Epidemiological Investigation Of PRRS And Pathogenicity Analysis Of Characteristic Isolates In Shandong Province

Porcine reproductive and respiratory disorders syndrome（Porcine reproductive and respiratory syndrome,PRRS）,was also known as blue ear disease.The disease is caused by porcine reproductive and respiratory syndrome virus（Porcine reproductive and respiratory syndrome virus,PRRSV）.The typical symptom of the disease is premature delivery of pregnant sows,weak fetuses,stillbirths and mummified fetuses,abortions,piglets and finishing pigs with severe respiratory symptoms.Since the isolation of PRRSV was first reported in 1995,PRRS has had a serious impact on China’s pig breeding industry.The outbreak of"High-Fever"in some areas of southern China in2006 was caused by highly pathogenic PRRSV（HP-PRRSV）,which directly lead to huge economic losses.In 2013,NADC30-like strain spread rapidly throughout the country,making the situation of prevention and control of the disease extremely serious.NADC34-like strain was first reported in Liaoning Province in 2017,and its epidemic status and pathogenicity became the focus.Up to now,the prevalence of PRRS is still widespread.It is prone to variation and reorganization,and the type of reorganization is complex and changeable,which brings great challenges to the prevention and control of PRRS.In order to better know the epidemic situation of PRRS,this study carried out pathogen detection of suspected samples from different sizes of pig farms in Shandong.We found that most of the suspected cases were respiratory symptoms of piglets and reproductive disorders of sows,and most of the pigs of all ages showed obvious respiratory symptoms;autopsy showed lung edema,interstitial widening and marble lesions of lymph nodes.The positive rate of PRRSV was 30.18%（223/739）in 739 suspected PRRSV samples,and the disease occurred seasonally and frequently in autumn and winter.The results of mixed infection of PRRSV showed that PCV2 was the highest,followed by mixed infection with HPS and SS.1593 serum samples were tested for PRRSN protein antibody,and the antibody positive rate was 80.6%（1284/1593）,which indicated that the positive rate of PRRSV antibody was higher in pig farms of different sizes in our province.The genetic evolution tree of Nsp2,ORF5 and ORF7 gene fragments showed that there was a certain genetic distance between most epidemic strains and the existing reference strains,and the nucleotide homology was less than 95%.It is inferred that there is a large variation between the current epidemic strains and the existing reference strains.Based on the sequencing results of the previous flow regulated virus strains,12 representative PRRSV positive samples were screened.Six stains were identified and isolated.The titer of F5generation virus was measured and the whole genome was sequenced.The results showed that the virus value of ZJW20210828X isolate was 10^4.63TCID₅₀/ml,the virulence of the other five isolates was greater than 10^5.0TCID₅₀/ml.The whole genome sequence alignment showed that the isolates LL20210319X1,SX20210319X and ZQ20210329X belonged to 1.9 sublineage,and the nucleotide homology with the reference strain of the sublineage was 88.0%～93.5%,86.7%～95%and87.1%～93.2%respectively.The nucleotide homology was relatively low,and the Nsp2 gene sequences of LL20210319X1 and SX20210319X belonged to pedigree 1 and 5,located in a new branch;LY20211216X6 isolate belongs to 5.1 sublineage,but ORF5 and ORF7 gene sequencing results belong to 1.5 sublineage and 1.9 sublineage respectively;407-20210415X isolate belongs to8.7 sublineage,but ORF5 gene belongs to a new branch of Lineage 8 and ORF7 gene belongs to Lineage 3;ZJW20210828X isolate belongs to 8.7 sublineage,but belongs to a single branch.Recombination analysis showed that there were two or three combinations of Lineage 1,3,5 and 8 in6 isolates,among which lineage 1 had recombination between NADC30-like and NADC34like/1-4-4,and Lineage 8 had recombination between HP-PRRSV like,CH-1a-like and intermediate subgroup.To explore the pathogenicity of the isolates,we selected 407-20210415X isolates with jxa1 as the skeleton recombination Lineage 3 and 8 and SX20210319X isolates with NADC30-like as the skeleton recombination Lineage 1,3 and 8（including NADC30-like and NADC34-like）for pathogenicity test.The results showed that the two groups of isolates induced body temperature rise and weight loss at different degrees.Commen autopsy findings included pulmonary edema and interstitial pneumonia.Pathological sections showed typical widening of lung stroma,alveolar wall thickening.And comprehensive analysis of viremia,specific antibody level,tissue viral load and other indicators.The two isolated virus groups had obvious pathogenicity in clinic,but due to the difference of recombination types,the pathogenicity of SX20210319X isolated virus was weak.This test provides a reference for the pathogenicity of PRRSV recombinant strain in our province.In conclusion,the current prevalent PRRSV epidemic strains in our province are recombinant strains,and the complexity and diversity of strains increase the difficulty of disease prevention and control.This study enriches the epidemiological data of PRRS and provides a scientific theoretical reference for the comprehensive prevention and control of PRRSV in Shandong.