Establishment And Application Of Getah Virus Detection And Isolation, Identification And Sequences Analysis Of Porcine Getah Virus

Porcine Getah virus(GETV),a kind arboviruses,can cause the its natural infection host-horse fever,rash,hind leg edema,and lymph node enlargement.born piglet tremor,skin redness,spiritual decadence,tan diarrhea with high mortality.Sows stillbirth,especially early pregnancy and specific GETV antibody has been detected in humain body.GETV first appeared in Malaysia in 1955,in the same species as Sagiyama virus(SAGV),which was separated from the triboscus in 1956.China isolated a GETV separator named M1 from the culex mosquito in hainan province in 1964.By means of the mosquito,libraries,midge,horses,pigs,birds,the fox and the person such as carrier or the host GETV epidemiological investigation,found that GETV now widely distributed in the eastern south Asian countries,Asia,northern Europe and the Pacific Ocean in northern Australia.the author isolated GETV in 2016 from China national pig for the first time in 2016.However,It is not yet clear about its pathogenicity to swine and that prevalence of pig in China.This study is based on the establishment of GETV detection method,the isolation and identification of epidemic strain,NSP3,Cap gene and whole genome sequence determination and analysis,to understand its characteristics and the direction of genetic evolution in Chinese swine populations,with a view to provide basis for research of GETV evolution process,pathogenic mechanism and scientific prevention and control.1.The establishment and application of RT-PCR method for Porcine GETVIn view of the fact that people know little about GETV as swines of emerging infectious diseases,this study established a RT-PCR detection method for pig GETV to increase understanding of it.According to the reported GETV gene sequence,the author designed a set of specific primers for the NSP3 gene,successfully establish a RT-PCR method for testing GETV,with a wide range of adaptability,good specificity and sensitivity,with a minimum content of 102.25TCID50/m L.Application of the technology to test the field's collection of 79 suspected GETV swine epidemic materials,results show that the clinical onset of pig GETV positive rate was 5.08%(4/79),of which three come from the intestinal of diarrhea vomiting piglets,one comes from a miscarriage fetus produced by a female swine during pregnancy.For the first time,RT-PCR was used to detect GETV from the infected swines and provide technical support to understand its prevalence in Chinese pigs.2.The establishment of the five RT-PCR methods for Porcine GETVIn order to distinguish various viral pathogens effectively from the diarrhea pig group,the authors established a five-weight RT-PCR detection method.In this experiment,5 pairs of specific primers were designed in the conservative areas of the Cap gene of GETV,E gene of PEDV,S gene of TGEV,VP6 gene of Po RV and N gene of PDCo V.By fixing affecting factors such as d NTP,Mg Cl2,ATP and dd H2 O RT-PCR,optimizing the primer concentration,annealing temperature and time extension,the results show that the molar concentration of each pair of primers than 30:10:3:2:2 join,annealing temperature 57 °C,extend the time of 80 s,40 loop built by methods of five heavy RT-PCR amplification effect is best.In this method,the detection of 93 samples of diarrhea symptoms from 6 farms in 3 cities of henan province show that the positive rate of PEDV was the highest(23.66%),and the detection rate of TGEV and GETV was low,and the positive rate of GETV was 6.45%.Compared with a single RT-PCR method,the coincidence rate of the two results was 100%,suggesting that the method applied in the clinical sample was reliable and practical,which could be applied to the clinical differential diagnosis.3.The prokaryotic expression purified and polyclonal antibody preparation of Cap protein in Porcine GETVFor preliminary understanding Cap protein properties,in this experiment the Cap full-length gene sequences of HNJZ-S1 strain is amplified,and cloned to the prokaryotic expression vector of p ET28 a carrying His label,then identifies by PCR,double enzyme digestion and sequencing,and recombinants p ET28a-Cap transformation plasmid to the E.coli BL21,with IPTG inducing the expression;SDS-PAGE detection,nickel column purification,Western-blot identification protein;The protein is purified by nickel column and immunes bal/c mouse to prepare polyclonal antibody.The experiment shows that the final concentration tendency for 0.1/L IPTG induced 20 ? after 12 h,high expression of Cap gene,the SDS-PAGE shows that the meringue relative to the molecular mass is 35 KDa.Western-blot shows the specific reaction of serum and fusion protein.The purified Cap protein and polyclonal antibody obtained in this study lays the foundation for further understanding of Cap structure and function,establishment of kit for detecting GETV antibody and research on GETV gene engineering vaccine.4.Detection and sequence Analysis of GETV NSP3 and Cap genes in 4 provinces of ChinaTo understand swine GETV epidemic in China and studay GETV's pathology and partial genetic sequence analysis,for the first time this study detectes 801 swine organs and tissues,from 231 swine farm in domestic four provinces Jin,Hebei,Henan,and Anhui,with RT-PCR method.The results show that,from the sample of the pigs in the four provinces,all of the samples from the 4 provinces are tested positive for 37 copies of the positive samples from the 32 positive farm,and the sample positive rate is 4.62%,the farm positive rate was 13.9%,and it is the first time to