Genetic evolution of class I Newcastle disease virus from poultry at live bird markets in Eastern China

Background: Live bird markets (LBMs) serve as a natural reservoir for class I Newcastle disease virus (NDV) and play an important role in viral evolution and spread. LBMs and commercial farms are the main sources of daily poultry products. However, limited studies are available for the class I NDV circulating at LBMs and commercial farms. In this study, signicant progress has been made in sample detection and genetic analysis at LBMs and commercial farms in Eastern China. Methods: A long-term epidemiological investigation at LBMs and commercial farms in Eastern China was conducted. We sequentially conducted the collection of samples, virus isolation, RNA extraction, RT-PCR, and phylogenetic analysis. We also analyzed class I NDVs deposited in NCBI during 2002–2018 from China in terms of the host, genotype, time, and functional domains. Results: Here we report that class I NDVs continue to circulate in LBMs. Class I NDVs were detected at a high prevalence in chickens (23/26) but were seldom detected in waterfowl (3/26) at LBMs. In contrast, class I NDVs were rarely detected in commercial chickens but were present in commercial waterfowl at a certain frequency before they were brought to the LBMs. This observation suggests that the mixture of poultry and the conventional housing at LBMs cause the spread of class I NDVs. Sequence analysis of Class I NDV genomes deposited in NCBI during 2002–2018 from China revealed a high prevalence of class I NDVs in terrestrial birds (82.7%), which was much higher than in water birds (17.3%). In addition, we found that the class I NDVs predominantly belonged to the sub-genotype 3c and rarely evolved into a new sub-genotype such as the 3d. Furthermore, the epidemic sub-genotype 3c seems to be under ongoing evolution. Conclusions: Our study provides evidence that the traditional feeding mode at LBMs causes the spread of class I NDVs and poses a great risk of NDV epidemics in the poultry industry in Eastern China. The sub-genotype 3c is circulating at LBMs and currently the terrestrial bird is the predominant host of class I NDVs.

poultry and the conventional housing at LBMs cause the spread of class I NDVs. Sequence analysis of Class I NDV genomes deposited in NCBI during 2002-2018 from China revealed a high prevalence of class I NDVs in terrestrial birds (82.7%), which was much higher than in water birds (17.3%). In addition, we found that the class I NDVs predominantly belonged to the sub-genotype 3c and rarely evolved into a new sub-genotype such as the 3d. Furthermore, the epidemic sub-genotype 3c seems to be under ongoing evolution.
Conclusions: Our study provides evidence that the traditional feeding mode at LBMs causes the spread of class I NDVs and poses a great risk of NDV epidemics in the poultry industry in Eastern China. The subgenotype 3c is circulating at LBMs and currently the terrestrial bird is the predominant host of class I NDVs.

Background
Newcastle disease virus (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) [1]. NDV is a single-stranded, negative-sense, non-segmented RNA virus of approximately 15.2 kb. From the NDV genome (3' to 5' terminus), six proteins are produced: nucleocapsid protein(NP gene), phosphoprotein (P gene), matrix protein (M gene), fusion protein (F gene), hemagglutinin-neuraminidase protein (HN gene), and the RNA-dependent RNA polymerase protein (L gene) [2]. Based on the genome size and phylogenetic analysis of the F gene, NDV can be divided into two groups: class I and class II [3]. Class I is exclusively avirulent and can be subdivided into nine genotypes (1)(2)(3)(4)(5)(6)(7)(8)(9). In comparison, class II is subdivided into eighteen genotypes (I-XVIII) based on the partial sequence of the F gene (47-420 nt). In addition to the classical classi cation methods, two modi ed classi cation methods have been introduced, rst in 2012 and then in 2019 [4,5].
The class I NDV, which with 15,198 nucleotides has the longest genome of APMV-1 [3], is present in domestic and wild waterfowl widely [2,6]. Compared with the full genome sequences of Class II NDV early lineage strains (15,186 bp), class I NDV has a 12-bp insertion in the phosphoprotein gene [7]. Waterfowl, the natural reservoir of NDVs, has the potential to spread the virus in the environment and within their susceptible hosts. It results in frequent ND outbreaks and subsequent economic losses [8][9][10]. Class I NDVs were originally recovered from both domestic and wild birds sampled in North America and Eurasia since at least the 1970s [6,11,12]. Then, it was reported that class I NDVs are also widely common in the USA and China [11,13,14]. The susceptible hosts of class I NDVs are quite manifold such as domestic poultry (chickens, ducks, geese, and pigeons) and wild birds (black swan, peafowl, egret, and heron) [6,11,13,15]. Also, the susceptible hosts in different genotypes are slightly different [3]. Class I viruses are generally lentogenic in chickens while there is a known mutant and historically it has been recovered from waterfowl (family: Anatidae) and shorebirds [16]. Furthermore, it is noteworthy mentioning that a virulent class I NDV causes the 1990 Ireland ND outbreak [11], and lentogenic NDVs, including class I, can enhance virulence through chicken or chicken embryo passages [17]. Most of the avirulent NDVs isolated from the LBMs belong to the class I clade [2,15,18,19]. Therefore, an in-depth epidemiological investigation of class I NDVs is signi cant to understand the spread and variation of the class I NDVs and predict future trends. However, little is known about the origin and the genetic evolution of class I NDVs at LBMs. Despite the epidemiology of NDV in several regions of China being well characterized [20][21][22], information for infection in Eastern China has rarely been reported in recent years.
In this study, we investigated the prevalence of class I NDVs at LBMs and commercial farms in Eastern China. LBM and farm poultry data were combined to analyze and their role in the transmission of class I NDVs was highlighted. Besides, we systematically investigated the genetic evolution of class I NDVs deposited in NCBI during 2002-2018 from China. Taken together, this study provides theoretical guidelines to evaluate the prevalence of class I NDVs in China.

