ABSTRACT
Newcastle disease (ND), caused by the virulent Newcastle disease virus (NDV), is one of the most important viral diseases of birds globally, but little is currently known regarding enzootic trends of NDV in northeastern China, especially for class I viruses. Thus, we performed a surveillance study for NDV in northeastern China from 2013 to 2015. A total 755 samples from wild and domestic birds in wetlands and live bird markets (LBMs) were collected, and 10 isolates of NDV were identified. Genetic and phylogenetic analyses showed that five isolates from LBMs belong to class I subgenotype 1b, two (one from wild birds and one from LBMs) belong to the vaccine-like class II genotype II, and three (all from wild birds) belong to class II subgenotype Ib. Interestingly, the five class I isolates had epidemiological connections with viruses from southern, eastern, and southeastern China. Our findings, together with recent prevalence trends of class I and virulent class II NDV in China, suggest possible virus transmission between wild and domestic birds and the potential for an NDV epidemic in the future.
INTRODUCTION
Newcastle disease virus (NDV) is synonymous with avian paramyxovirus type 1 and is a member of the genus Avulavirus within the family Paramyxoviridae (1). The virulent NDVs are the causative agents of Newcastle disease (ND), which is one of the most devastating diseases to the poultry industry worldwide (1). NDV is an enveloped virus containing a nonsegmented, single-stranded, negative-sense RNA genome 15.2 kb in size. The genome contains six genes, which encode the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), RNA-dependent RNA polymerase (L), and two additional nonstructural proteins (V and W, produced by a frameshift within the coding region of P) (1).
NDV is considered to be enzootic in both domestic and wild bird species (1). Based on the genomic length and phylogenetic analysis of the F gene, NDVs have been historically classified into two major groups, class I and class II, within a single serotype (2). Class I NDV is distributed globally, usually thought to be avirulent, and frequently isolated from wild birds and live bird markets (LBMs) (3). Class II NDV is widely distributed in multiple bird species. Some class II strains are avirulent or are vaccine viruses, but virulent strains of NDV also mainly group into class II and infections can cause significant economic losses to the poultry industry (2). Class I viruses were previously classified into nine genotypes (1 to 9), based on phylogenetic analysis using parts of the F gene (3, 4), but are now classified into a single phylogenetic group (genotype 1) based on the new classification criteria using the complete F gene sequence (5). Due to their high genetic diversity, class II viruses are classified into 18 genotypes (I to XVIII) (5, 6).
The scale and magnitude of ND outbreaks in China have been decreasing yearly since 2005 (Fig. 1). Interestingly, an increasing number of class I NDVs have been isolated from domestic poultry in southern, eastern, and southeastern China, as well as worldwide, in recent years (4, 7–10). However, no class I NDV was isolated in China before 2002, since they may escape detection by many conventional assays (2, 4). Furthermore, surveillance studies have demonstrated that the ecology of avirulent NDV in wild birds appears to be similar to that of low-pathogenicity avian influenza virus in that both viruses seem to enhance their pathogenicity in birds by accumulated mutations at the fusion protein cleavage site (3, 11–14).
ND outbreak events in China between 2005 and 2015. The asterisk indicates data cutoff on June 2015. All data were collected from the OIE official website (http://www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/statusdetail).
In this study, NDVs from wild birds and domestic poultries in LBMs were sequenced and analyzed in order to understand the current epidemiology and gene evolution of NDV in northeastern China. We then analyzed the epidemic trends of class I NDV in China and elucidated the potential ecological relationships between class I and class II NDVs, and the results are presented here.
MATERIALS AND METHODS
Sample collection, virus isolations, and identification.During the surveillance of NDV from April 2013 to June 2015, a total of 755 tracheal or cloacal swab samples were collected from clinically healthy birds in 16 LBMs and three wetlands in Jilin Province of northeastern China. Of these, 340 were from wild birds, and 415 were from domestic poultries. Details of collection for the NDV isolates are given in Fig. 2 and Table 1.
Sample collection sites in Jilin Province. Sampled cities or areas are indicated by color. The number of samples and positive rates of NDV are annotated.
Samples and isolates obtained from birds in different ecological groups in Jilin Province from 2013 to 2015a
All samples were inoculated into the allantoic cavities of 9- to 11-day-old specific-pathogen-free (SPF) chicken embryos (Beijing Merial Vital Laboratory Animal Technology Co., Ltd., Beijing, China) and incubated 96 h at 37°C. Allantoic fluids from inoculated eggs were harvested either when the embryos were killed or after the two passages; the presence of NDV was confirmed by hemagglutination, as well as hemagglutination inhibition (HI) assay, using La Sota-specific polyclonal sera (Harbin Weike Biotechnology Development Company, Harbin, China) according to the standardized OIE protocols for NDV (15).