check out the GETV from the seed of the pig semen.The sequence analysis of the amplification products of Cap and NSP3 of the virus shows that the GETV in China's pig population is closely related to the recent years in Japan,the source of the source and the source of the mosquito-borne virus,and the second is the Chinese mosquito source and the south Korean pig source.The results of sequence analysis are basically consistent with the results of Cap and NSP3 gene,all related to all Japanese strains since 2012.This study is the first to reveal that the existence of GETV in all four provinces of China and the existence of boar in pigs can be brought to the attention of people.In view of that fact that the virus is currently consider as human and animal common disease in China and abroad,it is necessary to carry out a thorough research on its infection spectrum,propagation path,epidemic regularity,pathogenicity and control measures in China,so as to understand its infection and prevalence in real time,and to provide scientific evidence for effective prevention and control measure.5.Comparative analysis of different GETV isolates from vaccine source and porcine source in the same locationDuring the clinical trial,a GETV is detected accidentally and isolated from the pig breeding and respiratory syndrome virus(PRRSV)live vaccine.For preliminary understanding of China's circulation of pig with live vaccine GETV pollution and the use of pigs in the contaminated vaccine field GETV epidemic situation,the author applies the RT-PCR and virus isolation technology,manufacturers from six 6 class 63 batches of commercial pigs with live vaccine checked out one manufacturer in a PRRS vaccine batches for GETV positive,batch positive rate 4.76%,identifies one individual plant GETV isolates,GETV-V1;To isolate and identify a GETV isolate from the aborted fetus in a pig farm with a GETV contaminated PRRSV vaccine,AH9192;The toxicity and sequence length of GETV-V1 and AH9192 are 106.32TCID50/m L and 106.89TCID50/m L,11689 bp and 11681 bp respectively.The similarity between the two GETV genes and the amino acid difference sites shows that the similarity of 16-I-599 was the highest(98.7%~98.9% and 98.2%~98.5% respectively)of the Japanese horse source GETV strains in 2016(98.7%~98.9% and 98.2%~98.5%).There is numbers of amino acid mutations in ns P1,ns P2,NSP3 ns P4,Cap,E2,6 k and E1 proteins,E1 region has the most mutations with a hydrophilic activities Cys insert at 969 aa,first discovers the NSP3 of AH9192 strains genome has three consecutive amino acid deficiency in 1818 aa.The whole gene sequence analysis shows that the isolated strains GETV-V1 and AH9192 are in the same branch in one large area,and in the recent years of Chinese mosquitoes,the strain of HB0234 and YN0540,and the Japanese mosquito of 2012,12IH26,the pig source 15-I-1105 2015 and horse source 14-I-605-C1 2014,which is in a large area,is the closest relationship to the Korean pig source South Korea in 2004.In general,the genetic distance between AH9192 and GETV-V1 of the field isolates is most recently,and the vaccine contamination strain GETV-V1 may be introduced into China through South Korea or Japan.6.Isolation and identification of 5 GETV and full sequence analysis of porcine source in Henan and Anhui provinces.Through RT-PCR detection,CPE and sequencing,five GETV,HNNY-1,HNNY-2,HNPDS-1,HNPDS-2 and AH9192 are isolated from the organs,tissues and abortion of the pigs in the two provinces of henan province from 2016 to 2017.The total length was 11689 bp,11689 bp,11689 bp,11689 bp and 11681 bp respectively.By comparing the sequence similarity and amino acid site differences between the first strain of GETV M1 strain,HB0234 and the first pig source of HB0234 and the first pig source,South Korea,12I26 and 15-I-1105,the HNNY-1,HNNY-2,HNPDS-1,HNPDS-2 and AH9192 are found to be the most similar to the first pig source GETV HNZJ-S1 in China(99.5%,99.8%,99.5%,99.5% and 97.1%),then are similar to the 15-I-1105 and South Korea.There are a number of amino acid substitutions in the poly(non-structural protein)and polystructural proteins,among which there are many substitutions in E1,E2,Cap and NSP3 regions,and it is worth noting that in the upstream of the NSP3 / ns P4 cut site,there are Q?R?A?T and T?amino acid replacement in the upstream of the NSP3 / ns P4 cutting site,and the three amino acids are missing.In addition,HNNY-1,HNNY-2,HNPDS-1,HNPDS-2 and reference strain HZJZ-S1 had a Ser insertion at the 34 aa of Cap protein,and 24 variation sites were found in the E1 protein region of 5 GETV strains.Phylogenetic analysis shows that the 5 isolates were in the same branch as the GETV isolates in Japan,China and South Korea in recent years,and they are closely related to the strains in the Japanese horse group(14-I-605-C1),pig group(15-I-1105)and mosquito(12IH26)in 2012~2016,and the genetic distance of HNNY-1 and the first pig source HNJZ-S1 in China was recently located in a small branch of HNNY-2,HNPDS-1 and HNPDS-2,and AH9192 was similar to South Korea's South Korea in 2004.The results of this study divides GETV into 3 gene groups or lineages(clade),and shows that its evolution have obvious time nodes and geographical features,while there are no obvious differences in species.The number of strains in the pedigree ?is highest(37/49),and the pedigree?is a transitional group,the pedigree?is the oldest pedigree.This study based on the five GETV isolates the whole genome sequence analysis,understand the organ of GETV latest popular dynamic and variation in pigs,also shows that GETV rapid expansion in China region and between species.