Screening Of Attenuated Vaccine Candidates Of Porcine Reproductive And Respiratory Syndrome Virus And Risk Analysis Of Reversion To Virulence

Porcine reproductive and respiratory syndrome(PRRS)first emerged in North America and Europe in 1990s has caused significant economic loss to global swine industry during the past thirty years.The hallmark clinical signs of PRRS are respiratory disease of piglets and growing pigs and reproductive failure of sows.Porcine reproductive and respiratory syndrome virus(PRRSV)was determined to be the causative agent of PRRS.Currently,vaccination is the most efficient strategy in the prevention of PRRS.In China,classic PRRSV was first isolated in 1996.Since 2006,a large number of new strains emerged in China,although many inactivated and live-attenuated PRRS vaccines were used to combat PRRS in China.Due to the high mutation rate and strong immunosuppression nature of PRRSV,the inactivated vaccines have failed to elicit enough protective immunity even against homologous virus.By the contrast,the live-attenuation vaccines are able to combat clinical disease of PRRS,although they are unable to provide enough cross-protection against heterologous strains.The safety concern is another drawback of live-attenuated vaccines,such as the spread of vaccine strains and the incidence of reversion to virulence.Thus,it is still huge challenge for the prevention and control of PRRS in China.The initial aim of this study is to isolate an epidemic strain of PRRSV and obtain an attenuated vaccine candidate by using traditional in vitro cell passage methods to reduce the virulence of virus and retain its good immunogenicity.Subsequently,animal experiments were carried out to verify the safety and efficacy of vaccine candidates.Two attenuated candidates PRRSV/0007-01-P85 and JXM105 did not cause any disease to piglets and provide 100%protection against HP-PRRSV JX143 strain.However,they both showed virulence reversion at different degree during in-vivo passage in piglets.Subsequently,we analyzed PRRSV quasispecies during in-vivo passage and identified several mutations that may be responsible for their virulence reversion.The correlation of these mutations with PRRSV pathogenicity was further assessed by reverse genetics and animal study.Finally,we discussed the mechanism of virulence reversion,which may provide a theoretical foundation for artificial attenuated PRRSV.1 PRRSV isolation,attenuation by passage and characterization in vitroOne PRRSV strain PRRSV/0007-01 was original isolated from a lung sample collected from a pig with PRRS-like symptom in 2012.This virus strain contains a HP-PRRSV characteristic 29+1 amino acids deletion and 21 amino acids deletion in the nsp2 coding region.In line with deletions in nsp2,the phylogenetic analysis showed that this strain located between HP-PRRSV strains and classical PRRSV strains.To attenuate this virus,we passaged PRRSV/0007-01 115 times in MA104 cells.Viral genomic sequences and viral titers were determined every 10 passages.These results suggested that the fitness of PRRSV/0007-01 in MA104 cells increased after serial passage.Of note,the number of mutations accumulated dramatically after passage 80.Based on our results on virus growth in vitro and genomic sequence analysis of passaged viruses,P85 and P115 were chosen as vaccine candidates.Both candidates showed similar growth property to parental strain in cells while passaged viruses showed higher viral peak titer than parental strain.Their full-length genomic sequences were determined using next-generation sequencing method.In comparison with their parental virus,P85 virus contains 50 nucleotide substitutions,while P115 virus contains 60 nucleotide substitutions.Among these mutations,21 mutations lead to amino acid substitutions in both viruses.In conclusion,we isolated PRRSV/0007-01,passaged it 115 times in MA014 cells.The P85 and P115 viruses were selected for further characterization in animal experiments.2 Evaluation of the safety and efficacy of attenuated PRRSV strainsThe live-attenuated PRRS vaccine candidates are required to be tested for their safety and efficacy in pigs and the risk of virulence reversion by back passages in piglets.To evaluate the safety and protective efficacy of live-attenuated vaccine candidates PRRSV/0007-01-P85 and PRRSV/0007-01-P115,the pathogenesis of parental strain was evaluated,and a challenge model was established by using JX143 strain.Three candidates included JXM105 strain were inoculated into four-week old piglets,respectively.The safety of these candidates was determined based on the clinical symptoms and lung lesion of infected animals during 21 days post infection.Some immunized pigs were challenged with JX143 on 28 days post immunization.The protection efficacy of candidates was evaluated primarily based on various indicators.The risk of virulence reversion of PRRSV/0007-01-P85 and JXM105 was assessed through passaging vaccine candidates in piglets.PRRSV/0007-01-P85 and serum collected from the piglet infected with PRRSV/0007-01-P85 serial passaged five times in piglet were inoculated to the pregnant sows to further evaluate their impact on the reproductive performance of sows.These results showed that PRRSV/0007-01-P3 resulted in 100%morbidity and 40%mortality,while JX143 showed 100%morbidity and 90%mortality.All candidates showed no pathogenic to pigs after serial passage in cells.PRRSV/0007-01-P85 and JXM105 could provide fully protection against JX143 challenge,while P115 strain provided partial protection.PRRSV/0007-01-P85 did not show virulence reversion within five passages in piglets but its 5th passage serum affected reproductive performance of sows seriously which resulted in no live piglets at the end day of experiment.JXM105 reverted to virulence at passage 4 in piglets.These results suggested that both strains were safe and effective in piglets,but both of them showed virulence reversion potential at different degrees.3 Study on the mechanism of virulence reversion of attenuated PRRSV strainsPoint mutations and viral quasispecies were considered to play critical roles in viral pathogenesis.In this chapter,PRRSV quasispecies diversity during viral passage was tracked and analyzed by next generation sequencing.Several potential virulence-related positions in JXM105 and PRRSV/0007-01-P85 were identified by comparing the mutations and their dynamic changes in consensus sequence.Our results showed that JXM105 had four potential virulence-related positions located at nsp1?-52?nsp10-275?nsp 10-313 and GP5a-2,respectively.PRRSV/0007-01-P85 reverted to virulent type at 7 positions including nsp1?-113?nsp2-722?nsp4-20?nsp9-246?nsp9-302?nsp9-670 and nsp10-72.The mutation frequencies of these positions were increased,and the virulent type of sequence became the dominant in viral quasispecies during passage.Using the reverse genetic system of PRRSV/0007-01,a mutant virus containing the attenuated type of amino acids at these seven residues was constructed.In comparison with parental virus,the mutant virus showed reduced pathogenicity in 3-week old piglets.These results showed that PRRSV evolves rapidly in animals,the accumulation of reverted mutations in virulence-related positions and becoming dominant in quasispecies is one of the critical causes for virulence reversion of PRRSV attenuated strains.Taken together,in this study,a high pathogenic PRRSV/0007-01 was isolated.Two attenuated viruses PRRSV/0007-01-P85 and P115 were obtained by passaging PRRSV/0007-01 strain on MA104 cells for 85 and 115 passages respectively.Both of them were safe in piglets.PRRSV/0007-01-P85 and JXM105 provided fully protection against JX143 strain,while PRRSV/0007-01-P115 only provided partial protection.PRRSV/0007-01-P85 did not show virulence reversion during five back passages in piglets,however its 5th back passage serum impacted sow reproductive performance seriously,while JXM105 strain reverted to virulence at passage 4 in piglets.Both attenuated strains showed the risk of virulence reversion.The evolution of PRRSV quasispecies diversity during in-vivo passage was analyzed by next generation sequencing and several potential virulence-related positions in JXM105 and PRRSV/0007-01-P85 were identified,and PRRSV/0007-01-P85 virulence mutants were further verified using reverse genetic system subsequently.The results of this study may provide a theoretical foundation for PRRS vaccine development and artificial attenuated PRRSV.