Identification And Phylogenetic Analysis Of Newcastle Disease Viruses Isolated In Eastern China During 2014-2016

Newcastle disease(ND)is an acute and serious infectious disease caused by virulent Newcastle disease virus(NDV),and can generate heavy economic losses in poultry industries.ND has experienced four epidemics in the world since its first report in 1926 and each epidemic was induced by new genotype virus.NDV is an evolving aetiology and possesses several different genotypes.The virulent NDV was firstly isolated in China in 1946,and the long-term existence and epidemiology of ND in China bring the consistent threat to the poultry breeding.Additionally,the antigenic variation of NDV has brought the challenge in ND control.To well know the NDV carrier and distribution status in the different poultry flocks in China and provide the important basis to control the ND,we isolated and identified the NDVs from different poultry flocks in Eastern China during 2014-2016.The phylogenetic analysis was then conducted according to the F gene fragments amplified with a novel one-step RT-PCR which can detect all the NDV strains.1.The establishment of one-step RT-PCR detection method for NDVsNDVs can be divided into two branchs,class Ⅰ and class Ⅱ,and the phylogenetic distance between them is relatively far.Usually,two specific pair of primers,targeting the class I and class II NDV F gene respectively,are required in the virus molecular identification by RT-PCR.In order to establish a novel one-step RT-PCR which can detect all the NDV strains,a pair of universal primers with broad target spectrum was designed based on the F gene sequences from different genotype NDVs.The one-step RT-PCR was optimized in terms of its reaction temperature,concentration of template,cycles and reverse transcription time.We also verify the sensitivity,specificity and feasibility of this novel method.Results showed the new method exhibited high sensitivity and specificity,and can detect all the tested NDVs both from class Ⅰ and class Ⅱ.Additionally,the amplified fragment could be used for virus genotyping and virulence analysis,therefore,the novel one-step RT-RCR provided a useful tool to identify the field NDVs rapidly.2.Isolation and phylogenetic characterization of NDVs at live bird markets in eastern China during 2014-2016In this study,we characterized the NDVs from different poultry flocks at live poultry markets in Eastern China during 2014-2016.Fifty-six NDVs were isolated and identified from live bird markets with a total isolation rate of 2.67%.75%of the NDV isolates(42/56)were identified as class Ⅰ with a total isolation rate of 2%.The rest strains belonged to class Ⅱ,with 4 genotype Ⅰ NDVs,8 genotype Ⅱ NDVs and 2 genotype VII NDVs.The genotype Ⅰ and Ⅱ NDV isolates shared high identities to the vaccine strain LaSota and V4 respectively.31 of the 42 class Ⅰ NDVs were isolated from the chicken flocks,and the other 11 strains were from the waterfowls.Phylogenetic analysis showed that 42 class Ⅰ strains all belonged to the branch 3c sub-genotype which has become the prevalent genotype circulating at LBMs in Eastern China.3.Isolation and phylogenetic characterization of NDVs from clinical specimens during 2014-2016Forty-seven class Ⅱ NDVs,with six genotype Ⅰ NDVs,29genotype Ⅱ NDVs and 12 genotype Ⅶd NDVs,were isolated from 185 clinical specimens during 2014-2016.The genotype Ⅰ and Ⅱ strains were similar to the vaccine strain V4 and LaSota,respectively.Further analysis showed that genotype Ⅶd NDVs all belonged to sub-genotype Ⅶd2,which indicated that this sub-genotype was still the prevalent virulent strains circulating in China.Phylogenetic analysis based on the F gene sequences showed that the Ⅶd NDV isolates shared 96-100%identities in nucleotide sequence and 97.5-100%identities in amino acid sequence.And based on the HN sequences,the Ⅶd NDV isolates shared 96.6-99.9%and 97.6-100%identities in nucleotide and amino acid sequence,respectively.Antigenic analysis displayed that 10 of 14 VIId2 NDVs presented the E347K mutation in the HN,which induced significant antigenic difference via the serum neutralization test.Additionally,we obtained and analyzed two whole genome sequences of sub-genotype Ⅶd2 strains.The results showed that genetic tree constructed by the full-length genome was highly similar with those constructed with each six gene sequence,which demonstrated that the NDV was evolved on the whole genome level.