Study On Genetic Variability Of PRRSV In China And Quantitative Interactome Of Prrsv Nsp2 Protien

Porcine reproductive and respiratory syndrome(PRRS) is a viral infectious disease leading to serious harm to the global pig industry caused by PRRS virus, which is typically characterized by apparent reproductive failure in pregnant sows, as well as severe respiratory disease and growth retardation in piglets. Especially, the “high fever” syndrome occurred in 2006 has been a huge hit to our country’s pig industry caused by highly pathogenic PRRSV(HP-PRRSV). After that, PRRSV occurred in sporadic outbreaks of varying degrees in 2009, 2011 and 2015. Moreover, the NADC30-like strains from North America led to sow abortion or premature birth in different provinces of China in 2015. Thus, it is very necessary to produce a tracking survey for the situation of PRRSV epidemiology, genovariation and homologous recombination, and to understand the genetic variation rules of PRRSV, providing fundamental basis for the prevention and control of PRRSV.PRRSV nsp2, as the largest nonstructural protein(nsp), is one of the highest mutation rates in PRRSV genome. The genetic evolution and variation rules for PRRSV could be grasped through monitoring the genovariation in nsp2 gene, and, more importantly, nsp2 plays vital roles in viral replication and immunoregulation. Therefore, this study has used the quantitative interactome methodology to identify the viral and cellular proteins that interact with nsp2 by LC-MS/MS. The main research works were as following: 1. The epidemiological investigation of PRRSV and the isolation and identification of novel PRRSV isolatesWe have collected 1909 clinical samples in 17 provinces of Southeast China from 2010 to 2015 for the epidemiological investigation of PRRSV. Our detecting results demonstrated that the positive rate of classical PRRSV infection was only 3.87%, while the positive rate of HP-PRRSV was up to 43.48% with decrease by degrees. The tendency of PRRSV epidemic situation of our country has gradually changed the dominant epidemic of HP-PRRSV strains to the dominant epidemic of NADC30-like strains, with the coinfection situation of these two subgenotypes. Meanwhile, we detected PRRSV in fecal sample with a high positive rate of 43.97%, and firstly isolated HP-PRRSV strain WUH4 from piglet stool samples, advancing the possibility of PRRSV transmission via the fecal-oral route. Moreover, the novel PRRSV strains: NADC30-like strains had been detected in clinical samples after 2014 with a higher positive rate of 63.04%. In the PRRSV NADC30-like positive samples, we have successfully isolated two isolates: WUH5 and WUH6. Through the sequence alignment analysis of complete genome and all viral proteins, there is a discontinuous deletion of 131 amino acids in the HV region of nsp2 both in WUH5 and WUH6 strains. Furthermore, the recombination analysis has revealed that WUH6 is a recombinant strain, with the result of recombination between strain NADC30 and strain JXA1. The sequence in middle recombinant region of WUH6 is highly homologous to that of JXA1, while the sequences in other regions of WUH6 are highly homologous to those of NADC30. 2. Quantitative interactome analysis of PRRSV nsp2In this study, a quantitative interactome approach based on immunoprecipitation and stable isotope labeling with amino acids in cell culture(SILAC) was performed to identify nsp2-interacting proteins under PRRSV-infected condition using LC-MS/MS. Nine viral proteins and 62 cellular proteins were identified as potential nsp2-interacting partners. And an interactome network of nsp2 was generated using Ingenuity Pathway Analysis(IPA) software.Our data demonstrate that the PRRSV nsp1α, nsp1β, and nucleocapsid proteins all interact directly with nsp2 both under PRRSV-infected and overexpression conditions, while nsp2 can interact with GP5 only under PRRSV-infected condition. Meanwhile, our results indicate that nsp2 specifically interacts with YWHAB, YWHAZ and MARK2 under both PRRSV-infected and overexpression conditions, while nsp2 can interact vimentin only under PRRSV-infected condition. However, interestingly, cellular vimentin, a known receptor for PRRSV, forms a complex with nsp2 by using viral nucleocapsid protein as an intermediate. Taken together, nsp2 and vimentin could interact indirectly with each other via the PRRSV N protein. Furthermore, knockdown of YWHAB, YWHAZ, MARK2 or vimentin could resulted in significant reduction of PRRSV replication in Marc-145 cells.