Epidemiological Investigation Of European Type PRRSV In China And Preparation Of Monoclonal Antibodies Against European Type PRRSV

Porcine Reproductive and Respiratory Syndrome(PRRS)is caused by porcine reproductive and respiratory syndrome virus(PRRSV),which is characterized by sow abortion,stillbirth and mummified fetuses,and respiratory disorders in piglets.PRRSV was divided into two genotypes named as the European type(genotype 1)and the American type(genotype 2),which represented by the European Lelystad virus strain and the American VR-2332,respectively.So far,only a few epidemiological studies on European type PRRSV in China were performed.In order to evaluate the epidemic situation of European type PRRSV,it is very important to strengthen the monitoring the prevalence of European type PRRSV in China.5 strains of European type PRRSV were detected from 350 samples suspected of PRRSV infection in domestic from 2016 to 2018,and were named as ZD1,WK14,WK141,WG9 and HN7.The whole genome sequence and genetic evolution of the 5 strains were analyzed.The nucleotide alignment showed that nucleotide homology between the 5 strains and the American type reference strain VR2332 was 60.0 %-60.4 %,and the European type reference strain Lelystad virus was 88.3 %-88.9 %.The nucleotide homology between ZD1,WG9,HN7 and LNEU12 strains was 93.5 %,93.3 % and 93.2 % respectively.The nucleotide homology between WK14 and BJEU06-1 strains was 89.7 %,and between WK141 and NVDC-NM3 strains was 93.5 % that showed the highest nucleotide homology.Sequence alignment of Nsp2,ORF3 and ORF4 showed that the ORF3 of ZD1 strain were terminated 26 aa in advance and the other 4 strains(WK14,WK141,WG9 and HN7)were missing 1 aa at position of 245-246 in ORF3.What’s more,all 5 strains had 1 aa deletion at the 65-66 position in ORF4 and had 4 and 1 aa deletions at the 357-360 and 411 positions in Nsp2,respectively.The deletion or early termination led to higher degree of variation of the European type PRRSV.However,the effect of these changes on the biological characteristics of PRRSV is unknown.As known,recombination is one of the main causes of PRRSV variation.There are frequent reports on recombination of PRRSV of American type,but few reports on recombination of PRRSV of European type.In our studies,the recombination analysis showed that WK14 train is the recombinant virus of BJEU06-1 and NVDC-FJ,and the recombinant region is located at the position of 7131~9111 bases.WK141 strain is a recombinant virus of NVDV-NM1-2011 and HENMD-10,and the recombinant region is located at the position of 7131~9111 bases.The reason why recombinant phenomenon is rare in European type PRRSV is complicated,which may be related to the characteristics of the strains.At present,most of detection methods for PRRSV antibodies are against the N protein in our country.Because the serum antibodies between European type and the American type PRRSV have cross-reaction,it is impossible to evaluate the prevalence of the European type PRRSV in China by detecting the antibody.Therefore,it is great significance to establish a specific method for detecting European type PRRSV.In this study,two hybridoma cell lines 2C5 and 2C6 were successfully prepared by using purified whole virus of PRRSV ZD1 to immunize the mice.Subtype identification showed that the heavy chain of monoclonal antibodies belonged to IgG2 a subclass,and the light chain was kappa chain in both of 2C5 and 2C6.The IFA titers of supernatant and ascites antibodies were 1:320 and 1:16000,respectively.IFA test showed that both of 2C5 and 2C6 could distinguish the European type from the American type PRRSV,indicating that the specificity of 2C5 and 2C6 is good.The binding of monoclonal antibody 2C5 and 2C6 to the virus can be blocked by pig positive serum using purified DV whole virus as the antigen.Over all,the 2C5 and 2C6 monoclonal antibody provides a useful tool for the establishment of a specific detection method for evaluating the prevalence of European type PRRSV in China.