Molecular Characterization Of Epidemic Prrsv Isolates And The Cultivation Of An Attenuated Vaccine Strain

Porcine reproductive and respiratory syndrome virus (PRRSV) is an emerging pathogen causing significant production and economic impacts on the swine industry worldwide. It mainly causes reproductive failure in the sows and respiratory distress in the piglets. The virus is an enveloped, single-stranded positive sense RNA virus, which has been classified in the family of Arteriviridaeand, and consists of9open reading frames. Different PRRSV isolates are antigenically, genetically and pathologically very heterogenic. In2006, there was a harrowing outbreak of PRRSV in the Mid-East China, which was characterized by durative fever (>41.0℃) and high mortality in herds of each age. The pathogen was identified as a new variant of PRRSV, with a unique hallmark in NSP2gene. In order to reveal the mechanism of pathogenicity differentiation among different PRRSV isolates and insight into the genetic variation and evolution of PRRSV, three PRRSV isolates were sequenced for complete genome and compared with each other and other American-type PRRSV isolates; PRRSV NT0801strain was using repetitive passaging of virus-infected cells and was evaluated the different pathogenicity between the NT0801F6and NT0801F70; the protective efficacy of avirulent PRRSV NT0801-F70strain was also evaluated.1. Molecular characterization of two different virulence Porcine reproductive and respiratory syndrome virus isolates from ChinaIn order to reveal the relationship between genetic variation and pathogenicity, highly pathogenic PRRSV BB0907and low pathogenic SY0909strains were sequenced, eight gene fragments of each isolate were amplified by RT-PCR and cloned into pEASY-Blunt vector for sequencing. Complete nucleotide sequence of genome of PRRSV BB0907and SY0909was obtained by splicing sequence of each fragment in order. The results showed that (1) The genomes of BB0907and SY0909isolates were15361bp and15369bp in length, containing nine open reading frames (ORFs), with189nt in the5prime UTR,191and199nt in the3prime UTR, including41and49nt poly (A) tail,GenBank:HQ315835, HQ315837.(2) The genome nucleotide sequence and derived amino acid sequence of SY0909were compared with other isolates in the world and phylogenetic trees were constructed. The results showed that SY0909and BB0907were in the same branch in phylogenetic trees. The nucleotide analyzes of SY0909showed that nt identity with BB0907is98.3%.(3) The alignment of amino acid sequence demonstrated that variations between S Y0909and BB0907were mainly focused on the Nsp2and GP5, the Nsp2gene of SY0909and BB0907have30amino acids deletion, GP5of SY0909isolate had a glycosylation site changed, which may be correlated with the variation of antigenicity and pathogenicity of virus. The above results suggested that variation of glycosylation site in GP5and the mutation in the whole genome may cause the virulence of SY0909strain was significantly lower than BB0907strain.2. Molecular characterization of a PRRSV Isolate NT0801from chinaPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the important pathogens causing serious economic losses to swine industry worldwide. PRRSV is genetically and pathologically heterogenous. PRRSV NT0801strain was isolated in a pig farm with clinical signs and had high pathogenesis in piglets. But its NSP2gene did not have30amino acids deletion as highly pathogenic JXA1strain. To elucidate the genetic characteristics of PRRSV NT0801strain, the full-length genome of NT0801isolates were sequenced and analyzed. The results showed that (1) the genome of PRRSV NT0801was15439bp in length, including29nt Poly(A) tail. Compared with the highly pathogenic JXA1strain, it had the nucleotide sequence identity of96.7%, amino acid sequence homology of97.2%and98.5%in GP3and GP5, respectively.(2) Phylogenetic analysis indicated that NT0801isolate was located between the traditional strain and the highly pathogenic strain. But no obvious recombination signal was observed, compared with other PRRSV isolates with different virulence.(3) The alignment of amino acid sequence of NT0801with other PRRSV isolates demonstrated that, three out of nine sites, being consistent with the highly pathogenic strain, were different from those in highly pathogenic while same as those in traditional strains and JXA1vaccine strain. And one out of9sites were same with JXA1vaccine strain exclusively, two out of9sites were different from all the strains. It indicated that PRRSV NT0801strain is closely related to highly pathogenic PRRSV, although there has no30amino acids deletions in NSP2region. The epidemic PRRSV strains variation results from the gene mutation. It should be useful for studying on the virulence genes located in different ORFs of PRRSV in the future.3. Passaging of PRRSV NT0801isolate in vitro and pathogenicity comparison of the different passages of the virusPRRSV NT0801isolate was repetitive passaged in Marc-145cells, CPE and stability of the virus titer was also observed. Twenty-four28-days old piglets free of PRRSV and PCV2were selected and randomly separated into4groups, six in each group. The first1-3were challenged individually with NT0801F6、F70and BB0907with intramuscular and intranasal injection. The group4were inoculated with DMEM and served as control. After challenged, the rectal temperatures of the pigs were recorded every day. The sera were collected for detection of the viremia. The dead and sacrificed pigs were autopsied and the lesions were examined. The results showed that (1) More than70%of cells showed CPE after passaged the virus to20generations, the TCID50was stable in10-70-10-7.25/ml.(2) NT0801F6and F70had relatively lower pathogenic ability and caused the pigs had1-2days fever with slight clinical signs compare with those in BB0907group, which caused apparent fever、clinical disease with great pathological changes and2out of6pigs were dead after infection.The pigs in the control group were quite healthy throughout the experiment.(3) The levels of virus load in serum showed that NT0801F6> NT0801F70> BB0907F6and the viremia duration of NT0801was longer than that of BB0907.(4) NT0801F6and F70infection groups produced a lower level of PRRSV ELISA antibody response than BB0907group.(5)The results of sequencing of Nsp2、GP3、GP5and N of NT0801F6and F70showed that a total of10amino acids were changed. It showed that the pathogenicity of NT0801F70was significant lower than NT0801F6, it lay the foundation for the research of PRRSV attenuated vaccine.4. Protective efficacy of avirulent PRRSV NT0801-F70strain in swine against challenge with highly pathogenic BB0907stainIn order to evaluate the protective efficacy of PRRSV NT0801F70strain which was attenuated in Marc-145cells against challenge with highly pathogenic BB0907stain, six28-days old healthy piglets were immunized with this strain and challenged with PRRSV BB0907virulent strain4weeks after vaccination, another six pigs which were not immunized were challenged with BB0907stain. The rectal temperature, virema, gross tissue lesion and microscope lesion were used to judgment the protective efficacy. The results showed that (1) All pigs after immunized with NT0801F70had no clinical signs; At 4days post challenged with BB0907, the rectal temperatures of pigs in the immunized group were up to40.3℃and lasted only2-3days. However, the temperatures of the pigs in no immunized group were up to41℃on the day post challenge and lasted12days.(2)The piglets could produce stable antibody after immunized with NT0801F70and the level of antibody was rapidly increasing after challenged with BB0907in the immunized group, which was also significantly higher than the no immunized group.(3) After challenged with BB0907, viremia wasn’t detected in the immunized group, but it was detected in the no immunized group.(4) After challenged with BB0907, the piglets in the no immunized group experienced the more serious tissue lesion compare with those in the immunized group. The results indicated that vaccination of NT0801-F70could provide a protection efficacy against PRRSV challenge in piglets. And this stain could be used as a new candidate stain of PRRSV attenuated vaccine.