Investigation On The Molecular Prevalence Of Porcine Reproductive And Respiratory Syndrome Virus In Some Areas Of Yunnan Province And Its Control Measures

Porcine Reproductive and Respiratory Syndrome?PRRS?is an important infectious disease in pig industry caused by Porcine Reproductive and Respiratory Syndrome Virus?PRRSV?.The mutation rate of PRRSV genome is very high and thus a large number of new epidemic strains are constantly appearing.Furthermore,safety and efficacy problems have been repeatedly disclosed due to the use of attenuated vaccines.How to effectively control the epidemic of the disease is a serious challenge and a major issue.This study first investigated the epidemiology of PRRS and analyzed the PRRSV genetic evolution in pig farms from some areas of Yunnan province.Then we evaluated the inhibitory effects of the Chinese herbal medicine‘Huang's Lanwenkang'on the replication of PRRSV on cellular and animal levels.Finally,further validation of the protective effects of‘Huang's Lanwenkang'in pig farms was conducted to provide a realistic basis for the prevention and control of PRRS.1.Between 2013 and 2018,2234 samples were collected from some pig farms with different scales in Yunnan province and tested for swine diseases.We found that594 samples?26.59%positive?were PPRSV positive and that the infection rate became higher over the investigation years.Among the PRRSV positive samples,the double,triple and quadruple infection rates were 38.72%,11.11%and 1.35%,respectively.The triple infection rate of PRRSV,PCV2 and CSFV was 6.06%;the triple infection rate of PRRSV,PCV2 and PRV was 3.37%;while the quadruple infection rate of PRRSV,PCV2,CSFV and PRV was 1.68%.Some positive samples were selected for PRRSV isolation in Marc-145 cells and further investigation.In total,20 Yunnan PRRSV isolates were obtained,with their TCID₅₀0 determined.2.Twenty PRRSV strains from Yunnan were isolated,with the whole genomes sequenced.We found that the length of the whole genome was between 15 230 bp and15 437 bp.The nucleotide homology with the JXA1 strain was the highest,ranging from 93.3%to 99.1%.Genetic evolution analysis showed that the 20 PRRSV Yunnan strains belonged to the domestic dominant strain sublineage 8.7,of which 6 Yunnan strains formed an independent branch between subgroup?and subgroup?,belonging to the Intermediate subgroup;6 strains are closely related to subgroup?,belonging to the highly pathogenic JXA1-like strain subgroup;the other 8 strains are closer to subgroup?,belonging to the HP-PRRSV vaccine-like subgroup.Compared with the reference strain,mutations and deletions were found at some key amino acid sites to some extent.3.Using Marc-145 cells as a cell model,the PRRSV inhibitory effect of the Chinese medicine"Huang's Lanwenkang"was evaluated in vitro.The results showed that the maximum non-toxic concentration?TC₀?of the drug was 1.56 mg/ml and the half-toxic concentration(TC₅₀)was 38.3 mg/ml.Chinese medicine can inhibit virus proliferation through three modes of action,i.e.,therapeutics,prevention and direct killing,with the IC₅₀0 at 0.265 mg/ml,0.300 mg/ml,and 0.282 mg/ml,respectively.The selection index?SI?is 144.53,127.67 and 135.82,respectively,indicating that the virus infectivity(TCID₅₀)can be reduced by more than 2 titers,with the best effect from the therapeutic mode.The results of real-time quantitative PCR showed that all the three modes of action of"Huang's Lanwenkang"could significantly inhibit the expression of the PRRSV nuclear protein gene?ORF7?,which was confirmed by indirect immunofluorescence assays on the PRRSV nuclear protein level.We demonstrated that"Huang's Lanwenkang"could inhibit the expression of NF-?B signaling pathway-related proteins,such as p-I?B?,p-p65,integrin?3 and RACK1.Animal experiments displayed that the score of clinical symptoms of the treatment group with"Huang's Lanwenkang"was lower than that of the control group.Furthermore,the expression of PRRSV ORF7 gene and N protein in the lung tissue of the"Huang's Lanwenkang"treatment group was significantly lower than that of the control group.Lastly,the pathological changes in the"Huang's Lanwenkang"group were mild;and immunohistochemical experiments showed that the PRRSV positive signals of all piglets in the group treated with"Huang's Lanwenkang"were significantly lower than those in the control group.4.The data from in vivo prevention and control experiments using traditional Chinese medicine"Huang's Lanwenkang"showed that the levels of vaccine immune antibodies in the three selected experimental sites were significantly improved,with an increase by 28.04%for the PRRSV immunization rate,an increase by 17.48%for the CSFV immunization rate and an increase by 15.89%for the PRV immunization rate after implementation of prevention and control measures for one year.The positive rates of the four diseases we investigated in this study decreased remarkably,with a reduction by 6.65%for PRRSV,by 6.35%for CSFV,by 10.64%for PCV-2 and by 3.03%for PRV.The average litter size of the sow was 11.8 heads/sow,which was0.43 heads/sow higher than that prior to implementation of prevention and control measures.The average size of live litter was 10.5 heads/sow,which was 1.57heads/sow higher than that prior to implementation of prevention and control measures.The weaning survival rate of piglets was 93.38%,showing an increase of11.61%.After the comprehensive measures were taken rol in pig farm A,the overall incidence of swine plague was decreased to 9.00%,the mortality rate was reduced to16.48%,the new net profit per head was 141.26 Yuan,the total economic benefit and the annual economic benefit was 313,000 Yuan.In pig farm B,the values were 7.44%,26.44%,114.44 Yuan,and 160,100 Yuan,respectively.In pig farm C,the values were9.36%,9.93%,135.41 Yuan and 357,100 Yuan,respectively.In summary,the prevalence of PRRS in pig farms with different scales in Yunnan province increased year by year from 2013 and 2018.Out of the complicated multiple infections,the dual infection with PRRSV and PCV2 was the main type in large-scale pig farms in some areas of Yunnan Province.Twenty Yunnan PRRSV isolates were isolated.The phylogenetic analysis based on the viral genome and deduced amino acid sequences revealed that the 20 strains belonged to the domestic dominant strain sublineage 8.7,indicating that the Intermediate strains,JXA1-like strains and HP-PRRSV vaccine-like strains were predominant in Yunnan.Compared with the JXA1-like strains in other provinces in China,the JXA1-like isolates from Yunnan showed high sequence homology.The mutations at some key amino acid sites may have exerted a certain impact on the current vaccine immune effect,which deserves further investigation.The traditional Chinese medicine"Huang's Lanwenkang"could effectively inhibit the replication of PRRSV through three kinds of actions of mechanisms:preventive,therapeutic and direct killing modes in vitro."Huang's Lanwenkang"showed certain protective effects in pigs from PRRSV infection.The application of"Huang's Lanwenkang"could significantly improve the immune level,control the main epidemic diseases,increase the average litter size and the survival rate of weaning piglets.As a result,the control measures showed recognizable economic benefits and are worth further implementation.