Phylogenetic characterization of Newcastle disease virus isolated in the mainland of China during 2001–2009

doi:10.1016/j.vetmic.2009.09.020

Veterinary Microbiology

Volume 141, Issues 3–4, 24 March 2010, Pages 246-257

https://doi.org/10.1016/j.vetmic.2009.09.020 Get rights and content

Abstract

Twenty Newcastle disease virus (NDV) isolates from infected chicken flocks during 2001 to 2009 in China were biologically and genetically characterized. All the 20 NDVs were categorized into velogenic (n = 17) and lentogenic (n = 3) viruses, respectively, according to the mean death time (MDT) of chicken embryos. Velogenic viruses carry the motif ¹¹²R-R-Q-K-R/F¹¹⁷ (n = 14) or ¹¹²G-R-Q-G-R/L¹¹⁷ (n = 3) at the F0 cleavage site, while all the lentogenic virus had a sequence motif of ¹¹²G-R-Q-G-R/L¹¹⁷ (n = 3) at the same site. Phylogenetic analysis revealed that at least three distinct genotypes (genotypes I, II and VII) existed in chicken flocks in China and VIId of genotype VII were mainly responsible for the present ND panzootic. Two natural recombinants (XD/Shandong/08 and QG/Hebei/07) were supposed and identified by the SimPlot program, and their two parental-like strains might be from the NDV vaccine lineage and VIId velogenic lineage respectively. The results indicate that recombination play a role in NDV evolution and the live vaccines have capacity to boost NDV evolution by homologous recombination with circulating virus.

Introduction

Newcastle disease (ND) is a highly contagious and widespread disease which causes severe economic losses in domestic poultry, especially in chickens (Alexander, 2003, Sinkovics and Horvath, 2000). ND is caused by avian paramyxovirus serotype 1 (APMV-1), also known as Newcastle disease virus (NDV), which belongs to the genus Avulavirus in the family of Paramyxoviridae, order Mononegavirales (Mayo, 2002). NDV strains can be classified as highly virulent (velogenic), intermediate (mesogenic) or non-virulent (lentogenic) based on their pathogenicity in embryonating eggs or chickens (Alexander, 1998, Alexander, 2003, Asahara, 1978).

NDV has a single-stranded, negative-sense, nonsegmented RNA genome of approximately 15,186 nucleotides in length, which contains six genes encoding the six structural proteins (from 3′ to the 5′ terminus): nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin–neuraminidase (HN) and RNA-dependent RNA polymerase (L) (Millar et al., 1988). Among these, HN and F are two major spike glycoproteins. The HN protein is responsible for virus attachment to sialic acid-containing receptors, and it also has a neuraminidase activity as well as a yet undefined role in promoting the fusion mediated by the F protein (Yusoff and Tan, 2001). The F protein can promote the fusion of host and viral cell membranes, resulting in penetration of the virus particle into the cell, which is an initial step in infection (Rott and Klenk, 1988). The F protein is synthesized as a precursor (F₀) which must be proteolytically cleaved into F₂ and F₁ polypeptides to activate F protein fusion activity (Collins et al., 1993, De Leeuw et al., 2005, Panda et al., 2004). NDVs are grouped into genotypes I to IX under one serotype based on the analysis of the restriction site and nucleotide sequence of the F protein gene. Genotypes VI and VII can be further divided into seven (VIa–g) and five (VIIa–e) subgenotypes, respectively (Bogoyavlenskiy et al., 2005, Liu et al., 2003, Liu et al., 2007a, Wang et al., 2006).

The genetic change of the virus has been reported as one of the reasons why NDV virulence changes (Gould et al., 2001). Except for the high mutation rates and large population sizes, recombination is also a potentially important means of generating more genetic diversity in RNA viruses (Lai, 1992, Worobey and Holmes, 1999). For negative strand RNA virus, the recombination, such as ambisense arenavirus (Archer and Rico-Hesse, 2002, Charrel et al., 2001), hantavirus (Klempa et al., 2003, Sibold et al., 1999, Sironen et al., 2001), influenza A virus (Gibbs et al., 2001), measles virus (Schierup et al., 2005), respiratory syncytial virus (Spann et al., 2003) and Newcastle disease virus (Chare et al., 2003, Qin et al., 2007) had been reported, but the rates of recombination in negative strand RNA viruses were much lower than those in positive strand RNA viruses.

