Hereditary Variation Of Newcastle Disease Virus From Wild Fowl And Swine And Their Pathogenicity In Different Hosts

Newcastle disease as “Asian fowl plague” is regarded worldwide as one of the mostdevastating diseases of poultry, waterfowl. And its pathogen is Newcastle disease virus(NDV). ND can cause the severe morbidity and mortality, so it is listed A disease by anOffice International des Epizooties (OIE).Wild fowl play an important role in the spread of the virus. Various wild migratoryfowls are considered as known reservoirs of ND. So strengthening the surveillance ofindependent activities wild fowl of NDV is of great significance. In this paper, some wildfowl were monitored for NDV, focusing on study of the molecular properties of ND. Weaimed to understand the epidemic condition of NDV in some wild fowl populations andanalyze their genetic characterizations.To understand the role of wild fowl as virus carriers, we collected1029fecal and20tissue samples from different areas in2010~2011for NDV surveillance and identification.Five strains were isolated, positive rate of about0.48%. Four NDV isolates were collectedfrom wild fowl fecal samples and named NDV1~NDV4, one NDV5strain was isolatedfrom pigeon. And their virulences were biologically analyzed through MDT, ICPI, IVPI.Through these pathogenicity index tests, NDV2and NDV5were confirmed as virulentstrain, NDV1, NDV3and NDV4were confirmed as avirulent strain.The cleavage site, genotype, antigen epitope and amino acid sequences of thecomplete coding region of F gene of five strains were characterized by sequencing their Fgenes. NDV2and NDV5, exhibited the cleavage site motif112R-R-Q-K-R-F117, which ischaracteristic of virulent NDV strain; other threes contained the112G-R-Q-G-R-L117motif,which is specific for avirulent or low-virulence strain. The results on virulence based onthe F protein cleavage site were consistent with those of the pathogenicity index tests.NDV2was clustered to genotype VII, NDV5was clustered to genotype VI, the other three were clustered to genotype II through F genotyping. Homology comparisons foundthese isolates with classic strains La Sota, V4, B1, TEX-48were different. Homologycomparison results showed that NDV1, NDV3and NDV4had higher homologies with LaSota vaccine strain, and the homologies with field strains were low. The homologies ofNDV2strain with domestic strains in2006-2011were very high, especially for VIIdsubtype strains,98.5~99.4%. The antigenic epitopes were predicted and the results showedthat there were22,24,21and19epitopes of F protein for NDV1, NDV2, NDV5and LaSota.Nine pairs of specific primers were designed by whole genomic sequence of NDVpresented in GenBank. The nucleotide sequences of NDV2strain were acquired by overlapRT-PCR reaction. The results showed complete genomic sequences of NDV2strain were15192nt. NDV2was6nt longer than15186nt NDVs, the additional6nt(ACCCTC) motifwas located in the non-coding region of NP gene and P gene. Its genome comprises sixORFs, encode Nucleocapsid protein, Phosphoprotein, Matrix protein, Fusion protein,Haemagglutinin-neuraminidase and Large protein. The length of each gene is respectively1753,1451,1241,1792,2004and6703nt.NDV2strain was classified into Class II, genotype VII; the homology comparison ofeach protein showed NDV2strains had high homology with domestic genotype VII strainsGX.11, WF.00, GD450.11in recent years. NDV2strain had the same start sequences withother reference strains, so the start sequences were conservative. As termination sequencesof NP, P, M and L, NDV2strain was consistent with the reference strains, but not the samein F and HN gene. NDV2had the same termination sequences with La Sota and Sweden95,but not the same with other field strains.Newcastle Disease Virus has been considered to only infect avian species. However,in recent years, there were some reports about swine NDVs. One suspected paramyxovirusnamed as Xiny was isolated from swine. By electron microscopy observation, serologicaldetection and sequence alignment, Xiny was proved as NDV. Physical and chemical testshowed that Xiny was sensitive to chloroform, ether and pancreatic enzyme; so the virushad envelope, was sensitive to heat and it can agglutinate red blood cells of many kinds ofanimals，but not sheep and cow. The differences of Xiny with another previous swine NDV (JL01) were compared on sequences of whole-lengthen F gene, biological characteristics,genotype and virulence.Xiny and vaccine strain La Sota both belonged to genotype II, as one of them, Xinyshared97.3%-98.7%homologies with genotype II NDVs, which were higher than othergenotypes. In addition, Xiny and La Sota owned the same12conserved Cysteine residuesand6potential glycosylation sites. As far as HN gene, the homology of Xiny with La Sotawas the highest,97.5%. The above data showed that the swine NDV Xiny may come fromLa Sota.At the same time, in order to understand the situation of swine carrying NDV,148serum samples and90tissue samples were tested. The results showed that all serum andtissue samples were negative except the Xiny. The results showed NDV infected swine wasonly local sporadic infection, not universal and epidemic.In order to understand infectivity of the wild fowl NDV or porcine NDV in differenthosts, we used wild fowl isolate NDV2, swine Xiny to attacking SPF chicken, duck,pigeon, swine and each attacking group had cohabitation infection group to observewhether the spread of the virus infection. By simulation under the natural state thepathogenicity of NDV on different hosts, NDV cross-species infection ability will beknown. Results: the pathogenicity of NDV2in SPF chicken and pigeon were higher thanduck, and NDV2strain can cause some cohabitation infected; Xiny strain was not enoughto cause attacking animal death, but most attacking animals were in the carrier-virus state.The virulent F48E9strain and Xiny isolate can not cause pig death, but the virus can bedetected in attacking swine.Innovation：1) The investigation about the situation of carrying virus of wild birds in China found thatwild birds in our country, especially the sea birds carried NDV strains with differentvirulences and molecular characteristics.2) One NDV was isolated from swine, and illustrated the swine NDV strain was likely tocome from La Sota.3) Through testing some swine serums and tissues, we found NDV infection in swine wasjust local sporadic infection, not into universality and epidemic. 4) These different host strains carried out mutual pathogenic analysis and cohabitedinfection in different hosts.