Isolation,Identification,Variation,Recombination And Pathogenicity Of Porcine Reproductive And Respiratory Syndrom Viruses

Porcine reproductive and respiratory syndrome virus(PRRSV)is an inf ectious disease of pigs.It mainly causes estrus and reproductive problems in sows an d causes respiratory symptoms in growing pigs.The disease was first discovered in th e United States,and followed outbreak in Europe.The initial onset of the disease spre ad rapidly.Nowadays,the disease have reported in most countries and regions in the world.PRRSV has evolved more than 30 years since it appearance and has not effect ively controlled.PRRSV was first isolated in 1996 in China and has also undergone m ore than 20 years evolution in China.It experienced three major epidemic peaks in Ch ina,bringing huge economic loss to Chinese pig industry.The prevention and control of PRRSV in China still rely on vaccination,but there are certain drawbacks to variou s vaccines.At present,the condition of domestic PRRS is complex in many types of st rains,and the mixed infections of various strains are serious,which in turn increases t he probability of recombination between different strains and bring new variant strain s.In addition,due to improper use of vaccines,non-standard management,and varyingquality,the problems which spread of vaccine poisons and the return of virulence hav e became increasingly prominent.The quarantine of breeders and introduction is not s trict and standardized,which increase the probability introduction of foreign strains.This study starts with the molecular detection and identification of pathogens.throu gh the pathogen isolation and identification of PRRSV?determination the antigenicit y and pathogenicity of the different types of isolated PRRSV,and systematically study the distribution? infection and prevalence of PRRSV? pathogenicity?and cross-pr otection among different strains of pigs in Henan and periphery areas,which expectin g provide a scientific reference for clinical diagnosis of PRRS,early warning of epide mics,vaccine development and effective control,etc.1.Establishment of Single and Duplex RT-PCR Methods for PRRSV1 and PRRSV2 StrainsTo understand and master the epidemic situation of PRRS in Henan Province,two pairs of specific primers were designed for duplex RT-PCR amplification of PRRSV1 and PRRSV2 against the ORF5 gene,and a pair of identification primers was designed to identify amplification of the Nsp2 gene of different types of PRRSV2(Classic,H P-PRRV,and NADC30-like).Through the optimization of the reaction system and con ditions,the simultaneous amplification of the same genotype target genes and differen t genotypes target genes of PRRSV1 and PRRSV2 were achieved in the same reaction system.The results showed that the minimum detection of PRRSV2 Nsp2 identificati on RT-PCR method was 2.2×10-1 to 3.1×10-1 ng for different types of PRRSV2,and t he minimum detection of duplex RT-PCR for PRRSV1 and PRRSV2 was 3.39×10-2 a nd 3.67×10-2ng.The above RT-PCR method is highly specific,sensitive and reproduci ble.The reliability and practicality of the method were further verified by the detection of 369 clinical suspected PRRS infection samples.A total of 47 clinically positive sa mples were detected by the identification RT-PCR of RRSV2 Nsp2 gene.In the 47 p ositive samples,the detection of HP-PRRSV and NADC30-like strains accounted for 93.62%,while the detection of classic PRRSV was only 6.38%.The duplex RT-PCR can specifically detect PRRSV1 and PRRSV2 simultaneously.From the 369 samples,114 were positive for a single PRRSV2,the positive rate was 30.89%;16 were positive for a single PRRSV1,and the positive rate was 4.34%;The number of PRRSV1 and PRRSV2 co-infected was 8(2.98%);Detection by single RTPCR and duplex RT-PCR was 100% consistent with each other in the triple detection test.In summary,the prevalence of PRRS in Henan Province is mainly dominated by N ADC30-like and HP-PRRSV strain.The results was found PRRSV1 strains infection i n Henan Province and mixed infection with PRRSV2 strains.The prevalence of PRRS V2 strains is diversified in the field;PRRSV1 and PRRSV2 co-infection increase the difficulty of clinical diagnosis.2.Isolation,Identification,Recombination and Variation Analysis of Different Types of PRRSV2 StrainUsing the established PRRS2 identification RT-PCR and PRRSV1 and PRRSV2 do uble RT-PCR,the positive samples were isolated for virus isolation and identification.A total 20 of clinical PRRSV strains were isolated and the genes of Nsp2?ORF5 an d the complete genome of some isolate strains were determined and sequenced.The re sults showed that the nucleotide similarity between the 9 isolates and the current dome stic NADC30-like strains was higher;the nucleotide similarities between the 6 isolate s and domestic HP-PRRSV strains were higher;5 strains is similar to HP-PRRSV vac cine strain JXA1-R strains.Whole genome sequence amplification,mutation and recombination analysis of HE NXX-8,HENXX-9 and HENJY-2 strains were performed.The results showed that th ere were obvious recombination events