Since the 1980s, vaccination has been the widely used method for prevention and control NDV infections in China, but the disease is still enzootic in some areas and remains a constant threat to domestic poultry. Epidemiological studies have revealed that genotype VII viruses circulating predominantly in China in the past decade were responsible for disease outbreaks in chicken flocks (Liu et al., 2003). The prevailing NDV strains have significant differences from the current vaccine strains in their biology, serology and genetics, which is considered as the main reasons for the outbreaks of this disease in vaccinated poultry flocks in recent years (Qin et al., 2008, Tsai et al., 2004). For further characterize and phylogenetically group clinical NDVs in China, 20 isolates were obtained from outbreaks in different regions of China during 2001 to 2009 for sequence comparison, recombinant identification and phylogenetic analysis in the study. The study revealed increased changes of circulating NDVs in China, highlighting aspects of NDV evolution acted by homologous recombination.

Section snippets

NDV isolates

Twenty isolates in the study were isolated from clinically diseased chickens in different regions of China during 2001 to 2009. Viruses were plaque-purified three times on primary chicken embryo fibroblasts and then grown in 10-day-old specific-pathogen-free (SPF) chicken embryonated eggs. Allantoic fluid samples were harvested and stored at −80 °C for further use. Allantoic fluids were used for RNA extraction, hemagglutination inhibition (HI) and mean death time (MDT) (Alexander, 1998, Collins

Virus isolates

Totally 20 NDV isolates, from outbreaks in different chicken flocks, dates, and regions in China between 2001 and 2009, were collected and summarized in Table 3.

Nucleotide sequencing and sequences alignment

The amplified F gene coding sequence for each isolate was 1662 nucleotides, directing the synthesis of a protein predicted to be 554 amino acids in length. The HN protein of 4 NDV isolates had 577 amino acids possessing a single arginine at position 572 and 16 isolates had 571 amino acids, which encoded by 1731 nucleotides and 1713

Discussion

Newcastle disease is a most important disease of poultry throughout the world because of its high morbidity and mortality (Alexander, 2003). In China, although an intensive vaccination policy has been implemented, virulent NDV can still be frequently isolated in well-vaccinated flocks. Genetic analysis of NDV strains have proved its utility in determining pathotype, recent and past history of NDV strains and epidemiological relationships in geographically widely separated areas. In this study,

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Phylogenetic characterization of Newcastle disease virus isolated in the mainland of China during 2001–2009

Abstract

Introduction

Section snippets

NDV isolates

Virus isolates

Nucleotide sequencing and sequences alignment

Discussion

Virology

Virology

Virus Res.

Virology

Vet. Microbiol.

J. Virol. Methods

Virology

Microb. Pathog.

J. Clin. Virol.

Virology

Vet. Microbiol.

Newcastle disease virus and other avian paramyxoviruses

Newcastle disease

Study on the virulence of Newcastle disease virus

Kitasato Arch. Exp. Med.

Molecular characterization of virulent Newcastle disease virus isolates from chickens during the 1998 NDV outbreak in Kazakhstan

Virus Genes

Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses

J. Gen. Virol.

Deduced amino acid sequences at the fusion protein cleavage site of Newcastle disease viruses showing variation in antigenicity and pathogenicity

Arch. Virol.

Evaluation of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed “pigeon PMV-1 viruses”

Arch. Virol.

Probing the sialic acid binding site of the hemagglutinin-neuraminidase of Newcastle disease virus: identification of key amino acids involved in cell binding, catalysis, and fusion

J. Virol.

Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein

J. Gen. Virol.

Recombination in the hemagglutinin gene of the 1918 “Spanish flu”

Science

Genomic sequence of an isolate of Newcastle disease virus isolated from an outbreak in geese: a novel six nucleotide insertion in the non-coding region of the nucleoprotein gene

Arch. Virol.

The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence

J. Virol.

Genetic interaction between distinct Dobrava hantavirus subtypes in Apodemus agrarius and A. flavicollis in nature

J. Virol.

Automated phylogenetic detection of recombination using a genetic algorithm

Mol. Biol. Evol.