Collection of samples
We administered a long-term survey of settled LBMs and commercial farms in Eastern China during 2017-2018. The avian samples mainly came from several provinces in Eastern China, such as Jiangsu, Anhui, Shandong, Zhejiang, and samples from other areas were sporadic. Monthly, we collected pooled oropharyngeal and cloacal swabs from randomly-selected poultry at each LBM. We also collected a few environmental samples at LBMs. Similarly, we collected samples from commercial poultry regularly. Then samples were transported at 4°C, immediately aliquoted, and stored at −80°C until testing.
In addition to collecting the above samples, our laboratory conducted a long-term epidemiological investigation in Eastern China during 2002-2016 and isolated several class I NDVs. For the comprehensiveness of data, the previous data were combined and analyzed together.
Virus isolation, RNA extraction, RT-PCR and sequencing of the F gene The viruses isolated from LBMs and commercial farms were identi ed, and the virus isolation method was previously described [19]. The swab samples were frozen, thawed three times on dry-ice, and squeezed. Then the supernatants were collected after centrifugation (5 min, 8,000 rpm) at 4°C. The supernatant was inoculated with 10-day-old speci c-pathogen-free (SPF) chicken embryos (Beijing Merial Wetong Experimental Animal Co., Ltd.), and the allantoic uid was collected from incubated eggs at the appointed time as described previously [19]. Next, viruses were identi ed by hemagglutination (HA) and hemagglutination inhibition (HI) experiments (chicken NDV standard positive serum prepared by our lab). The virus stock was stored at −80°C until use. Sample collection and all experiments in the present study were performed in negative-pressure isolators with HEPA lters in a biosafety level 3 (BSL3) animal facility by the institutional biosafety manual.

Phylogenetic evolution analysis
Nucleotide sequence editing, analysis, and alignment were performed using the Clustal W multiple alignment method in the BioEdit (BioEdit Sequence Alignment Editor, version 7.2.5) and MEGA 7 software (Molecular Evolutionary Genetics Analysis, Version 7.0.21). We used the classical classi cation method because the collected sequences were concentrated in 47-420 nt. Phylogenetic trees were constructed using the neighbor-joining method in MEGA 7 based on the F partial gene (374 bp). To estimate evolutionary rates and distances of different sub-genotypes of genotype 3 in F genes, the molecular analysis was performed based on the pairwise distances implemented in MEGA 7 (nucleotide: maximum composite likelihood).
There are 241 strains deposited in NCBI, and the information about these nucleotide sequences is given in additional le 1. The F gene sequences of NDVs (n = 241) from the NCBI database (https://www.ncbi.nlm.nih.gov/) were added to the phylogenetic analysis.
Analyses of class I NDVs in terms of the host, genotype and time We elucidated the relationship among the host, genotype, and time of class I NDVs. Class I NDVs isolated in China during 2002-2018 were compared according to the host, genotype, and time. Stacked bar charts were drawn by the GraphPad Prism 7.00 software. We also studied the distribution of the host and time in different genotypes presented as phylogenetic trees. The evolutionary history was inferred by the neighbor-joining method and evolutionary analyses were conducted in MEGA 7. Then, phylogenetic trees were edited and decorated by the Dendroscope (3.5.7) software.

