Etiology Surveillance On Genotype Ⅸ Newcastle Disease Virus And Study On Genetic Evolution And Virulence Of Islolates From Different Fowl

Newcastle disease is known as the primary infectious diseases in poultry due to its worldwidedistribution and devastating effects, the causative agent Newcastle disease virus （NDV）,designated as avian paramyxovirus serotype1（APMV-1）, belongs to the Avulavirus genusand the Paramyxoviridae family. The enveloped NDV contains a negative-sensesingle-stranded RNA genome of¹5.2kb. F protein cleavage site motif and HN were themost important determinant for NDV virulence. NDV isolates are categorized as being highlypathogenic （velogenic）, intermediate （mesogenic）, or apathogenic （lentogenic） strainsbased on their pathogenicity in chickens.The ‘early’ genotype Ⅸ NDV with15,192nt in the genome, containing the Chinesestandard F48E9strain, represents a unique geographic expansion of the NDV. This genotypevirus has been endemic in China since1946and a strict vaccination policy was adopted in the1980s, in the past30years, it have been implicated in sporadic ND cases in poultry flocks,however, recent reports suggest that genotype Ⅸ strains have been accidentally isolated fromwaterfowl. Although different of NDV genotypes have simultaneously evolved at variousgeographical locations throughout the world, the epidemiology and evolution of genotype Ⅸviruses remain an enigma due to several isolates from different hosts that have been reported.Thus, evaluate the epidemic and evolution status of genotype Ⅸ NDV from the wild andpoultry will be of great significance to prevent and controll the outbreak of ND, which olsoillustrate the general evolution status in genotype Ⅸ NDV and the exactly function of wildbird in the evolution and transmittion of virulent genotype Ⅸ NDV. The main result as follow:1. Etiology surveillance and virulence analysis of genotype Ⅸ NDV strains isolated fromwild birds in northern Qinling Mountains.This study aimed to evaluate the epidemiological status of genotype Ⅸ Newcastledisease virus （NDV） from healthy wild birds and virulence of isolates in the northernQinling Mountains and Guanzhong Areas from August,2008to July,2011.374larynx andcloaca swabs from healthy wild birds were used to isolate and identify genotype Ⅸ NDV,which were subsequently characterized pathotypically and genotypically.5NDV isolates were isolated and named the Peafowl/China/08, WhooperSwan/China/08, Blackbird/China/08,SpottedDove/China/08and White-cheekedStarling/China/08, respectively. Sequences analysisof partial F gene（535bp）revealed that all the5isolates shared common F0protein cleavagesite motif112R-R-Q-R-R-F117equivalent to virulent NDV. The5isolates including2frommigrants exhibited different pathogenicity indices, with MDT, ICPI and IVPI ranging37.2h～64.8h,0.425～1.638,2.04～2.76, respectively. One novel isolate from theSpotted-necked Dove（family Columbidae）exhibited discordance between its lentogenic ICPIand its virulent proteolytic cleavage site motif¹¹²R-R-Q-R-R-F¹¹⁷. Homology analysis basedon partial F gene （535bp） revealed that the5isolates shared homology as high as99.8%～100%with each other. Phylogenetic analysis based on variant region of F gene（42～420nt,374bp） revealed all the5isolates clustered to genotype Ⅸ NDV, together with the Chineseearly prevalent strain F48E9（homology99.4%～99.6%）, distinguished the traditional La-Sota and V4vaccine strains, or currently prevalent genotype Ⅶ NDV in poultry. This studyrevealed that asymptomatic wild bird species had natural resistance to genotype Ⅸ NDV, andacted as natural carriers of virulent genotype Ⅸ NDV, which may charge NDV "aircraft" inaccelerated spread of these viruses. The result indicated that these genotype Ⅸ NDV isolatesfrom wild bird had been circulating in Qinling Mountains and Guanzhong area for someperiod of time, also posed threat for poultry and enviroment.2. Etiology surveillance and virulence of the genotype Ⅸ NDV strains isolated fromcommercial poultry flocks in Shaanxi Province.This study was conducted to evaluate the status of genotype Ⅸ Newcastle disease virus（NDV） from commerical poultry flock in Shaanxi.160tissue samples, larynx and cloacaswabs were involved in34flocks in the duration of Jun,2009and July,2012.2isolates wereabtained from layer flocks ND outbreaks in Chang’an and Yuling, namelyLayer/China/Yulin/10and Layer/China/Changan/10, which were subsequently characterizedpathotypically and genotypically. The2isolates exhibited different pathogenicity indices, withMDT, ICPI ranging37.2h～56.5h,1.603～1.838, respectively. Sequences analysis of partialF gene （535bp） revealed that the2poultry isolates shared common F protein cleavage sitemotif112R-R-Q-R-R-F117equivalent to virulent NDV, which shared as high as99.8%homology with each other,99.6%～99.8%with the previous5wild bird isolates.Phylogenetic analysis based on variant region of F gene（42～420nt,374bp） revealed all the2isolates clustered to genotype Ⅸ NDV, together with the Chinese early prevalent strainF48E9（homology99.3%～99.4%）, distinguished the traditional LaSota and