of the three isolates,and the recombination pat terns were inconsistent.The HENXX-9 strain is a recombinant strain between HP-PR RSV and NADC30-like strains,the HENXX-8 ?HENJY-2 strains are recombinant st rains between NADC30-like strain and HP-PRRSV.The growth curve showed that the three strains had a proliferative capacity of HENXX-9> HENXX-8> HENJY-2.The r ecombination among different strains further exacerbated the difficulty of prevention and control of clinical PRRS.3.Isolation,Identification,Complete Sequence Analysis and Molecular Epidemiological Investigation of PRRSV1 StrainsIn this study,16 samples of PRRSV1 positive samples were obtained through the detection of clinical samples,and 6 ORF5 genes of PRRSV1 were obtained.One strain of PRRSV1 was isolated and identified named HENZMD-10.The phylogenetic tree constructed by 6 strains of PRRSV2 ORF5 gene showed that the genetic distance between the six ORF5 genes was far.The nucleotide sequence similarity between HENZMD-10 and the American type representative strain VR2332 and European type strain Lelystad virus strain was 62.1% and 89.1%,respectivly.Phylogenetic analysis showed that HENZMD-10 had the closest genetic relationship with domestic isolated PRRSV1 strains NCDC-NM1-2011,LNEU12,BJEU06-1 and FIEU13 strains.Through analysis of the Nsp2 coding region of HENZMD-10 found 27 nucleotides deletion in the Nsp2 region,whereas the BJEU06-1,NVDC-NM1-2011,LNEU12,and FJEU13 strains reported in domestic were deletion 15 nucleotides in the region.The nucleotides of foreign isolated KUN-07,Euro PRRSV,MLV-DV PRRSV1 strains are deletion 50 to 222 nucleotides in this region.This study first time isolated PRRSV1 strains from Henan.There no recombination was found in HENZMD-10.Although there no recombinant strains have been found between PRRSV1 and PRRSV2,the existence of the two strains in the field herds bring risks to the current prevention and control of PRRSV.So,it is necessary to continue to expand the area for monitoring of PRRSV1 and PRRSV2 in the field,grasp its epidemic dynamics,and to provide a meaningful scientific basis for its clinical prevention and control.4.Cross neutralization test of different PRRSV strains in vitroIn this experiment,we have prepared a commercial vaccine and a field-resistant stra in of rabbit anti-hyperimmune serum against the current situation of diverse field epid emic strains and complex conditions,and verified the cross neutralization test of differ ent PRRSV by serum neutralization tests in vitro.The experimental results showed th at the prepared rabbit anti JXA1-R hyperimmune serum had higher cross immunity pr otection against different types of strains than VR2332 R and JXA1R;the level of imm une protection against classic strains was highest,with a neutralization titer of 1:114;The VR2332-R serum had the lowest neutralizing potency against the HP-PRRSV stra in(1:11);Rabbit anti HENXX-8 hyperimmune serum can provide good immune pr otection for recombinant strains HENJY-2 strain,but the immune protection of JXA1-R is low;Rabbit anti JXA-R hyperimmune serum and rabbit anti VR2332-R hyperim mune serum can provide better immune protection against NADC30-like strains.5.Development and Application of a Duplex RT-PCR Assay for Detection of PRRSV and GETVGetah virus(GETV)is an infectious pathogen affecting humans and animals.The GETV mainly causes the disease in pigs with symptoms similar to those caused by the porcine reproductive and respiratory syndrome virus(PRRSV).It is difficult to distinguish co-infection of pigs with GETV and PRRSV by clinical means.Several studies based on multiplex polymerase chain reaction(PCR)for detection of PRRSV and other viruses have been reported,but a simultaneous detection methods for PRRSV and GETV have not.Therefore,based on the whole genome sequences of PRRSV and GETV published in Gen Bank,two pairs of primers for amplification of PRRSV ORF7 and GETV Cap were designed,and a duplex reverse transcription PCR(RT-PCR)assay was established in China for detection of GETV and PRRSV in a single amplification reaction.This method was used to amplify a 633 bp specific fragment of PRRSV ORF7 and 316 bp specific fragment of GETV Cap gene.The nucleic acid amplification,however,was negative for all the genes for the swine fever virus,porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus,porcine parvovirus,porcine pseudorabies virus and porcine circovirus.The limit of detection(in ng)of GETV and PRRSV was 2.48 ×10-4 and 2.50×10-5,respectively.A total of 136 clinical samples collected from 92 farms in 15 cities in Henan Province were tested by duplex RT-PCR.The number of positive samples from 58 farms with PRRSV infection only and with GETV infection only was 44(32.4%)and 7(5.15%),respectively.The number of samples co-infected with both pathogens was 8(5.88%).Detection by single RT-PCR and duplex RT-PCR was 100% consistent with each other in the triple detection test.This new method could be used for the rapid detection,differential diagnosis and molecular epidemiology research of the diseases caused by PRRSV and GETV.