Analyses of key functional domains in F protein of epidemic genotype
To further investigate the current epidemic genotype, we analyzed the amino acids in key functional domains of sub-genotype 3c NDVs. The functional areas included the signal peptide, fusion protein, transmembrane domain, and N-linked glycosylation sites. Protein sequences were predicted and analyzed in MEGA 7.

Prevalence of class I NDVs in chickens at LBMs but not in commercial chickens
We collected 1,618 samples from poultry at LBMs during 2017-2018, including chicken swab samples, duck swab samples, goose swab samples, pigeon swab samples, and environmental samples. Twentysix class I-positive samples were isolated from LBMs, and the other three strains were highly homologous to the vaccine strain La Sota. We detected class I NDVs in 23 (1.42%) isolates of chicken and 3 (0.19%) isolates of goose. The results revealed that the positive samples mainly belong to the class I branch at LBMs. We also collected samples from commercial farms, including commercial chickens and ducks. There was a certain separation rate from commercial ducks, and Muscovy ducks were more susceptible to NDV, while there was no NDV isolated from commercial chickens (Table 1). In addition to the recent isolates, our lab isolated 308 class I strains at LBMs in Eastern China during 2002-2016, and 106 strains were further studied. Therein, the 106 strains came from chickens (52/106), ducks (45/106), and geese (9/106) ( Table 2). Besides, the isolation rate in the colder months would be higher than in the hotter months, which was similar to the in uenza virus isolation changes because of seasonal changes (relevant information was not displayed).
To avoid repetitive sequences, we screened a fraction of the sequences (n = 87) isolated by our lab for the evolutionary genetic analysis. The phylogenetic tree showed that class I isolates were further divided into the genotype 2, the sub-genotypes 3b and 3c (Figure 4), and the cleavage site sequence of F genes was 112 E/G-R-Q-E/G-R-L 117 (unlisted).
The terrestrial birds and sub-genotype 3c have become the popular host and genotype, respectively, and a new sub-genotype was con rmed In pursuit of data reliability, we conducted a synthetic analysis of class I NDVs during 2002-2018 in China. The class I NDVs isolated in China were distributed throughout the country, but the class I NDVs focused on the eastern coastal areas (Figure 1). There was a pattern on the host and genetic distribution. For hosts, water birds are thought to provide a natural reservoir for lentogenic NDVs, but the result showed that class I NDVs gradually transitioned from water birds to terrestrial birds (Figure 2 and 5A). For genotypes, the result showed that the isolation rate of the genotype 3 increased with time, and the subgenotype 3c became the most popular genotype in recent years (Figure 3 and 5B).
According to the phylogenetic tree, the class I NDVs isolated from our laboratory could be divided into the genotype 2 (n = 13), 3b (n = 34), and 3c (n = 40). The homology rates among three genotype groups were 87.3%-92.3% (2 vs 3b), 85.8%-93.2% (2 vs 3c), and 84.3%-94.1% (3b vs 3c). Further analysis showed that the homology rate of the genotype 2 isolates ranged from 94.1% to 100%, and their homology rate was 93.4%-99.2%. The genotype 2 isolates were close to the genetic relationship of the isolates from 2007 to 2011. The homology rate of the sub-genotype 3b isolates ranged from 94% to 100%. Compared with the isolates from 2005 to 2010, their genetic relationship was relatively close, and the homology rate was 93.7%-99.7%. The homology rate of the sub-genotype 3c was 91.3%-100%, and their homology rate was 90.7%-100%. The sub-genotype 3c isolates were close to the genetic relationship of the isolates in 2009-2018, The phylogenetic tree based on the partial F genes of class I NDVs in China is shown in Figure 4. At present, the genotype 3 could be divided into 3a, 3b, and 3c, while the genetic evolution analysis of the F gene revealed a new branch in genotype 3. The two strains (HQ398796, HQ398797) published in NCBI formed a separate branch. The mean evolutionary distances between the new branch and three established branches, i.e., 3a, 3b, and 3c were 0.044, 0.071, and 0.084, respectively (Table 3). This conformed to the classi cation criteria for sub-genotypes. Therefore, the new sub-genotype was provisionally named as 3d. Moreover, the sub-genotype 3d might evolve from 3a because of the higher homology with the genotype 3a than 3b and 3c.
Epidemic genotypes or sub-genotypes constantly change through host and time alternation Genotypes or sub-genotypes were associated with host and time. According to the analysis of the phylogenetic tree, the prevalent genotypes of class I NDV in China were the genotype 2 and 3 during 2002-2018. From the host perspective, the waterfowl was the susceptible host of the genotype 2. In comparison, the susceptible hosts of the genotype 3 were much more abundant. Each sub-genotype of the genotype 3 was widely found in terrestrial birds, and the sub-genotype carried by water birds was concentrated in the sub-genotype 3b. Besides, the sub-genotype from wild birds was concentrated in the sub-genotype 3c ( Figure 5A). From the time perspective, the epidemic sub-genotypes were the sub-genotype 3a and 3b during the early stages of surveillance (during 2002-2005). At the middle stage of surveillance (during 2006-2009), the popular genotypes were the genotype 2 and sub-genotype 3b. At the end of surveillance (during 2010-2018), the epidemic sub-genotype turned into the sub-genotype 3c (Figure 3 and 5B).
Concerning sub-genotype 3c, terrestrial birds increasingly became the susceptible host, and exogenous viruses disrupted the sequences According to the phylogenetic tree, the class I NDVs isolated from 2011 to 2018 all belong to the subgenotype 3c. Although the early isolation rates of water birds and terrestrial birds each accounted for a certain percentage, the terrestrial birds gradually replaced water birds. Besides, the result showed that the homology among the isolates from 2009 to 2018 was higher than with isolates from other years, and there was also a high homology in the isolates with distant years (Figure 4). According to the epidemiological surveillance of the settled LBMs in recent years, most of the NDV isolates were classi ed into a uni ed branch. However, some isolates were also merged into a branch with exogenous NDVs, and there was a higher homology among these strains. For example, six isolates in 2018 belong to the same branch with the exogenous strains in 2013. This phenomenon had been observed since 2009 (Figure 4). These results show that exogenous NDVs were constantly invading the LBMs, and class I NDVs were evolving at LBMs.
The tendency for amino acids variation in the signal peptide of the sub-genotype 3c NDVs We analyzed the evolution of functional regions in the F protein of the sub-genotype 3c NDVs, including the signal peptide, fusion region, transmembrane domain, and N-linked glycosylation sites. The tendency for amino acid variations in these functional domains existed only in the signal peptide of the F protein (Table 4). In detail, there was a variation of amino acids at the 8th, 14th, and 18th sites in the signal peptide: S→G, P→L, M→V.

