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Citation: Xiaolong Lu, Xiaoquan Wang, Tiansong Zhan, Yifan Sun, Xin Wang, Naiqing Xu, Tianxing Liao, Yu Chen, Min Gu, Shunlin Hu, Xiaowen Liu, Xiufan Liu. Surveillance of Class Ⅰ Newcastle Disease Virus at Live Bird Markets and Commercial Poultry Farms in Eastern China Reveals the Epidemic Characteristics [J].VIROLOGICA SINICA, 2021, 36(4) : 818-822.

Surveillance of Class Ⅰ Newcastle Disease Virus at Live Bird Markets and Commercial Poultry Farms in Eastern China Reveals the Epidemic Characteristics

  • Corresponding author: Xiufan Liu,, ORCID: 0000-0002-9924-6646
  • Electronic supplementary material The online version of this article ( contains supplementary material, which is available to authorized users.
  • Received Date: 25 October 2020
    Accepted Date: 21 January 2021
    Published Date: 15 March 2021

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    Surveillance of Class Ⅰ Newcastle Disease Virus at Live Bird Markets and Commercial Poultry Farms in Eastern China Reveals the Epidemic Characteristics

      Corresponding author: Xiufan Liu,
    • 1. Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou 225000, China
    • 2. Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou 225000, China
    • 3. Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou 225000, China


    • Dear Editor,

      Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), is a highly contagious and economically devastating viral disease of birds (Habib et al. 2018). NDV, also termed as avian paramyxovirus type 1 (APMV-1), belongs to the genus Orthoavulavirus in the family Paramyxoviridae according to the International Committee on Taxonomy of Viruses (ICTV) (Amarasinghe et al. 2019). According to the latest phylogenetic classification system, NDVs can be further divided into two groups: class Ⅰ and class Ⅱ. There are three sub-genotypes in a single class Ⅰ genotype 1, including sub-genotype 1.1.1, 1.1.2, and 1.2 (Dimitrov et al. 2019). Class Ⅰ NDV, with the genome size of 15, 198 nucleotides, is distributed globally and isolated frequently from wild birds and domestic poultry (Miller et al. 2010). Waterfowl can harbor lentogenic NDV strains and act as a natural reservoir for NDV (Kim et al. 2007; Wang et al. 2016), and class Ⅰ NDVs have been reported to transfer from waterfowls to chickens and circulated in chicken flocks extensively in China (Zhu et al. 2014; Chen et al. 2020). Although class Ⅰ NDVs are generally avirulent, they are likely to increase their virulence. For instance, class Ⅰ NDVs can enhance virulence through several passages in chicken due to the mutations at the F cleavage site (Yu et al. 2002), and they can also evolve into a virulent virus through only a few point mutations (Collins et al. 1998). Furthermore, the 1990 Ireland ND outbreak was caused by a virulent class Ⅰ NDV (Alexander et al. 1992). However, owing to its avirulence, class Ⅰ NDVs are often under-reported or neglected within other surveillance efforts. To better evaluate the prevalence of class Ⅰ NDVs, we performed the continuous surveillance of class Ⅰ NDVs and revealed the epidemic characteristics of class Ⅰ NDVs at live bird markets (LBMs) and commercial poultry farms in eastern China.

      During the active surveillance program of NDV from January 2017 to September 2018, we randomly collected samples from LBMs and commercial poultry farms from different provinces in eastern China, including Jiangsu, Shandong, Hebei and Liaoning. Cloacal and oropharyngeal swabs were collected monthly from each flock. Herein, a total of 1608 swab samples were collected from poultry at settled LBMs, including chicken samples (639/1608, 39.7%), duck samples (380/1608, 23.6%), goose samples (582/1608, 36.2%), and a few pigeon samples (7/1608, 0.4%). Furthermore, we also obtained samples from commercial poultry farms regularly, including commercial chickens (n = 400) and ducks (n = 800). Briefly, commercial chickens consisted of layers (n = 252) and breeders (n = 148), and commercial ducks were separated into Muscovy ducks (n = 200), meat ducks (n = 200), and mixed ducks (n = 400). Samples were diluted in phosphate-buffered saline (PBS) containing penicillin (1000 U/mL) and streptomycin (1000 U/mL), then frozen and thawed three times on dry-ice to facilitate the release of the virus, squeezed, and centrifugated. The supernatant liquid was collected and inoculated into the allantoic cavity of 10-day-old specific-pathogen-free (SPF) chick embryos for 96 h post-inoculation (hpi). The allantoic fluid was harvested and identified by standard hemagglutination activity (HA) test and hemagglutination inhibition (HI) test according to the World Organization for Animal Health (OIE) protocols for NDV. After purified by end point dilution method using embryonated chicken eggs, the virus stocks were grown in allantoic fluids and collected after 96 hpi at 37 ℃.