V4vaccinestrains, or currently prevalent genotype Ⅶ NDV in poultry. This study confirmed that virulentgenotype Ⅸ viruses were responsible for ND outbreak in commercial poultry flocks around the location of wild bird samples collection, also suggested that genotype Ⅸ viruses had everbeen epidemic in poultry in the northern Qinling Mountains.3. Genetic evolution analysis of the virulence gene of genotype Ⅸ NDV isoates.The ORF of F and HN gene of the7isolates were amplified by RT-PCR, sequenced andanalyzed for theri homology, deduced amino acid residue substitution，genetic relationship byperforming the alignment and constructing phylogenetic tree based on the sequences. Thehomology analysis within genotype Ⅸ NDV revealed that they share homology of99.6%～99.9%with each other, and94.4%～99.9%with other genotype Ⅸ NDV reference strains inF gene, while they shared homology of99.3%～100%with each other,99.3%～99.9%withother genotype Ⅸ NDV reference strains in HN gene. Most of the previous genotype Ⅸstrainshared homology more than99.0%, which indicated interspecies risk. The F gene distanceestimation analysis involving23sequences in genotype Ⅸ NDV, including the7isolates and16reference strains, showed the overall average value for the estimated evolutionary distancewas0.012, and each estimated average evolution distance of the7isolates was＜theoverall average value （0.012）. The HN gene distance estimation analysis involving14sequences in genotype Ⅸ NDV, including the7isolates and7reference strains, showed theoverall average value for the estimated evolutionary distance was0.007. The each estimatedaverage evolution distance of the7isolates was approximate the overall average value （0.012） in HN sequences except the Layer/China/Changan/10. Even though the7isolatesunderwent several amino acid mutations in the fusion protein, evidence of continuousevolutionary divergence did exist in the genotype Ⅸ NDV, which was always regarded as aconservative genotype, and in which the larger evolution divergence indicated the waterfowlproduced positive effect on evolution within this genotype.4. Genomic analysis of genotype Ⅸ NDV isolates from different fowl.To futher evaluate the genetic evolution realationship of genotype Ⅸ NDV isolates fromwild and poultry, Two complete genome sequence of the wild bird isolate Blackbird/China/08and the poultry isolate Layer/China/Yulin/10were obtained by RT-PCR and genomic analysiswas conducted using DNAStar5.0and MEGA5.2software, which was referenced againstprevious genome sequence of SpottedDove/China/08and8genotype Ⅸ NDV sequencesavailable in GenBank. Genomic sequence showed that both isolates consist of15,192nt, andgeneomic arrangement is3’Leader-N-P-M-F-HN-L-Trailer-5’. Both the characteristics of Fprotein cleavage site motif and length of HN protein of the two isolates were consistent withvirulent NDV. Phylogenetic analysis of complete genome indicated the both isolates stillclustered to the genotype Ⅸ clusters. The results of genetic evolutionary distance showed thatthere were slightly difference in evolutionary distance among each gene and whole genome evolution, HN gene and V gene is slightly higher than that of other genes. The results suggestthat the genomic evolution is highly conservative among genotype Ⅸ NDV viruses, andwaterfowl may play an important role in NDV evolution, the hypothesis that V gene as a markof NDV virulence needs further validation. Each genome evolution levels of these isolatesBlackbird/China/08,Layer/China/Yulin/10and SpottedDove/China/08were below （nearly） theestimated average evolution level with genotype Ⅸ NDV. The genome evolution levels ofpoultry were higher then the wild,due to the immune pressure selection against by poultryflock. Wild birds were confirmed nature reservoir of virulent genotype Ⅸ NDV strains, whichaccelerated the proliferation and spread up.In conclusion, this work was conducted for genotype Ⅸ NDV isoltates from differentfowl in the duration of Aug,2008and July,2012,7isolates from different fowls exhibiteddifferent virulence in despite of pocessin the common F protein cleavage site motif112R-R-Q-R-R-F117. Remarkable, a novel lentogenic NDV isolate from the Spotted-neckedDove （family Columbidae） exhibited discordance between its lentogenic ICPI and itsvirulent proteolytic cleavage site motif112R-R-Q-R-R-F117. The results suggest that thegenomic evolution is highly conservative among genotype Ⅸ NDV viruses, and waterfowlmay play an important role in NDV evolution, the hypothesis that V gene as a mark of NDVvirulence needs further validation. Each genome evolution levels of these isolatesBlackbird/China/08, Layer/China/Yulin/10and SpottedDove/China/08were below （nearly）the astimate average evolution level with genotype Ⅸ NDV. The genome evolution levels ofpoultry were higher then the wild, due to the immune pressure selection against by poultryflock. It was supposed that wild birds were nature reservoir of virulent genotype Ⅸ NDVstrains, and accelerated the spread of genotype Ⅸ NDV viruses. The hpothesis that V gene asa mark of NDV virulence need further validation.