Nucleic acid extraction, PCR, and sequencing.For genetic characterization of NDV, total RNA was extracted from infectious allantoic fluid using TriPure RNA isolation reagent (Hoffmann-La Roche, Ltd., Basel, Switzerland) according to the manufacturer's instructions. The extracted RNA was used for reverse transcription with random hexamer primers and Moloney murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI) according to the manufacturer's instructions.
The amplification for partial F genes of class I and II strains are performed as described in previous studies (16, 17). Conditions for PCR of complete F genes was as follows: 95°C for 3 min, followed by 35 cycles at 95°C for 1 min, 51°C for 45 s, and 72°C for 2 min 30 s, with a final extension step at 72°C for 10 min. The primer pair sequences used are listed in Table 2. Sequencing of PCR amplicons was conducted by Majorbio (Shanghai, China).
Primers used in this study
Phylogenetic analysis.In this study, the sequences of class I NDV reference strains of 1a, 1b, and 1c subgenotypes were obtained from GenBank (http://www.ncbi.nlm.nih.gov/GenBank). The accession numbers of the reference NDVs are shown in the phylogenetic trees. Alignment and comparison of the nucleotide and amino acid sequences were performed using the MegAlign program in the Lasergene package (DNASTAR, Inc., Madison, WI). A maximum-likelihood tree was generated using MEGA 6.06 (18).
Pathogenicity tests.The intracerebral pathogenicity index (ICPI) in 1-day-old SPF chickens and mean time to death (MDT) in 9- to 11-day-old SPF embryonated chicken eggs (Beijing Merial Vital Laboratory Animal Technology Co., Ltd., Beijing, China) were determined according to standard OIE procedures (15).
RESULTS AND DISCUSSION
A total of 755 samples were obtained from 16 LBMs and three wetlands from April 2013 to June 2015. Ten NDVs (six from LBMs and four from wild birds) were isolated; five isolates from LBMs were found to cluster with class I subgenotype 1b, two isolates (one from wild bird and one from LBMs) grouped with the vaccine-like class II genotype II, and three (all from wild birds) belonged to class II subgenotype Ib, based on complete F gene sequences (Fig. 3). No virulent NDV strain was isolated. Isolation rates for class I and II NDV were 0.66% (5/755), indicating that both viruses were equally prevalent in the sampled bird population.
Phylogenetic analysis of complete F gene sequences (1,662 nucleotides). Class I and II sequences are indicated as gray and white circles, respectively. Only bootstrap values of ≥50% are shown. The evolutionary history was inferred by using the maximum-likelihood method based on the general-time-reversible model. The tree with the highest log likelihood (−11262.9423) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using the maximum-composite-likelihood approach. A discrete gamma distribution was used to model evolutionary rate differences among sites (four categories [+G, parameter = 0.6973]). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 0.0000% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 58 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1,662 positions in the final data set. Evolutionary analyses were conducted using MEGA 6.06.
All five class II isolates showed the typical lentogenic sequence motifs 112GRQGR*L117 and 112GKQGR*L117 (the asterisk indicates the cleavage site of the F0 precursor protein into its F1 and F2 subunits) (Table 3). The genotype II strain Swan goose/China/Jilin/CC01/2015, which was isolated from a swan goose in the wetlands of Changchun, Jilin Province, in 2015, has a high similarity of complete F gene sequence compared to the duck-origin NDV strain Duck/China/Jilin/SY01/2014 (nucleotide sequence homologies of 100%), which was isolated from LBMs in Songyuan, Jilin Province, in 2014. Both strains share a 99.9% nucleotide similarity in complete F gene sequence to two commonly used live vaccine strains in China, La Sota and clone 30, suggesting that the swan goose and duck strains may be reisolation of a vaccine strain. The remaining three class II NDVs (Swan goose/China/Jilin/CC02/2013, Hooded crane/China/Jilin/BC01/2015, and Hooded crane/China/Jilin/BC02/2015), isolated from wild birds in Baicheng and Changchun of Jilin Province, were clustered into subgenotype Ib, which phylogenetically closed to both wild waterfowl isolates in the Far East (Russia, South Korea, and Japan) and recent isolates from domestic ducks in Asia (Eastern China, Japan, and South Korea) (8, 9, 19–21). Highly similar genotype II NDVs isolated from distinct species in different cities of Jilin Province suggested that the virus may be transmitted between wild birds and domestic poultries in the region and that the NDV isolates from wild birds detected in this study will likely be detected from domestic birds in the future.
