Study On The Pathogenicity Of Porcine Reproductive And Respiratory Syndrome Virus NADC30-like Strain And The Function Of Nsp7β

Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease,caused by porcine reproductive and respiratory syndrome virus(PRRSV).In 1995,PRRSV was first confirmed and reported in China.Since then,it has been prevalent for nearly 26 years in China and caused huge economic losses in pig industry,especially the outbreak of highly pathogenic PRRSV(HP-PRRSV)in 2006.Due to its high infection rate,high morbidity and high mortality,HP-PRRSV rapidly become an epidemic strain.Since 2012,PRRSV NADC30-like strains have been reported and isolated in many places of China.The characteristic of its genetically extensive recombination further increases the complexity of the prevention and control of PRRSV.In recent years,NADC30-like strains have gradually become epidemic strains in the field.The recombinant PRRSV strains exhibits different genomic characteristics,which leads to the difference on pathogenicity among different strains.The proliferative ability of viruses is one of the key factors that affect its pathogenicity and virulence.It is of important scientific significance to analyze the pathogenicity and functions of proteins of different PRRSV strains.In this study,the pathogenicity of NADC30-like strain HNhx to piglets with different weeks of age was analyzed,and the function of PRRSV Nsp7β was explored.The results of this study will provide insights into the study of prevention and control of PRRSV.1.Study on the pathogenicity of PRRSV NADC3 0-like strainIn order to analyze the pathogenicity of NADC30-like strain HNhx,PRRSV-negative piglets of different weeks old were inoculated with HNhx.The clinical manifestations,body temperature changes,weight gain,viremia and serum antibody levels were assessed.The results show that compared with the control group,the piglets inoculated with NADC30-like strain HNhx exhibited persistent high fever,decreased weight gain and even negative growth,as well as anorexia and dyspnea.The mortality rates of 5-week-old,6-week-old,7-week-old,and 8-week-old groups were 60%,80%,40%and 33.3%,respectively.After 3 days of inoculation,the viremia in serum of each groups with HNhx inoculation was significantly increased,and the viral load of tissues were positive.After 14 days of inoculation,the level of specific antibody in serum turned to be positive.It is worth noting that the mortality rate of piglets in the 6-week-old group was as high as 80%,fever above 40.5℃ on the first day after inoculation and lasting for 18 days,weight gain negative in the first week after inoculation.Moreover,the viremia in serum was always at a high level,and the viral load in lung tissue was significantly higher than that of other groups.However,the mortality of piglets in the 8-week-old group was lower than that of other groups,fever above 40.5℃ on the third day after inoculation and lasting for 11 days,and the level of viremia in serum on the 3rd day was significantly lower than that in other groups.Therefore,the age of inoculated piglets might affect the pathogenicity of the virus to some extent.In summary,NADC30-like strain HNhx can cause high death rates of piglets at different weeks of age,and showed high pathogenicity.2.Study on the function of PRRSV Nsp7βPRRSV ORFla and ORF1b regions encode at least 16 different non-structural proteins(Nsps).Nsp1α/β,Nsp2,Nsp4 and Nsp11 have been reported to inhibit host innate immune responses.Nsp12 is involved in viral RNA synthesis and inhibits IFN-I expression through RIG-I/NF-kB pathway.The researches on the function of Nsps is helpful to understand the biological characteristics of PRRSV.But the function of PRRSV Nsp7β protein remains unclear.At present,it is known that Nsp7 is one of the highly conserved non-structural proteins of PRRSV.Studies have shown that the deletion of PRRSV Nsp7 and Nsp7α/β are fatal to virus.It is speculated that Nsp7β plays an important role in the life cycle of the virus.Our experiment will focus on the function of Nsp7β.In this study,Pull down,SDS-PAGE electrophoresis,silver staining and mass spectrometry were used to analyze the proteins that may interact with Nsp7β.It was found that Nsp7β interacted with host protein annexin2(AnxA2),which was further confirmed by co-immunoprecipitation.Then,it was found out that PRRSV infection up-regulated the expression of AnxA2,and knockdown of AnxA2 inhibited the proliferation of PRRSV.Secondly,antibody inhibition test and recombinant protein competitive test confirmed that the host protein AnxA2 promoted virus proliferation.Finally,we explored the possible role of Nsp7β in the innate immune response.It was found out that nsp7β could significantly inhibit IFN-α-mediated ISRE promoter activaties by using dual luciferase reporter methods.Furthermore,it was confirmed that PRRSV nsp7β could significantly inhibit the mRNA expression of ISG54 and ISG56 using fluorescent quantitative PCR.In conclusion,these results demonstrate that NADC30-like strain HNhx shows high pathogenicity to piglets of different weeks of age.PRRSV Nsp7β interacts with the host protein AnxA2,which promotes virus proliferation.Nsp7β inhibits the expression of IFN stimulated genes such as ISG54.These findings are helpful to understand the pathogenesis of PRRSV and the molecular mechanism of immunosuppression,provide the basis for the prevention and control of PRRSV.