Surveillance Of NDV In Some Areas In China During 2016-2017 And Establishment Of Two Methods For Rapid NDV Detection

Newcastle Disease(ND)is a kind of avian acute and highly contagious infectious disease caused by virulent Newcastle Disease virus(NDV),which is very harmful to the poultry production worldwide.Although the disease is under control in China now,the pathogen still exists in some areas in the country and there were some new genotype viruses reported in recent years.In order to prevent and control more effectively and know more about the distribution of pathogens and pathogenic characteristics of the agent,surveillance were carried out,the isolates were identified,the genetic charecteristics were analysed and 2 methods which could identify virulent strain of NDV quickly and used for NDV strains differentiated were established in the study.1.Surveillance of NDV in Some Areas in China during 2016-2017Epidemiological surveillance ofNewcastle Disease virus were carried out during 2016-2017 in some areas in China.3674 samples were collected and 62 strains of NDV were isolated and identified.46 NDV strains were obtained in 2016,27 strains were from chicken,14 strains were'from ducks,4 strains were from geese and 1 strain was from pigeon;16 NDV strains were isolated in 2017,11 strains were from chicken,3 strains were from environment and 2 strains were from pigeons.Parts of the F gene of isolates were amplified and sequenced,the genetic evolution were analysed and the result showed that 39 strains among the 62 belong to genotype 3 of Class ?,13 strains belong to genotype ? of Class ?,3 strains belong to genotype? of Class ?,1 strain belongs to ? of Claaa ? and 6 strains belong to novel genotype of Class ?.Genotype 3 of Class? and genotype ?of Class ? were found to be dominant genotype in this survey.In order to learn more of the pathogenic characteristics of new genotype strains and dominant genotype strains of the isolates.Some strains of NDV(1 genotype 3 of Class ? strain and 1 genotype ? of Class ?strain and 4 novel genotype of Class ? strains)were chosed and used for full F gene amplification and further homology analysis.The result of the homology analysis showed that the genotype 3 virus had close relationship with the genotype 3 isolates in China,the genotype II isolate were 99.9%homologous with La Sota.NDV F gene analysis results of 4 novel.genotype strains showed that the cleavage sites of F gene were 112E-RWQGE?R-L'Vn1 conformed to the cleavage sites sequence of avirulent NDV,both have 12 cysteine and 5 glycosylation site.By comparing F protein with other strains of Class I,no protein replacement were found in all 4 viruses.Phylogenetic tree based on F gene coding region sequence revealed 4 isolates had a distante genetic relationship of genotype 1-10 of Class I with the bootstrap<75,they were new genotype strains of Class I.Phylogenetic tree with full length sequence of F gene showed that the isolates belong to genotype lc,were highly homologous with North American isolates.In order to make the genetic molecular characteristics of novel genotype strains in China more clear,the whole genome sequences of Ningxia isolates duck/Ningxia/2209/2016(referred to NX2209 hereinafter)were amplified and analysed,NX2209 has a genome with full length of 15198nt and it belonged to Class 1.2.The establishment and application of double RT-PCR method for virulent and avirulent strains ofNDV.2 pairs of specific to virulent or avirilent NDV respectively were designed and a one-step double RT-PCR for virulent and avirulent strains differentiated were established.2 products of 353 bp and 228 bp could be amplified for virulent NDV,only one product of 353 bp could be abtainded in the test.The sensitivity test indicated that the minimum number of nucleic acid copies could be detected by this method was 1.1 ×105 and 1.7 ×105,while the concentration of RNA is 0.02 pg/?L.9 others poultry viruses in veterinary clinic(avian influenza virus(subtype H5,H7 subtypes and H9 subtype),infectious bronchitis virus,infectious bursal disease virus,infectious laryngotracheitis virus,chicken egg-drop syndrome virus,avian encephalomyelitis virus,reticuloendotheliosis virus)could not get any specific product by amplification.A parallel tests was conducted to compare the double RT-PCR established in the study with the rapid identification of C ? and C ? multiple RT-PCR method of NDV established before in our lab and the RT-PCR method recommended by national standard,718 clinical samples aboved were chosed to test randomly,and it confirmed that the diagnosis sensitivity of this method was 98.36%,specificity was 97.0%and better than the other two methods.Compared with the gold-standard method,the double RT-PCR method has a 90.54%positive coincidence rate.3.The establishment and application of NASBA detection method of virulent NDVBy primers screening and condition optimizing,a NASBA method for virulent strains of NDV were established successfully.The sensitivity of test showed that it could get a positively results of 5 copies of NDV nucleic acid.7 other kinds of viruses(H5 subtype of avian influenza virus,H7 subtype of avian influenza virus,H9 subtype avian influenza virus,infectious bronchitis virus,infectious bursal disease virus,infectious laryngotracheitis virus,avian encephalomyelitis virus)were tested for its specificity,and any products were obtained in the tests.53 strains of NDV were used to compare the charecteristics of the NASBA test and the one step double RT-PCR established above,the results showed sensitivity of this method was 97.22%,specificity was 94.11%.The NASBA test was better than the one step double RT-PCR test.According to the sequencing results(gold-standard method),the NASBA method has a 97.22%positive coincidence rate,better than the RT-PCR method,the RT-PCR test with a virulent conformity rate of 86.11%.The results showed that the NASBA test were more sensitive and specific than the RT-PCR method.