Identification And Sequence Analysis Of F Gene Of Newcastle Disease Viruses Isolated From Apparently Healthy Duck Flocks

Newcastle disease(ND),which is caused by Newcastle disease virus(NDV),is one of the most menacing highly contagious viral diseases of poultry and could cause large economic losses in poultry production worldwide.Aquatic birds,such as goose and duck, were usually considered to be resistant even to most virulent NDV strains.However,the goose flocks in some regions of South and East China have experienced severe outbreaks of NDV infection since 1997.Since 2000,ND have taken place in duck flocks from Anhui, Shandong,Fujian,Zhejiang,Hebei and other places,which characterized by digestive tract and immune organ damage,and its mortality rate can be as high as 90 percent.Although measures have been taken to control the disease,it still spreaded progressively and became another major viral disease of poultry industry.Lots of NDVs were isolated from outbreaks since late 1990s,and some biological characters of those viruses were studied.However, there are rarely researches about the NDV from apparently healthy duck till now.In the present investigation,we performed isolation and characterization of NDV from apparently healthy domestic duck in different regions of Hebei province.By studying the biological and molecular characteristics of these NDV isolates,the status about NDV infection in apparently healthy domestic duck and the origin of those duck-pathogenic ND virues were discussed.1.Isolation and characterization NDV from apparently healthy domestic duck flocksCloacal swabs from the apparently healthy domestic duck flocks were collected to characterize NDV.A total of 15 NDV isolates were derived from 260 samples during 2006-2008.The mean death time(MDT)in embryonated chicken eggs and intracerebral pathogenicity index(ICPI)in 1-day-old chickens of those 11 isolates were determined.Of the 16 isolates,11 viruses(TS/1/06/Dk,FR/3/06/Dk,FR/7/06/Dk,FR/8/06/Dk,FR/9/06/Dk, FR/10/06/Dk,LF/11/07/Dk,AX/12/07/Dk,FR/14/06/Dk,BY/15/06/Dk BY/16/08/Dk)the MDT for 72.00-132.00 h,ICPI 0-1.063 found for the weak strain of NDV;three FR/4/06/Dk,FR/5/06Dk,FR13/06/Dk,for the MDT for 69.60-84.00 h,ICPI 1.215-1.415 found for NDV medium virulence strains;a virus FR/2/06/Dk the MDT for 48 h,ICPI was found to 1.913 for the virulent NDV. 2.Virulence analysisChicken pathogenicity were studied on the basis of assay of the virulence indicators (MDT and ICPI)of FR/2/06/Dk,FR/13/06/Dk,LF/11/07/Dk,which were belonged to velogenic virus,mesogenic virus and lentogenic virus,respectively.Gross lesions and pathohistological changes were observed including 20 kinds of organs,such as trachea, lung,proventriculus,etc.The results showed that velogenic viruses can cause the typical symptoms of ND,with 100 percent incidence rate and 50 percent mortality rate.Evident gross lesions and pathohistological changes were observed from the chickens infected with velogenic virus.The lesions of digestive tract,respiratory tract and some immunal organs were particularly serious,largely for the performance of major diseases intestinal mucosa hemorrhage or necrosis,hemorrhage of thymus,necrosis of spleen and pancreas. Organizational changes for the various characteristics of epithelial cells and lymphocytes significant degeneration and necrosis.Mesogenic virus could cause clinical symptoms,but no deaths.The chicken digestive and respiratory infections are obvious pathological changes.The chickens infected with lentogenic isolate had no obvious symptoms,but with a slight gut changes.3.Sequence analysis of the fusion protein(F)gene of NDV isolates of duck originThe above-mentioned NDV duck isolates were examined for the features of their fusion protein(F)genes.A 450 nucleotides(nt)fragment of the F gene(1705nt)of each of the isolates was sequenced,and analyzed.The results revealed that these isolates shared common transcriptional sequence and translational start position with the hitherto reported NDV strains.The results showed that TS/1/06/Dk,FR/3/06/Dk,FR/4/06/Dk,FR/7/06/Dk, FR/8/06/Dk,FR/9/06/Dk,FR/10/06/Dk,LF/11/07/Dk,FR13/06/Dk,FR/14/06/Dk,BY/15/06/Dk, BY/16/08/Dk F Protein cleavage site sequence for the¹¹²G-R-Q-G-R-L¹¹⁷with the typical characteristics of attenuated strain of NDV;The nucleotide identities in the 450nt portion of F gene among 10 lentogenic viruses and 2 mesogenic viruses were 97.9%～99.5%.12 isolates in this study NDV F nucleotide sequence with the standard strain of Newcastle disease at home and abroad in recent years and the strains of chickens and other origin NDV sequence to compare results show that the virus between 12 to homology 97.9%to 99.5%.And from 2001 to the domestic geese origin of the disease NDV between the homology of 68.4 percent respectively to 96.7 percent with the 2003 separation of ducks at home and abroad to source the pathogenicity of homology between the NDV were 52.4% To 97.6%with the 1996 separation of pigeons to the source of strain homology were 52.4%-93.4%Source standards and the chicken attenuated strain LaSota identity were 97.2%-97.6%and 97.4%-97.7%;Domestic separation in recent years and the source of some of the chicken NDV identity were 68.0%-96.5%and 68.1%-87.2%.In addition,the comparisons of the N-terminal the amino acid sequences of 27 residue sites in the variable portion of the F protein revealed that the 12 isolates used in this study shared highly comparability with some ND viruses isolated from chicken recently.Phylogenetic analysis revealed that the10 lentogenic isolates and 2 mesogenic ones were highly homologous to the reference strains LaSota.