Epidemiological Investigation Of Lineage 3 PRRSV In China And Preparation Of Monoclonal Antibodies Against Lineage 3 PRRSV

Porcine reproductive and respiratory syndrome?PRRS?is caused by porcine reproductive and respiratory syndrome virus?PRRSV?.Piglet respiratory and sow reproductive disorders are the main clinical symptoms.PRRSV was first isolated in our country in 1996,causing huge economic losses to the pig industry in our country.With the continuous evolution of PRRSV in China,its diversity has become more complicated.Lineage 1 and 8 PRRSV were the main epidemic strains,and Lineage 3 PRRSV continued to exist.Lineage 3 PRRSV?QYYZ-like?has been prevalent mainly in southern China since it was first discovered in 2010,but there have been recent reports of isolation of this type of virus in other parts of China.The early clinical manifestations of Lineage 3 PRRSV strains are mild,so they have not received enough attention.Subsequent research reports found that the virulence of Lineage 3 PRRSV after recombination with other lineage strains increased significantly,so it is very import ant to strengthen the monitoring of Lineage 3 PRRSV.Monoclonal antibodies play an important role in animal disease treatment,diagnosis,and pathogen biology research.The screening of monoclonal antibodies against PRRSV with blocking effect is an essential material for establishing PRRSV antibody blocking ELISA.Currently,most PRRSV monoclonal antibodies reported in China are directed against HP-PRRSV and Lineage 3 PRRSV monoclonal antibodies have not been reported.Therefore,the preparation of Lineage 3 PRRSV monoclonal antibodies has been used to understand the structure and function of the PRRSV genome and further use of monoclonal antibodies to prepare PRRSV antibodies Blocking kits are important.From 2017 to 2019,this study performed RT-PCR on 325 suspected PRRSV disease materials from 16 provinces,municipalities and autonomous regions.The test results showed that there were103 positive PRRSV samples,of which 16 were Lineage 3 samples,accounting for about 16%of the total positive samples.In this study,ORF5 gene sequencing was performed on 16 Lineage 3PRRSV positive samples,and 7 samples?LCL15,XNF10,XNF53,XNF60,XNF74,XNF78,XNF229?were selected for whole genome sequencing,and the obtained sequences were compared and analyse.The nucleotide sequence homology comparison results showed that the nucleotide homology of the ORF5 gene of 16 PRRSV strains was 62.9%to 65.0%with that of the European representative strain Lelystad virus,and was homologous with the representative American str ain VR2332.It has the highest homology with QYYZ strain?Lineage 3?among the representative strains in the Americas,ranging from 91.2%to 92.0%.Whole-genome homology analysis found that they have low homology.LCL15 and Lineage 1 represent the highest homology of NADC30,XNF53,XNF60,XNF78 and Lineage 8 represent the highest homology of JXA1,XNF10,XNF74,XNF229 and Lineage 3 represents the highest homology of QYYZ.The analysis of genetic evolution based on the ORF5 gene revealed that 16 PRRSV strain s belonged to the epidemic virus sublineage 3.5 in China,and the genome-wide genetic evolution analysis showed that XNF10,XNF74,and XNF229 belong to sublineage 3.5.XNF53,XNF60,XNF78 belong to sublineage 8.7,and LCL15 belong to sublineage 1.8,which is similar to the results of homology analysis.The Nsp2 gene deduced aa sequence analysis found that seven new Lineage 3 PRRSV strains had a large number of complex deletions and insertions in the Nsp2 region.The deletion and insertion patterns of XNF10,XNF74,XNF78,and XNF229 on the Nsp2 protein reported in this study were first reported.ORF5 deduced aa sequence alignment results showed that Lineage 3 PRRSV had unique amino acid mutations in amino acids at Y³⁸,S³⁹,C⁶⁶,S⁹²,F¹¹⁷,I¹⁵²,R¹⁹⁶,and H¹⁹⁹.The new Lineage 3 PRRSV contains a large number of amino acid mutations in the linear antigen region,and mutations in these positions may cause vaccine immunization failure.The results of recombination analysis showed that 7 new Lineage 3 PRRSV strain s were all recombinant viruses and had a very complicated recombination relationship.Recombination between PRRSV strains would have a greater impact on virulence.Recombination analysis showed that 7 strains of Lineage 3 PRRSV had complex recombination patterns,and the recombination hotspots were located on NSP2,NSP7,ORF2a and ORF3.Virus isolation of the newly developed Lineage 3PRRSV successfully isolated XNF10 strain.This study provides a reference for further exploration of the variation and genetic evolution of Lineage 3 PRRSV strains in China.In this study,BALB/c mice were immunized with ultra-purified Lineage 3 PRRSV XNF10strain three times,and IFA was detected 7 days after the third immunization.The spleen of the positive mouse was removed under sterile conditions,and the spleen cells were washed out and fused with SP2/0 cells.After four subclonings,the stability of the antibodies secreted by the selected hybridoma cells was verified,and the results showed that two positive hybridoma ce ll lines capable of stably secreting anti-PRRSV MAb were successfully obtained,named DD-1 and DD-2.The obtained positive hybridoma cells were cultured in large numbers,and mice were inoculated to prepare ascites.The titers of DD-1 and DD-2 cell supernatants determined by IFA were 1:64 and 1:128,respectively.Ascites titers of DD-1 and DD-2 are 1:5000 and 1:10000,respectively.Broad-spectrum detection of monoclonal antibody cell lines using the IFA method showed that both DD-1 and DD-2 were compatible with the representative strains of American-type PRRSV SD53-1603?Lineage 1?,Hu N4-F112?Lineage 8?,and JXA1-R?Lineage 8?,MLV?Lineage 5?,but not with the European representative strain DV.Therefore,DD-1 and DD-2have good broad-spectrum properties in American PRRSV.Western blot was used to identify the binding proteins of DD-1 and DD-2.It was found that both of the newly prepared monoclonal antibodies could specifically bind to GP3 of PRRSV.Using purified XNF10 whole virus as the coating antigen,the binding of monoclonal antibody DD-2 to the virus can be blocked by PRRSV-positive pig serum.The DD-2 monoclonal antibody prepared in this study provided the basis for the establishment of an American PRRSV antibody method.