Surveillance of avirulent Newcastle disease viruses at live bird markets in Eastern China during 2008–2012 reveals a new sub-genotype of class I virus

Newcastle disease (ND) is caused by Newcastle disease virus(NDV)which has caused severe economic losses all around the world [1],[2]. NDV is a member of avian paramyxovirus (APMV), which belongs to the genus Avulavirus, sub-family Paramyxovirinae, family Paramyxoviridae, and order Mononegavirales[3]. The genome of NDV is a single-stranded, negative-sense, nonsegmented RNA of approximately 15.2 kb. The viral genome is comprised of six genes: nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin–neuraminidase (HN), and RNA-dependent RNA polymerase (L) [4]. The strains of NDV can be divided into two distinct clades: class I and class II, and based on the older classification system, both clades could be divided into 9 genotypes, and the class II clade can be divided into fifteen genotypes when based on the new classification system, while the class I clade can be divided into three sub-genotypes [5]–[7].

Liu et.al has found that the class I virus strains circulating among Eastern China were genotype 2 and 3, while the genotype 3 can be divided into two sub-genotype: 3a and 3b [8]. In 2012, one class I virus isolated in Eastern China was ascribed to genotype 4 [9], which means that the class I viruses circulating in Eastern China were mix. As Liu et al. [8] only concerned about the class I virus circulating in ducks at LBMs in Eastern China, and deals of chickens were also existed at the live bird markets (LBMs), however, the circulation of the class I virus among chickens has not been well elucidated. In this study, we collected 7106 cloacal swabs from 600 flocks including both chickens and waterfowls for virus isolation from 2008 to 2012 and 34 class I isolates were characterized phylogentically while and 15 of the 34 NDVs were pathologically characterized.

Currently, most of the avirulent NDVs isolated from the LBMs in America and Korea belonged to class I clade. [10]–[12]. Liu et.al have reported that the class I viruses circulated in waterfowls from 2002 to 2007 at LBMs in Eastern China were genotype 2 and 3 which could be divided into two separated groups: 3a and 3b [8]. In this paper, we conducted the continuous surveillance and characterization of class I clade NDVs both from the chicken and waterfowl flocks at LBMs in Eastern China during 2008 to 2012 and found a new separate cluster in genotype 3 which possessed its own characteristic amino acid substitutions on the F protein (Tables 1 and 2).

During May 2008 to April 2012, 247 NDVs were isolated from the LBMs in Eastern China and 95.5% (236/247) were class I NDVs, which indicated that the class I NDVs were still frequently isolated among the birds at LBMs. Researchers have already ensured that the class I clade NDVs mainly circulated among waterfowls [5],[6],[8], however in this research, 21 of the 34 classI NDV-positive flocks were the chicken-origin, which indicates that the cross-species transmission has been appeared and class I NDVs have already become frequently isolated among the chickens. In china, deals of chickens and waterfowls are made together at the LBMs, therefore the LBM has been one of the important sites for pathogens inter- or cross-species transmission. Considering the higher isolation of the class I NDVs at the LBMs, much attention should be paid to the transmission of class I NDV at this site.

All the isolates in this study were clustered into genotype 3 which was divided into two sub-genotypes 3a and 3b in the previous study [6],[8]. However, a separate cluster was identified here based on the phylogenetic analysis of the partial F genes. As is illustrated by Diel et al. [7], different genotypes should have an average distance per site >10% (0.1) and different sub-genotypes should have an average distance per site between 3 (0.03) and 10% (0.1), while its evolutionary distance with 3a and 3b was 0.068 and 0.099, respectively, which has met the requirement to form a new sub-genotype and was designated here as 3c. Based on the two classification methods, the isolates from 2008–2010 are more similar to older previously studied isolates and the more recent strains (2011–2012) are clustering together and the waterfowl-origin isolation rate is higher than the 2011–2012 isolation rate, which means that the class I NDV is keeping evolution in the poultry flocks and can affect the chickens more efficiency. Comparing the partial F genes between the three sub-genotypes, we found that the sub-genotype 3a and 3c shared the same cleavage site EQQERL and 3c viruses showed both higher nucleotide and amino acid homologous with sub-genotype 3a when compared with 3b. Therefore the sub-genotype 3c viruses might evolve from the sub-genotype 3a.

Previously, seven neutralizing epitopes positioned at residues 72, 74, 75, 78, 79, 157 to 171, and 343 of the F protein have been identified [13]–[16]. When compared the F genes of 3c cluster with the vaccine strain LaSota, four of these epitopes were changed as follows: A75Q, A79S, V168I, and D170S, which displayed the antigenic difference between them and was confirmed by the results derived from the cross-HI and -neutralization tests in this study. Class I NDVs are the avirulent strains usually isolated from waterfowls [5],[8], however, Yu et. al found that the class I avirulent virus could be generated into virulent one through consecutive passages in the chicken only by two residues alteration at the F cleavage site [17]. As is reported that, the virulent class I viruses which were phylogenetically closely related to those found in WS have previously been found [18],[19], and the risk of ND epidemic would be introduced due to its high infection rate in the poultry flocks, the significant antigenic difference and low genetic homology with the vaccine strain [20]. Therefore, continuous surveillance for the class I NDVs both from the LBMs and poultry farms should be performed and strict control strategies should be developed to eliminate the potential risk caused by these NDVs.

The NDVs isolated at LBMs during 2008–2012 belonged to sub-genotype 3b (or 1a), and a newly identified cluster 3c (or 1b) which has already transferred from waterfowls to terrestrial birds and has been frequently isolated in the chicken flocks extensively.