the low rate of protection.5.For 2006-2008 isolates,we employed codeml program in the PAML package for phylogenetic analysis based on dN/dS(nonsynonymous-to-synonymous rate ratio) ratios(ω). All of these ORFs[ORF1a(coded for Nsp2),ORF2a,ORF2b,ORF3,ORF4,ORF6,ORF7] except ORF5,gave negative selective results using different codon substitution models of PAML.For ORF5,Branch models(model = 2) detected significant positive selection of the 2006-2008 isolates group,with the result ofω= 3.50(background = 0.23;likelihood ratio test = 7.49,df = 1,P＜0.01).We submitted these ORF alignments to Datamonkey webserver and analyzed online.Integrative selection analysis based on the SLAC(0.1＜p＜0.2),FEL(P = 0.1) and REL(Bayes factor = 50) methods with the fit model HKY85 found 1 potential positively selected codons of ORF5.Results show that this site is under positive selection pressure, suggesting that this may allow PRRSV to adapt to new host cells,and ultimately new host species.6.A recombinant strain BL21(DE3)(pET-N) was constructed via molecular technology. The SDS-PAGE and Western blotting indicated that the N protein was highly expressed in Escherichia coli and the molecular weight of the N protein was about 17 kD.The recombinant strain was induced of different concentrations of IPTG and different temperature. Accumutation of the N protein was about 52.635%of total cellular protein.The N protein has been purified after SDS-PAGE,will be used for the further preparation of immunocolloidal gold test strip or the provision of basic ELISA KitIn summary,in this experiment we finished the analysis of Nap2 and other main sturctural genes of HPPRRSV from different areas of China,clarified the source,genetic background and evolution direction of HPPRRSV.The results set biological foundation of vaccine reseacrh,molecular diagnosis methods and measures of prevention.