Molecular Epidemiology Of The Porcine Reproductive And Respiratory Syndrome Virus In Henan Province From 2018 To 2019

Porcine reproductive and respiratory syndrome(PRRS)is an infectious disease caused by PRRSV,characterized by reproductive disorders such as miscarriage,premature delivery,stillbirth and mummified fetus,and respiratory symptoms of pigs at various stages.Since the disease was first reported in China in1996,China’s pig industry has been affected by PRRS for a long time,especially the highly pathogenic PRRSV outbreak in 2006 has brought a huge blow to the Chinese pig industry.At present,there are 6 types of PRRSVII in China,namely Sublineage1.8(NADC30-like strain),Sublineage 1.5(NADC34-like strain),Sublineage 3.5(QYYZ-like strain),Sublineage 5.1(Resp-PRRS MLV-like)Strains),Sublineage 8.7(HP-PRRSV)and Lineage 9,of which NADC30-like Strains and HP-PRRSV strains are the main epidemic strains in China.Henan is located in the Central Plains and has convenient transportation* As an important province of pig breeding and transportation in China,Henan is facing a more serious risk of importing and spreading animal diseases.The current research results show that NADC30-like strains are the main epidemic strains in Henan area,and this kind of strains are constantly mutating in pigs,which brings great difficulties to PRRS prevention and control.There are two main difficulties in the prevention and control of NADC30-like strains.On the one hand,the homology between different strains is relatively low? and the similarity of strains in different regions and different farms is also relatively large;The research results show that most of the detected NADC30-like strains have some genetic recombination,and the recombination patterns are complex and diverse,which brings great resistance to the epidemiological studies of PRRS and the genetic evolution of viruses.In this study,we conducted preliminary PRRSV molecular epidemiological surveys in18 prefectures and cities in Henan Province to initially understand the prevalence and distribution of PRRSV strains in Henan area.Long-term investigation and research,tracking and analysis of the PRRSV strains circulating in the farm under closed conditions,in order to understand the prevalence of PRRSV in the farm.From horizontal survey to vertical tracking,from the point of view,to further study the genetic evolution and variation of PRRSV strains,to provide a more effective scientific basis for the clinical diagnosis,prevention and control of PRRS and vaccine development.1.The prevalence ofPRRSV in Henan Province in 2018-2019In order to understand the prevalence and variation of PRRSV in Henan,this study conducted RT-testing on 613 suspected PRRS clinical samples(pig lung,spleen,and lymph node tissue)collected from 94 pig farms in 18 prefectures and cities in Henan during 2018-2019.PCR detection,ORF5 and NSP2 gene amplification,sequence determination and genetic evolution analysis of positive samples confirmed by the test.The results showed that a total of 151 PRRSV positive samples were detected in 613 samples,with a positive rate of 24.63%;among them,the detection rate was highest in northern Henan;83 ORF5 and 31 NSP2 gene sequences were amplified from 151 positive samples;Genetic evolution analysis based on 83 strains of PRRSV ORF5 gene sequence showed that 74 strains of PRRSV out of 83 strains belonged to NADC30-like strains,6 strains of PRRSV belonged to QYYZ strains of epidemic strains in South China,and 3 strains had PRRS commercial live gene sequences Highly homologous.The amino acid sequence comparison results of the ORF5 gene derivation of 74 NADC30-like strains found that the homology of the ORF5 gene amino acid sequences between the strains was quite different.Among them,there were insertions or deletions in the ORF5 sequence of the 10 strains in the hypervariable region.The amino acid sequence alignment results of 31 strains of PRRSV NSP2 gene found that 29 strains of PRRSV NSP2 sequence had 131 consecutive amino acid deletions,1 strain of PRRSV NSP2 sequence had the same deletion as the highly pathogenic PRRS vaccine strain JXA1-R,1 The NSP2 sequence of the PRRSV strain is 99% similar to the classical vaccine strain RespPRRS MLV.This shows that in 2018-2019,the PRRSV in Henan region was dominated by NADC30-like strains,and the NSP2 and ORF5 genes were greatly mutated;at the same time,the appearance of QYYZ-like strains in Henan and the continuous detection ofPRRSV live vaccines made PRRSV The popularity is more complicated.Therefore,it is particularly important to strengthen the continuous monitoring of the epidemiological changes and genetic variation of PRRSV.This study can provide an effective clinical basis for the clinical diagnosis,prevention and control ofPRRSV and vaccine development.2.Tracking and monitoring ofPRRSV in a large-scale farm in Henan ProvinceTo further understand the evolution and variation of PRRSV in specific farms,this experiment regularly monitored PRRSV from a large-scale farm in Henan from 2017 to 2019.By performing RT-PCR detection on the suspected PRRSV samples collected from the field in different months,and performing ORF5,NSP2 and full gene sequence amplification on the detected positive samples,the genetic sequence analysis of the obtained strain sequence information was performed,and the nucleotides were similar Sexual comparison,prediction of glycosylation sites of encoded proteins and recombination analysis.A total of 46 PRRSV sequence information was obtained.The results of genetic evolution analysis based on the ORF5 gene showed that the strains detected in the farm were all NADC30-like strains,belonging to Sublineage 1.8.Combined with the analysis of amino acid variation pushed by the ORF5 gene,All the strains detected were subdivided into 5 groups(A-E).The amino acid variation of the strains in the ORF5 gene was similar between the same group.The analysis found that the longer the existence of each group of strains in the farm,the greater the genetic distance between the strains within the group;and found that the ORF5 gene between the B and D strains have the same amino acid variation site,suggesting that these two There is a certain relationship between the PRRSV mutation process between groups.The analysis results of GP5 protein glycosylation sites showed that the glycosylation sites of GP5 protein in each group of strains had a certain increase or displacement.The amino acid sequence alignment results of 10 PRRSV NSP2 genes showed that all of the 10 PRRSV NSP2 sequences had 131 consecutive amino acid deletions.In addition,through the recombination analysis of the entire genes of the amplified SP41810-30 strain and SP41904-39 strain,it was found that both strains recombined with the NADC30 strain and the JXA1-PI00 strain,and there was the same recombination,mode.This experiment reveals the characteristics of continuous propagation and genetic evolution of PRRSV in large-scale farms,and provides a reference for the PRRSV mutation and recombination mechanism and immune prevention and control.3.Isolation and identification of PRRSV Henan epidemic strainsIn order to study the biological characteristics of PRRSV field epidemic strains,in this experiment.50 PRRSV positive tissue sample treatment solutions were inoculated into Marc-145 cells.After blind passage for 3 generations,RT-PCR identification and indirect immunofluorescence tests were conducted.PRRSV strains were subjected to whole-gene amplification,genetic evolution analysis and mutation analysis.Results Two PRRSV strains were successfully isolated,named HENSQ-3 and HENXX-12,with genome-wide sizes of 15319 bp and 15014 bp respectively;the infection titers of the two PRRSV fifth-generation viruses on Marc-145 cells were]q6.33 XCID50 / ml and 10515 TCID50 / ml.Through genetic evolution analysis,it was found that HENSQ-3 belongs to Sublineage8.7(HP-PRRSV),and the nucleotide sequence is highly homologous to the JXA1 strain,with a similarity of 98.6%;HENXX-12 is a NADC30-like strain,which is similar to the NADC30 strain The similarity is 93.6%.Recombination analysis found that HENXX-12 was a recombinant strain with NADC30 strain as the main parent and highly pathogenic strain HK13 as the secondary parent,with a recombination node of 5723bp-6378 bp.This test provides a reference for studying the genetic evolution and pathogenicity of PRRSV.