      Next, viral genomic RNA was extracted using Trizol reagent following manufacturer's instructions, and the initial RT reaction was conducted using a 6-nt random primer with M-MLV reverse transcriptase. Based on the available NDV sequences from GenBank, primers (forward: 5'-CTATTGCCAAATACAACCCGTTC-3' (4365–4387) and reverse: 5'-GCTGACCCCTCTCTCCAT-3' (6424–6441) were designed by Primer Premier (Version 5.00) and synthesized by TSINGKE Biological Technology (Beijing, China). PCR was performed to amplify the complete F gene sequences, and all PCR products were finally sequenced by Sanger sequencing. The sample information of LBMs and commercial poultry farms is shown in Table 1. For LBMs, most of the NDV-positive samples (26/1608, 1.6%) belonged to class Ⅰ clade besides three vaccine-like class Ⅱ strains. In class Ⅰ clade, we isolated most of strains (23/26, 88.5%) from chickens except 3 isolates from geese. Interestingly, our study showed that climate change had a significant impact on the spread of class Ⅰ NDVs. The isolation rate of class Ⅰ NDVs in the cold months was relatively high and stable, with the highest rate in March (8.74% and 10.29%) (Fig. 1A). This finding was consistent with previous studies (Liu et al. 2009; Zhu et al. 2014), as they reported that the isolation rates in colder months (from November to March) would be higher than in warmer months (from April to October). For commercial poultry farms, class Ⅰ NDVs were isolated mainly from commercial waterfowls, including Muscovy ducks (n = 7) and mixed ducks (n = 4), while no class Ⅰ NDV was detected in commercial chickens. In short, class Ⅰ NDVs were circulated in chickens at LBMs extensively but not at commercial poultry farms, and they have transferred from waterfowls to chickens at LBMs. Also, great importance should be attached to monitor class Ⅰ NDVs in cold months.

      Sample type Collected samples No. (%)a NDV-positive samples No. (%)b
      Class Ⅰ Class Ⅱ
      Live poultry markets
      Oropharyngeal and cloacal Speciesc 1608 (100%) 26 (1.6%) 3 (0.2%)
      Chickens 639 (39.7%) 23 (1.4%) 3 (0.2%)
      Ducks 380 (23.6%) 0 (0.0%) 0 (0.0%)
      Geese 582 (36.2%) 3 (0.2%) 0 (0.0%)
      Pigeons 7 (0.4%) 0 (0.0%) 0 (0.0%)
      Farms (chickens)d
      Layers Farm-A 84(21.0%) 0 (0.0%) 0 (0.0%)
      Farm-B 84(21.0%) 0 (0.0%) 0 (0.0%)
      Farm-C 84(21.0%) 0 (0.0%) 0 (0.0%)
      Breeders Farm-D 74(18.5%) 0 (0.0%) 0 (0.0%)
      Farm-E 74(18.5%) 0 (0.0%) 0 (0.0%)
      Farms (ducks)e
      Muscovy ducks Farm-A 200(25.0%) 7(3.5%) 0 (0.0%)
      Meat ducks Farm-B 200(25.0%) 0 (0.0%) 0 (0.0%)
      Mixed ducks Farm-C 200(25.0%) 3 (1.5%) 0 (0.0%)
      Farm-D 200(25.0%) 1 (0.5%) 0 (0.0%)
      Samples of each flock and Newcastle disease virus (NDV)-positive samples by RT-PCR were collected from LBMs and commercial poultry farms in Eastern China during 2017–2018
      aPercentage of samples collected
      bPercentage of samples positive for NDV
      cPercentage of samples collected from each flock at LBMs
      dPercentage of samples collected from each flock at commercial chicken farms
      ePercentage of samples collected from each flock at commercial duck farms

      Table 1.  Information for samples at live bird markets (LBMs) and commercial poultry farms in Eastern China during 2017–2018

      Figure 1.  The isolation rate and phylogenetic tree of class Ⅰ NDV isolates in Eastern China during 2017–2018. A The bar graph is colored according to different years and shows the isolation rate of class Ⅰ NDVpositive samples for each surveillance month at LBMs. The data was collected over a period of 21 months (from January 2017 to September 2018). B The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model. The tree with the highest log likelihood (- 9171.1384) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+ G, parameter = 0.6281)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 55 nucleotide sequences. There were a total of 1662 positions in the final dataset. Evolutionary analyses were conducted in MEGA6. Bootstrap values < 50% are not shown. The class Ⅰ NDVs from LBMs and commercial poultry farms detected in this study are marked with black triangles and circles, respectively

      In order to further assess the phylogenetic analysis of class Ⅰ NDVs from LBMs and commercial poultry farms, nucleotide sequence assembly, editing, and alignments were performed by BioEdit (BioEdit Sequence Alignment Editor, version 7.2.5) and MEGA6 software (Molecular Evolutionary Genetics Analysis, Version 6.06). For LBMs, phylogenetic trees were constructed based on the complete F gene (1–1662 nt) sequence using the maximum likelihood method with bootstrap analysis (n = 1000), implemented by the T92+G +I model. The sequences used for phylogenetic analysis were downloaded from GenBank, and the GenBank accession numbers are shown in the phylogenetic tree. The phylogenetic analysis showed that 26 class Ⅰ isolates from LBMs were categorized as subgenotype 1.1.2 (Fig. 1B) which was the epidemic genotype of the class Ⅰ NDV isolates from China (Chen et al. 2020). For commercial poultry farms, we selected the representative class Ⅰ NDVs (n = 3) to conduct the phylogenetic analysis. As shown in the phylogenetic tree, class Ⅰ NDVs isolated from commercial poultry farms were also divided into sub-genotype 1.1.2 (Fig. 1B). All these 29 class Ⅰ NDVs showed typical avirulent sequence motifs 112A/E-RQ-E-R-L117 and shared high genetic identities (nucleotide sequence homologies of 97.1%–100%).

