Epidemiological Investigation Of European Type PRRSV Analysis,Isolation And Identification Of ORF5 Gene Mutation

Porcine reproductive and respiratory syndrome(PRRS)is a virulent infectious disease that occurs in pigs.Our country divides it into a class of infectious diseases.The diseased sow mainly causes reproductive disorders such as stillbirth,abortion,mummification,etc.and the pathogen is porcine reproductive and respiratory syndrome virus(PRRS).Inter-American porcine reproductive and respiratory syndrome virus(NA-type)and its mutant strains have been popular for decades in our country and spread to various regions and the like.However,the data from epidemiological investigation in recent years have shown that the European-type porcine reproductive and respiratory syndrome virus(EU-type)has been detected in some parts of our country,which do not cross-protect but can be mixed with infection and the virus can be recombined.Therefore,the prevention of blue ear disease is not only to control the widely-popular American type,but also to prevent the introduction of the new European type.The prevalence of this laboratory is mainly for the past two years,PRRSV China's Southern China Region conducted epidemiological investigation of pathogens,9 strains were obtained.The positive samples selected 3 strains were amplified by RT-PCR,and the whole ORF5 gene genetic variation analysis.After using PAMs cells successfully isolated a strain of European type PRRSV named PRRSV CN-2016.The ORF5 reading frame has the most obvious gene mutation in all open reading frames.The homology between European and American ORF5 genes is only 55 % ~70 % by comparison of nucleotide sequence.The PCR products of European and American strains are 1296 bp and 198 bp,respectively.The primers are named EU-ORF5 and NA-ORF7.After the optimization of reaction conditions and the detection and analysis of a large number of samples,we have successfully established PCR and respectively.By using the method,310 suspected PRRS from Guangdong Province from 2015 to2017 were detected.The average positive rate was 28 %,and the positive rate was 2.9 %.Among all the positive isolates,10.4 %,the homology of the three strains was 82.8 %,which was the same as that of 10 strains isolated from European region(Netherlands,Denmark,Belgium,Spain).%~88.9%.phylogenetic tree analysis showed that 3 PRRSV strains were located in the G2 group,with group G2 strains were from Belgium and Chinese,suggesting that in Chinese,the epidemic strain may be derived from Belgium.Three strains tested as positive were isolated and filtered by centrifugation and filtered by bacteria,and inoculated on the prepared porcine alveolar macrophages to continuously carry out the blind transmission.After the transfer to the 3rd generation,the cells were isolated and stably transferred to the 6th generation.According to the degenerate bases(including part of ORF4 and ORF6 genes)of the virus strains in GeneBank,a fragment of about 1296 bp was amplified from the cell culture by RT-PCR,and cloned into pMD18.Sequencing analysis showed that isolates T vector,and 13V117 and Lena of Belgium strains phylogenetic tree in the same branch,86.8% nucleotide homology and 82.4%isolates may be derived from Belgium.This study showed that there was a European type of PRRSV invasion in mainland China,and the cross-protection of the widely used American strain vaccine against European type PRRSV was weak in China.Therefore,the disease prevention and control strategy needs to cause further thinking.