Detailed information of NDVs isolated from northeastern China from 2013 to 2015
The complete F gene of four class I NDVs (Chicken/China/Jilin/CC02/2015, Chicken/China/Jilin/CC03/2014, Duck/China/Jilin/CC04/2015, and Chicken/China/Jilin/CC05/2015) isolated from LBMs shared high genetic identities (nucleotide sequence homologies of 99.7 to 100%) and formed a cluster within subgenotype 1b, suggesting that these isolates had a similar ancestor (Fig. 3). The remaining F gene sequence characterized in this study (Chicken/China/Jilin/SY02/2015) was also nested within subgenotype 1b. Chicken/China/Jilin/SY02/2015 had a typical cleavage site of avirulent class I subgenotype 1b strains, 112ERQER*L117, while Chicken/China/Jilin/CC02/2015, Chicken/China/Jilin/CC03/2014, Duck/China/Jilin/CC04/2015, and Chicken/China/Jilin/CC05/2015 displayed a motif of 112ARQER*L117 due to a nonsynonymous adenine-to-cytosine substitution at nucleotide position 335. All class I strains belonged to the subgenotype 1b cluster found mainly in domestic ducks and chickens in eastern, southern, and southeastern China (7, 8). Interestingly, similar subgenotype 1b strains were not detected in poultries in northeastern China, even during more intensive sampling periods during 2013 to 2014, suggesting these strains may have been introduced into northeast China from eastern, southern, or southeastern China via the poultry trade. To our knowledge, wild birds are the natural reservoirs of NDV (1). Hence, NDV transmitted between migratory birds and domestic poultries also cannot be excluded (22), even though class I NDV was not isolated in Japan and South Korea after 2006 (20). These countries, including China, are all in the East Asian-Australasian flyway, which provides the opportunity for NDV transmission from wild birds to domestic waterfowl via the stopover wetland sites and vice versa (20, 23).
Class I NDV was first isolated in Fujian and Zhejiang Province in 2002 (9) and since then has been spread into most regions of China, including the south, east, and southeast regions, as well as Hong Kong (Fig. 4) (4, 7–9). Class I NDVs are always considered avirulent for chickens; however, a waterfowl-source avirulent virus, goose/Alaska415/91, became virulent after serial passaging in SPF chicken eggs (24, 25). Therefore, class I NDV has the potential risk to virulent by the viral amino acid substitutions and may need to be monitored in the future (24, 25). In addition, previous study indicated that vaccination with avirulent class II NDV vaccine strains, such as La Sota and V4, may fail to prevent virus shedding of ducks infected with class I virus due to considerable differences in their antigenicity (8). Thus, a killed vaccine based on class I NDV may be necessary to protect the domestic poultry industry from potentially virulent strains of class I NDV.
Isolation sites and years (A) and percentages (B) of class I NDV clusters in China. Sampled provinces in this study are indicated by a star in panel A. The asterisk in panel B indicates the data cutoff on 20 November 2015. All data were obtained from GenBank (http://www.ncbi.nlm.nih.gov/GenBank/).
Based on hemagglutination inhibition (HI) and virus neutralization (VN) tests, class I NDV showed a broader cross-antigenicity to other genotype NDV strains (8, 26) and displayed higher HI and VN titers against both class I and class II virulent NDV strains than the current class II vaccine strain La Sota (26). Therefore, to some extent, the wide distribution of class I NDV may protect infected poultry against disease caused by virulent class II NDVs and may be one of the reasons for the decreased incidence of ND outbreaks in China from 2005 to 2015.
NDV recombination has also been reported in some reports during recent years (27–32), though even these events are quite controversial (30, 33). A high rate of NDV coinfection will increase the chance for recombination; therefore, the growing prevalence of class I NDV in wild waterfowl and domestic poultries may recombine with other class I and II circulating viruses to generate new strains with unpredictable phenotypes. More research is needed to elucidate the frequency and impact of NDV recombination among wild and domestic birds.
In conclusion, both class I and II NDVs were prevalent in northeastern China from 2013 to 2015. Some class II viruses of wild bird origin were closely related to the NDV from domestic poultry, suggesting interspecies transmission and the potential for virus recombination into more virulent strains, necessitating the demand for constant surveillance.
ACKNOWLEDGMENTS
This study was partly supported by two grants from National Science Foundation of China (31402195 to R.Y. and 31472195 to Z.D.), two grants from Jilin Provincial Science Technology Department (20140520171JH and 20160414029GH to R.Y.) and one grant from Chinese Special Fund for Agri-scientific Research in the public interest (201303033 to Z.D.), three grants from Jilin University (4305050102TS and 450060501486 to R.Y. and 4305050102S4 to X.L.). None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
FOOTNOTES
- Received 19 October 2015.
- Accepted 16 December 2015.
- Accepted manuscript posted online 28 December 2015.
- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