      To confirm the virulence predicted by the cleavage site sequence analysis of the F protein, 11 representative class Ⅰ NDV strains isolated from different hosts and months were selected for the pathogenicity tests. The intracerebral pathogenicity index (ICPI) in 1-day-old chicks and the mean death time (MDT) in 9 to 11-day-old SPF chicken embryos were determined for these representative isolates using the OIE standard procedures. As the results showed, these isolates had an ICPI value 0 and an MDT > 120 h (Supplementary Table S1). Therefore, they were all classified as avirulent NDV according to the OIE definition.

      Since 1926, ND has experienced four pandemics worldwide. As is well-known that ND outbreaks in China have only been ascribed to class Ⅱ NDVs (Kim et al. 2007; Wu et al. 2011; Chen et al. 2020), while once a virulent class Ⅰ NDV caused the ND outbreak in Ireland (Alexander et al. 1992). Due to most of the class Ⅰ NDVs with low virulence, few efforts over time have been focused on these viruses. However, class Ⅰ NDVs have the potential to increase the low virulence and cause the threat to the poultry industry. To date, LBMs play an important role in the spread of class Ⅰ NDV, and the commercial poultry farm is the main source of poultry at LBMs. It is reported that most of the lentogenic NDVs from LBMs belong to class Ⅰ clade (Liu et al. 2009), which is consistent with the result of our study. Furthermore, this study showed that all the class Ⅰ isolates from LBMs belonged to sub-genotype 1.1.2. Utilized on the new naming criteria, sub-genotype 1.1.2 corresponds to the past epidemic sub-genotype 1b (Dimitrov et al. 2019). Previous studies have suggested that sub-genotype 1b NDVs are the most frequently isolated class Ⅰ strains at LBMs (Zhu et al. 2014; Zhang et al. 2015). This further indicates that the epidemic genotype over recent years has been concentrated in sub-genotype 1.1.2 (former 1b) at LBMs. Besides, class Ⅰ NDVs can be isolated from commercial poultry farms, the common provider of live birds (Hu et al. 2010; Zhu et al. 2014). In this study, class Ⅰ NDV was detected in commercial waterfowls but not in commercial chickens. Significantly, sub-genotype 1.1.2 has also become the epidemic genotype in commercial waterfowls. Nowadays, Southeast Asian farmers have been upgrading from the backyard to confined all-in-all-out systems (Carrique-Mas et al. 2015; Van et al. 2020), but some traditional LBMs do not popularize this feeding mode in eastern China, therefore providing an opportunity for virus spreading. The strict isolation prevents NDV from spreading among various hosts at commercial poultry farms, while the lack of strict isolation and disinfection facilitated the infection of class Ⅰ NDVs at LBMs. After poultry was introduced to LBMs, waterfowls, a natural reservoir for lentogenic NDVs, carried class Ⅰ NDVs to further infect the poultry from commercial poultry farms. Chickens have currently replaced waterfowls as the dominant host of class Ⅰ NDV at LBMs. Moreover, avirulent viruses have been reported to become velogenic after transmission and circulation in chicken populations (Yu et al. 2002). Thus, it is necessary to monitor the prevalence of class Ⅰ NDVs both at LBMs and commercial poultry farms.

      In conclusion, our findings suggest that class Ⅰ NDVs are widely circulating in chicken flocks at LBMs but not at commercial poultry farms, and sub-genotype 1.1.2 is the epidemic genotype at the domestic poultry in eastern China. Besides, the strict surveillance and scientific feeding mode at LBMs should be developed to eliminate the potential risk caused by class Ⅰ NDVs. And the cold season is a critical time for virus prevention and control. However, due to limitations of the sample collection and detection in this study, further research will be required to gather more various hosts and conduct the phylogenetic analysis of class Ⅰ isolates at commercial poultry farms.

    • This work was supported by grants from the National Key Research and Development Program of China (2017YFD0500101-3, 2017YFD0500802), the National Natural Science Foundation of China (31873021), the National Key Technology R & D Program of China (2015BAD12B03), the Earmarked Fund for China Agriculture Research System (CARS-40), the National Key Research and Development Program of China (2017YFD0500702), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

    • The authors declared that they had no competing interests.

    • The protocols for all animal experiments were approved by the Jiangsu Administrative Committee for Laboratory Animals (Permission number: SYXK-SU- 2017-0044) and accorded with the guidelines of Jiangsu Laboratory Animal Welfare and Ethics of Jiangsu Administrative Committee of Laboratory Animals.

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