Isolation,Identification And Molecular Epidemiology Characteristic Of Newcastle Disease Virus Causing Clinical Disease In Geese

Newcastle disease(ND),is an acute, highly contiguous avian infectious disease brought by Newcastle disease virus (NDV). Newcastle disease is A violent contagion classified by OIE. Newcastle disease virus is a member of Rubulavirus, Para-myxovirinae, Paramyxoviridae, and a negative RNA virus which has vesica membrane. It exists in all tissues, organs, body fluid, secretion and excrement. For the time being, Waterfowls were considered to be the natural reservoir for certain viral pathogens in-cluding avian influenza virus (AIV) and Newcastle disease virus (NDV). Goose and duck, were usually considered to be resistant to virulent NDV strains. However, the geese South and East China occured severe outbreaks of NDV infection since 1997. The disease has spreaded quickly and has become a nationwide threat to goose farming in China. It is important to study ND molecular epidemicology because of higher variability of NDV.In the present study, eight NDVs isolated from disease-infected geese flocks of Ji-angsu provience from 2005 to 2006, labeled as JS/1/05/Go,JS/2/05/Go,JS/3/05/ Go,JS/4/05/Go,JS/5/06/Go,JS/6/06/Go,JS/7/06/Go and JS/8/06/Go,These strains were cloned and purified by the method of limited-point dilution with egg embryos, The mean death time (MDT) in embryonated chicken eggs and intracerebral pathogenicity index (ICPI) in day-old chickens of these 8 isolates were determined. 8 isolates gave MDTs ranging from 50.4～60 h and ICPI values between 1.75～1.91,All strains were considered to be velogenic.The above mentioned NDV goose isolates were examined for the features of their fusion (F) protein genes. A 1700 nucleotides (nt) fragment of the F gene of each of the isolates was cloned, sequenced, and analyzed. The results revealed that 6 potential glycosylation sites and 12 cystenine residues were highly conserved through the se-quences. The cleavage site of F0 proteins showed 112R(K)-R-Q-K-R-F117 sequence typical of the virulent NDV strains. The nucleotide identities in the 1700nt portion of F gene among 8 isolates except JS/2/05/Go were 97.5～99.8%, The deduced amino acid sequences identities were 97.8～99.8%,while the identities between isolate JS/2/05/Go and the remaining 7 strains were 86.3～86.9%. The amino acid sequences identities were 90.4%～91.1%, F48E8, a most frequently used reference strain in China, shared identities of 86.9%～92.3%, with these 8 goose isolates. the amino acid sequences identities were 91.3%～94.2%, the nucleotide and deduced amino acid sequences identities among 7 isolates used in this study and those goose-pathogenic ND viruses isolated from late 1997 were higher than 97%,or JS/2/05/Go strain's nucleotide identi-ties were lower than 87.6%,the amino acid sequences identities were 90% or so, The amino acid sequences identities among 8 isolates and those apparently healthy goose ND viruses isolated during 2003～2004 were 84.2%～88.6%, The amino acid se-quences identities were 88%～89.8%.On the phylogenetic tree based on sequences of F gene variable region 374nt(from47～420nt), and The distribution of cleavage sites with 3 restriction en-zymes (HinfⅠ,BstOⅠand RsaⅠ) in 75% region of the F gene (between 334～1682nt), which was used to group NDV strains into different genotypes, was also analyzed with the aid of computer program MegAlign. the 7 velogenic virus were located in the clus-ter with subgenotypeⅦd . JS/2/05/Go located in the same cluster with AUS/32 strain within genotypeⅢ.At positions 52, 71, 176, 272, 314, 402, 489, 7 out of 8 goose isolates contained residues which were seldom found in chicken and pigeon isolates. It is noteworthy that several chicken isolates, derived between 1996～2001 in China, also shared high similarities with goose isolates, and in some or all of the above mentioned positions, they possessed the same residues as goose isolates.Partial sequence analysis of hemagglutinin-neuraminidase (HN) genes was carried out to further identify the genetic characteristics of goose isolates of NDV. The results revealed that the coding regions of the HN genes of these isolates were all 1716 nu-cleotides long, typical for virulent strains of NDV, These 8 isolates were highly ho-mologous in terms of nucleotide (96.4%～99.7%) and amino acid sequences (94.9%～99.7%) of the HN gene and protein. At the nucleotide and amino acid levels, they were very closely related to some chicken isolates and goose-pathogenic isolates of recent years. In contrast, they were less closely related to F48E8, because comparisons demon-strated the highest nucleotide sequence homology was 81.8%, and amino acid se-quence homology was 87.6%, respectively.Thirteen cysteine residues of the HN protein of these isolates were highly con-served throughout the deduced amino acid sequences, and the number of potential glycosylation sites was 5. No differences were found in the receptor-binding regions of HN proteins between the goose and chicken isolates. However, there were 6 residues changes at positions 48, 54, 77, 266, 340 and 384, characterized by Met48→Thr, Ser54→His, Asn77→Ser, Ile266→Ala,Tyr340→His and Glu384→Lys substitutions when compared with most of other NDV strains. 6 out of 8 virus showed the same substitutions at the positions 48 as the chicken isolates.In conclusion, this study suggested that the goose isolates of NDV that caused clinical disease in recent years were might be evolved from a common progenitor, and it is most likely that they were originated from virulent strains of NDV in chickens, However, more intensive studies are necessary to confirm this hypothesis.