Molecular Epidemiology Of Newcastle Disease Virus Isolated In Partial Regions Of China Between 2001 And 2007 And The Pathogenicity Of Representative Isolates

Newcastle disease (ND) was caused by Newcastle disease viruses (NDV), which pose a major threat to the avian species worldwide particularly to domestic poultry and caused serious economic lose. The disease characterized mainly in alimentary tract and respiratory tract lesion. Although a series of combination control measures which focus on mass immunization had been adopted and the epidemic had been under control to some extent, however, ND was one of the most devastating infectious diseases that threatened the poultry industry in China also. At present, ND show some new epidemical characterizations such as atypical ND, emergence of new (sub)genotypes, enlargenment of host range and enhancement of virulence to NDV, which complicated the control of ND. Therefore, monitoring the molecular epidemiology and identifying the pathogenicity differences of distinct originated isolates would illustrate the tendency and variation of NDV, understand the ecological contribution of different birds, which contribute practically for the adjusting of combination control measures for ND.After specific-pathogen-free (SPF) chicken embryos inoculation and serological identification, 37 NDV isolates were identified and collected from the ND suspected specimens. The time spanned 2001-2007, and the hosts were chicken, duck, goose, pigeon, mandarin duck and wild bird.The isolates were purified once by CEF plaquing or by limiting dilution firstly. F gene fragment (860bp, contained the cleavage site) were amplified from 3-5 clones by RT-PCR, then the PCR product were sequenced directly. After ensuring no mixed infection, conserved clone was chosen to clone the full length of F gene, then sequenced and analyzed. The results showed that 37 F genes contained an open reading frame (ORF) in length of 1662nt, coding 533 amino acids, no insertion or deletions were founded. Phylogenetic tree was constructed using F gene nucleotide sequences of these 37 isolates and 20 reference strains from GenBank. The 37 isolates were classified into genotypeâ… ,â…¡,â…¢,â…¥b andâ…¦d. The isolates included in genotypesâ… (4/37),â…¡(6/37)andâ…¢(1/37)were all vaccine-like strains, which share 99% nucleotides homology with vaccine strains. Pigeon-origin isolates were classified into subgenotypeâ…¥b(2/37), shared more than 96% nucleotides and 94% amino acids homology with the early Pigeon-origin isolates in China, they showed little variation. Most of the isolates were classified into subgenotypeâ…¦d(24/37), of which in account 65%, shared more than 96% nucleotides homology with the earliest goose-origin isolate QY97 belonged to subgenotypeâ…¦d, they may share a common ancestor. F protein cleavage sites motifs of 37 isolates were ¹¹²GKQGRL11(7genotypeâ… ),¹¹²GRQGRL¹¹⁷(genotypeâ…¡),¹¹²RRQRRF¹¹⁷(genotypeâ…¢),¹¹²KRQKRF¹¹⁷(subgenotypeâ…¥b )and ¹¹²RRQKRF¹¹⁷(subgenotypeâ…¦d)respectively. Most strains contained pairs of dibasic amino acids excluding strains belonged to genotypeâ… andâ…¡, indicated that most strains were mesogenic at least, while the rests were lentogenic. The neutralization sites, glycosylation sites and cysteine residues of F proteins were highly conserved, indicated that the realized significant functional domains of F proteins of 37 isolates in our research changed little. The pathogenicity of subgenotypeâ…¦d isolates didn't change comparing with F48E9 indicated that the epidemic of subgenotypeâ…¦d viruses were not caused by the increasing of virulence.To elucidate the pathogenicity of distinct originated isolates or genotypes, eight isolates from different genotypes (â…¡,â…¢,â…¥b andâ…¦d ) or hosts (chicken, mallard duck, goose, pigeon, mandarin duck, wild bird) were chosen to challenge SPF chicken intranasally in dose of 10^3.0 EID₅₀/0.1mL. After challenged, strains of subgenotypeâ…¦d caused almost 100% death ratio, only one bird challenged with wild bird-origin survived. Postmortem signs indicated they were viscero-tropic velogenic strains. While strain of genotypeâ…¡caused no clinical signs was a lentogenic strain. Strains of genotypeâ…¢andâ…¥b, however, caused clinical signs such as ruffled feathers and depression, but no death cases.Tissues were collected at 2 days interval after challenged from 3 chickens each time. Viral quantities were detected to compare the viral load and tissue tropism. SYBR Greenâ… Real-time RT-PCR was constructed targeting at M gene conserved regions. Using Real-time RT-PCR, viral load in Brain, Harderian gland, Trachea, Lung, Bursa of Fabricius and Cecal tonsils at 48hpi, 96hpi, 144hpi were detected. The results showed that strains of subgenotypeâ…¦d, could be detected in immune organs at 48hpi, the viral load was 10²copies/μL at least. The viral load reached a peak at 96hpi, was 105copies/μL at least. Even the moribund birds and those deaths in different times, viral load changed little except a few tissues fluctuated 102-104 times. Viral load of strains of genotypeâ…¢and subgenotypeâ…¥b at 96hpi was low even negative at 48hpi, viral load at 144hpi was 10 times higher than that at 96hpi, but the maxlmum was only 104 copies/μL. The nearer the tissues with nasal, the higher the viral load rising. Viral load of strains belonged to genotypeâ…¡was undetected.Pathogenesis analysis found that the genotypeâ…¡isolate was avirulent (lentogenic) strains, the genotypeâ…¢and subgenotypeâ…¥b isolates were moderate strains (mesogenic), subgenotypeâ…¦d was viscero-tropic velogenic strains (velogenic) and showed high pathogenicity to SPF chickens. Subgenotypeâ…¦d viruses showed the same pathogenicity as F48E9 indicated that the epidemic of them did not cause by the increase of virulence.Our research showed that multiple genotypes were co-circulated in China at present. Isolate belonged to genotypeâ…¡was lentogenic which correlated with vaccine strains. Genotypeâ…¢and subgenotypeâ…¥b isolates were mesogenic, caused onset but no death. Subgenotypeâ…¦d strains were dominant strains, shared more than 95% homology with QY97, indicated that the virus changed little in recent 10 years. The realized significant functional domains of F proteins of 37 isolates in our research changed little, point mutation was the main causes of the NDV F gene variation. Those subgenotypeâ…¦d strains, isolated from duck, goose and health wild bird, were viscero-tropic velogenic strains, but the virulence did not increase. The ecocycle of NDV threatened the birds potentially, and renewed the function of waterfowl and wild bird on NDV ecology, thereby, made the control measures adjusted effectively.