Identification The Main Gene Determing The Infectivity Of HP-PRRSV HuN4-F112 Strain On PAMs In Vivo

Porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory problems in piglets, is a widely disseminated and economically important disease for the swine industry. Since its emergence in America in 1987, porcine reproductive and respiratory syndrome (PRRS) has been reported in many pig producing regions. Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), characterized by high fever (41℃), high illness rates (50-100%) and high death rates (20-100%) for pigs of all ages, emerged in central China in the year 2006 at first and spread quickly to almost every province of China and other Southeast Asian countries. For the great threat of HP-PRRSV, it is classified as Class A animal disease by People’s Republic of China Ministry of Agriculture which is needed for execution of compulsory immunization in China.After the outbreak of HP-PRRS. HP-PRRSV attenuated vaccine HuN4-F112 was developed by our laboratory. With the universal application of the HP-PRRSV vaccine in our country, HP-PRRS in China has been effectively controlled, however, the attenuated mechanism of the vaccine was unclear. It recently has been demonstrated that PRRSV tropism for macrophages and porcine alveolar macrophages (PAM) was the main host for replication of PRRSV in vivo. Surprisingly, in terms of the infection efficiency of PAM, there is a huge difference between HP-PRRSV HuN4-F112 and its parent strain. When the vaccine was passaged twice in PAMs nucleic acid couldn’t be detected by RT-RCR. Besides three PRRSV vaccine strains MLV, CH-1R and JXA1-R were also evaluated using the same method, and the results showed that they were deficient in infection to PAMs. The apoptosis of PAMs would be found after the host was infected by PRRSV, and then causing the typical symptom called interstitial pneumonia. So infection efficiency to PAMs is a key indicator to distinguish the virulence of PRRSV. These vaccines were passaged in Marc-145 cells for more than 80 generations. It’s possible that the mutations in the key gene leaded to a decrease of their capacity on infection to PAMs. It is very likely that it is the reasons why they are attenuated. It’s significant to identify the key gene in order to promote the prevention of PRRS.To determine the key genes about its virulence, two infectious clone cHuN4-F5 (HP-PPRSV wild strain) and cHuN4-F112 (HP-PRRSV vaccine strain) were used as the framework, and 10 chimeric viruses which exchanging the corresponding gene of the two strains were constructed and rescued. Copies of these chimeric viruses were detected by fluorescence quantitative PCR to make sure that the PAMS were infected by the same dose of each chimeric virus in the infection assay. After the PAMs were infected for 24 h, these cells were collected respectively, and PPRSV M protein monoclonal antibody and FITC labeled goat anti-mouse IgG were incubated with them in succession. Flow cytometry was utilized to evaluate the proportion of infected PAMs. The result revealed that the infection efficiency of chimeric virus which exchanging the gene NSP2 changes a lot. So NSP2 may be the key factor of the attenuated vaccine.To determine the key amino acids affecting the function of NSP2, infectious clones cHuN-F112 and RQNSP2 (the nsp2 gene of cHuN-F112 was replaced for the NSP2 gene of HuN-F5) were used as skeletons, and site-directed mutagenesis was performed, generating 10 chimeric viruses. Infection assay of these viruses were performed in PAMs, and the 893th and 979th amino acids in ORFla were verified to be the key amino acids.To explore the mechanism of the NSP2 gene on impacting the infection efficiency, PAMS were infected by four interchangeable recombinant virus NSP2 or ORF2-7 gene and their two parent HuN4-F5 and HuN4-F112 respectively at Moi= 2.0.2 h,12 h,24 h,36 h and 48 h post infection they were collected and washed several times.600 ng RNA were extracted and transcribed into cDNA. Fluorescence quantitative PCR were carried out to detect viral RNA copies. The results revealed that all these viruses invaded into PAMs 2 h post-infection. There is no significant difference in the quantity, indicating vaccine strain (HuN4-F112) still have the capacity of binding with PAM’s receptors. And 12-36 h post-infection, there is a rapid increase in the quantity of 6 viral RNA, suggesting the mutation in the NSP2 gene of HuN4-F112 also synthesized don’t affect the ability of RNA synthesize. Previous experimental results indicate that there were significant differences on the expression of virion proteins when PAM cells infected by HuN4-F5 and HuN4-F112 respectively.12 h post-infection, a large number of viral N protein could be detected by Western blotting with the PAM infected by HuN4-F5. But a trace amount of N protein could be detected until 36 h, and the expression of N protein was still in a small amount until 60 h. Thus it might be the mutation in nsp2 significantly reduced or delayed the Transcription of viral RNA into structural proteins.A long period of viremia was detected after the piglets were immunized by HP-PRRSV vaccine HuN4-F112. However, a few PAMs were infected in vitro, indicating in addition to PAM there must be other major tissues for viral replication. In this study,5 piglets were injured by HuN4-F112 and 3 piglets were infected by Hun4-F5. After 10 d,11 d,14 d and 16 d, they were sacrificed for collection the cells of heart, liver, spleen, kidneys, submandibular lymph nodes, hilar lymph nodes, inguinal lymph nodes, PBMC, tonsils, thymus. The proportion of these cells infected was evaluated by flow cytometry. And we found that tonsils were the main tissue for viral replication, there are some differences and similarities between the two groups. Both of the proportion of infected PBMC was almost zero and the liver cells and kidney cells was not significant infected. All the lymph nodes were infected by HuN4-F5 and HuN4-F112 in two groups. However,, in the vaccine group the heart was significantly infected which was not found in the pig infected by HuN4-F5. At the same time thymus which is the immune organs was infected by HuN4-F5 only. The results revealed that tonsil is the principal tissues for replication of PRRSV, which may be associated with persistent infection of PRRSV.In this research the key gene NSP2 which is related to PRRSV replication in PAM was identified and the attenuated underlying mechanism of vaccine was partly revealed. It is significant for the development of basic research and next generation of PRRSV vaccine. We also identified the tissue for storage and replication of PRRSV, it’s meaningful for clinical diagnosis of the PRRSV persistent infection.