Isolation,identification And Genetic Analysis Of Peste Des Petits Ruminants Virus In Guizhou Province

Peste des petits ruminants(PPR)is an acute and fatal infectious disease that is caused by peste des petits ruminants virus.It spreads rapidly with high morbidity and mortality,which seriously did a great harm to the development of small ruminants animal husbandry.It was first discovered in West Africa in 1942,and so far there are more than 40 countries which have found it,as well as Tibet in China.It outbroke in many provinces in China since 2014,and 8 regions in Guizhou Province were also affected,which has caused many economic losses of the breeding industry.In order to understand the small ruminants animal disease epidemic characteristics and pathogenic molecular characteristics of Guizhou Province,this paper carried out the researches about the rapid detection methods,epidemiology,pathogenic molecular characteristics,which could help to provide theory references for understanding the PPR Guizhou epidemic characteristics,pathogen characteristics as well as to offer theoretical basis fo comprehensive prevention and control measures.1.Establishment of one step RT-PCR detection method for PPRV:Synthesized specific primers was designed based on PPRV N gene sequence,taking small ruminants beast disease virus vaccine strain RNA sample as the template and to establish Peste des petits ruminants one-step RT-PCR detection system,through the performance evaluation established to check the reliability of one step RT-PCR assay for detection of Peste des petits ruminants.The results showed that the fragment of the N gene of 447 bp could be amplified and the size was consistent with what had expected;the optimal annealing temperature was 60?;the best template final concentration was 6.76 x10^-3g/L;the minimum PPRV RNA detectable concentration was 6.76 x 10^-6g/L,the method was specific with high sensitivity,which could be used for detection of Peste des petits ruminants clinical samples.specific primers and probes were designed based on PPRV P gene,taking small ruminants beast disease virus vaccine strain RNA samples as a template for preparation standard plasmid product,and then to establish Taq Man probe real-time fluorescence quantitative RT-PCR assay for detection of reaction system and the standard curve and the established by the method of evaluation and determine the reliability built by Taq Man probe real-time fluorescence quantitative RT-PCR detection method.The results showed that Taq Man probe real-time fluorescence quantitative RT-PCR detection method could effectively detect plasmid standard and form typical amplification curve and standard curve;the positive standard minimum detectable concentration was 1.7 x10²copies/?L,which was more sensitive than conventional RT-PCR;the coefficient of variation between the group that repeated the experiment and the group that repeated the test was less than 2%,proving stability and good specificity,which could be used in the screening of small ruminants animal disease clinical cases of various samples detection and optimal detection samples.2.Epidemiological survey of small ruminants animal disease in Guizhou Province:722 serum samples and122 samples of suspected cases of goat lung and lymph node tissue samples were collected in Guizhou Province Bijie City,Qiandongnan Prefecture,Qiannan Prefecture,Tongren City,Liupanshui City,Zunyi City,Guizhou,Anshun City eight city(state,county),and were detected by ELISA method for serum epidemiological investigation and RT-PCR methods epidemiologic survey.Molecular epidemiological investigation was carried out based on the N,H gene.The results showed that the positive rates of PPRV antibody were 26.3%(93/354),and the positive groups of PPRV latent infection were found in most months of the whole year,which were the highest in 3-5 months.The average antibody positive rates were 59%(217/368),and the whole herd immunity was not good.PPRV nucleic acids were detected in all samples from 8 regions of the province,and the average positive detection rates were 18.9%(23/122).The PPRV contents of different samples were different,and the lung samples could be used as the best material for the detection of this disease.The full length of N gene was 1578bp.Encoding 525 amino acids and the homology of 8 Guizhou Province PPRV epidemic strains of N gene reached92.2%?100%,and 97.7%?99.6%with the domestic strains,88.5%?97.6%with the foreign strains.The variation of amino acid sequences deduced from N genes was mainly concentrated in Zone IV(421?525 the carboxyl terminal region);and H gene full-length was 1830bp,encoding 609 amino acids.H gene homology of 8 Guizhou Province PPRV strains was 88.9%?99.8%,and 97.5%?99.8%with other Chinese PPRV strains,89.0%?98.2%with foreign strains.Point mutations of amino acid sequences deduced from H gene were 203 leucine(L)mutated to methionine(m),315 lysine to arginine(R),450 arginine(R)to lysine(K)and 546 glycine(G)to serine(S).PPRV N and H gene genetic analysis showed that PPRV Guizhou epidemic strains belonged to the fourth branch and were different from the first branch of the vaccine strain Nigeria-75-1.The results of the study showed that PPR had a wide range of popular areas in Guizhou Province and a high incidence from March to May.PRRV Guizhou epidemic strains had regional characteristics and a certain variation.3.Isolation,Identification of PPRV in Guizhou province:PPR positive samples of lung and lymph node were collected,and cultured in Vero cells.The viruses were identified by morphological observation,the viral nucleic acid detection,indirect immunofluorescence test and SEM test.The results showed that PPRV Guizhou strains could cause CPE for Vero cells.RT-PCR identification showed that there were differences between the first generation and the fifth generation of virus cell culture,and the existence of viral nucleic acid could be detected from the sixth generation.Indirect immunofluorescence assay showed that the specificity of green fluorescence in cell culture reflected the presence of virus antigen in cells.In the transmission electron microscope,the PPRV particles could be observed in the cytoplasm of Vero cells.The experimental results showed that the PPRV were isolated from the samples of clinical positive cases,and 2 strains were named as PPRV-GZPX strain and PPRV-GZSY strain respectively.4.Genetic Analysis of Peste des petits ruminants virus in Guizhou province:Two strains of PPRV Guizhou strains were as the research objects and 7 pairs of primers were designed.RT-PCR was used to amplify seven gene fragments of PPRV genome-wide.The bioinformatics software was used to analyze PPRV sequence S.The results showed that the total length of PPRV in Guizhou was 15954bp,and the homology of 2 Guizhou isolates was99.7%,and 92.1%compared with the vaccine strain Nigeria75-1,97.2%?99.7%compared with Chinese strains,88.1%?97.1%compared with foreign strains.RNA chain from 3'to 5'were N-P(C/V)-M-F-H-L six genes which encoded the nucleoprotein(N),phosphoprotein(P),matrix protein(M),fusion protein(F),the haemagglutinin(H),large protein(L)6 structural proteins and 2 non structure protein(C/V).The protein amino acid sequence had different degrees of variation and H gene mutation were the most.The results of phylogenetic analysis showed that the PPRV stains of Guizhou were in the same branch with other Chinese strains isolated in recent years,all belonged to PPRV IV,which was different from that of the vaccine strain.The results of the study showed that the whole genome length of PPRV was stable and there were obvious mutation and point mutation.Vaccine strains had significant difference should be given more attention in choosing the vaccine.