Molecular Epidemiology Of Newcastle Disease Virus Isolated In Partial Regions Of China During2010to2012

Newcastle disease virus （NDV） is the causative agent of Newcastle disease （ND）, a highlycontagious disease. Outbreaks of ND may be devastating, with flock mortality approaching100%infully susceptible chickens. From the1920s to the present, it has distributed worldwide, causing severeeconomic losses due to disease and, for countries that export poultry or poultry products, lossesresulting from trade restraints and embargoes. Due to a strict vaccination policy, outbreaks of ND weremild and sporadic, which show some new epidemical characterizations such as atypical ND, emergenceof new genotypes, and enlargenment of host range. Therefore, monitoring the molecular epidemiologyof ND would illustrate the variation tendency of NDV, and the ecological contribution of different birds,which contribute to the combination control measures for ND.By specific-pathogen-free （SPF） chicken embryos inoculation and serological identification, thirtystrains of Newcastle disease virus （NDV） were isolated from outbreaks in fowl in partial regions inChina during2010–2012, among them,27strains were obtained from the northeast of China.30NDVstrains were purified and classified by F genotype. Phylogenetic analysis revealed that all of30NDVstrains belonged to Class Ⅱ, including genotype I （2）, genotype II （3）, which had the lentogenic motif¹¹²RRQKRF¹¹⁷at the F0cleavage site and should be classified into vaccine-like strains.2strains isolatedfrom pigeons in Inner Mongolia and HeiLongJiang province belonged to genotype VIb, which showedhigh homology with some strains isolated from ShanDong, JiangSu province and Europe, indicated thatthese prevalen strains in pigeons in China in recent years may share the common ancestor with Europepigeons strains. Amonge30strains,23strains belonged to genotype VIId, which had the velogenicmotif¹¹²RRQKRF¹¹⁷at the F0cleavage site, indicated that genotype VIId NDV was mainly responsiblefor the present ND panzootic in Northeast China. Phylogenetic analysis revealed that no host-specific orregion-specific characteristics were found among the twenty NDV isolates comparing to NDV strainsisolated from other regions in China.185strains of NDV were isolated from1788swabs samples collected from healthy poultry andenvironment in17provinces of China in the winter of2011, provided friendly by State Avian InfluenzaReference Laboratory, Harbin Veterinary Research Institute, CAAS.172strains were analyzed by Fgene sequencing. The phylogenetic tree based on the partial sequences of the F gene revealed that the172isolates of NDV could be divided into Class Ⅰ（148） and Class Ⅱ （24）.24Class Ⅱ NDV isolateswere divided into three genotypes, including genotype VII （5）, genotype I （1） and genotype II （18）.148strains of Class ⅠNDV isolates were were determined to be of genotype3, shared more than96%nucleotides homology with each other. These148Class ⅠNDV strains were collected from differentclinical specimens, including chickens, ducks, geese and environment, were also found to beremarkably similar to the NDV strains isolated from live-bird markets in Hong Kong in recent years.All of the Class Ⅰisolates had the F protein cleavage motif¹¹²GRQGRL¹¹⁷at the F0cleavage site，whichis the characteristic to lentogenic NDV strains. Class ⅠNDV was originally obtained from waterfowl,shorebirds （WS） or poultry in live bird markets （LBMs）, the close phylogenetic proximity of the Class INDV obtained from some WS, LBMs and China mainland suggests that transmission may occurbetween waterfowl and poultry, also, no host-specific or region-specific characteristics were foundamong the Class ⅠNDV isolates.As the high infection rate of Class Ⅰstrain in flocks and environment, it is necessary to analysis ofthe influence of Class ⅠNDV in ND epidemiology, so the replication characteristics of Class Ⅰvirus in SPF chickens and ducks were analyzed firstly. Two-week-old SPF chickens and SPF ducks wereinoculated with ClassⅠ strainNDV/Env/CH/SC144/2011（MDT>120h，ICPI=0.275） in a dose of106EID50/0.1mL by intranasally and eye way, the parameters including morbidity, mortality, tissue tropismof virus, virus shedding, serum antibody level and cross-protective were detected. The results showedthat no clinical signs or mortality was observed in chickens and duck that had been vaccinated withSC144strains. Only intermittent, shedding was detected from a small number of ducks （cloacal） untilDPI14; No virus was isolated from any ducks from oropharyngeal during the experiment; No virus wasdetected from group organs; Serum antibody HI titers of ducks was5.67log2post infection7days, butreduced to3.3log2post14days. The serum antibody levels of control group and experimental groupshowed no obvious difference, which indicated that the Class Ⅰvirus can horizontal transmission inwaterfowl and replicate in vivo of duck. Initial virus shedding was detected on DPI1via the two routesand peak oropharyngeal and cloacal shedding occurred on DPI1-5, followed by a sharp decline in thenumber of positive chickens. No further shedding was detected from either route on DPI14; Strainscould be detected in caecum tonsil of one chicken; HI titers of Class Ⅰantiserum was5.43log2afterinfection in one weeks, and5.8log2after infection in two weeks. The serum antibody levels of controlgroup and experimental group showed no obvious diffenence, which indicates that the Class Ⅰcanspread in chickens. After challenge by LL01（genotype Ⅶ）and F48E9（genotype Ⅸ）, Class Ⅰvirusescan provide a100%protection rate, but they cannot block virus shedding upon challenge with virulentstrains.Class Ⅱ genotype VII viruses were responsible for disease outbreaks in chicken and goose flocksand circulated predominantly in China in recent years. Two strains isolated from pigeon were belongedto genotypeⅥb. The infection rates of Class Ⅰis higher than vaccine strain and virulent strain. Chickensvaccinated with Class Ⅰwere fully protected from disease and death against challenge by LL01（genotype Ⅶ）and F48E9（genotype Ⅸ）. Our research showed that the infection of Class Ⅰis benefitto the protection of chicken against ND. However, the high infection of Class Ⅰenhanced the interactionbetween the NDV isolates and the variants of virus. Therefore, it is urgently to enhance and insist theepidemiology and molecular epidemiology research of Newcastle disease.