Discussion
Newcastle disease went through four pandemics, and there have been variant genotypes documented as the epidemic genotype in China and some other Asian countries since the 1990s [24]. Lentogenic NDVs gradually spread through poultry at LBMs, and most of the lentogenic NDVs isolated from LBMs in America and Korea belong to class I clade [25,26]. In recent years, the isolation rate of class I NDVs at LBMs has signi cantly risen, and the sub-genotype 3c has gradually become an epidemic strain [22,27].
Although class NDVs are genetically conserved and avirulent, these viruses are likely to cause variation and epidemic of ND without any control measures [11].
Class I NDVs, frequently isolated from LBMs [6], were originally found in waterfowls such as ducks and geese, which were the natural reservoirs of avirulent NDVs [15,20]. In general, class I NDVs were more susceptible to wild birds and water birds [28]. The existence of genetically related class I NDVs in domestic birds and wild birds implied that there were epidemiological connections among these populations [29]. Interestingly, this study showed that class NDVs were isolated principally from chickens rather than water birds at LBMs. According to the epidemiological survey at LBMs from 2002 to 2018, terrestrial birds gradually became the popular host of class I NDVs after 2014. This result con rmed that the susceptible host of class NDVs had transitioned from water birds to terrestrial birds at LBMs as a previous study illuminated [11]. We speculated that the emergence of this phenomenon might be related to the polyculture of different birds at LBMs, which may have promoted the circulation of class I NDVs. In response to this problem, we should strengthen the monitoring of the isolated feeding of different poultry ocks at LBMs. However, we were surprised to nd no class I NDV isolated from commercial chickens while there still was a certain virus isolation rate in commercial ducks. This phenomenon interestingly showed that commercial chicken farms were not the cradle of class I NDVs. The monitoring results of LBMs and commercial farms showed that the mixed breeding model at LBMs facilitated the spread of class I NDVs, and water birds carried a steady stream of class I NDVs to infect newly introduced chickens. Many Southeast Asian farmers have been upgrading from the backyard to con ned all-in-all-out systems [30,31]. But some traditional LBMs did not popularize the "all in and all out" model in Eastern China, which might provide an opportunity for virus spread. Therefore, a scienti c raising model at LBMs would help control the epidemic of ND.
To obtain a comprehensive analysis of class I NDVs, we also selected all class I NDVs published in NCBI during 2002-2018 from China. Although different classi cation methods have been introduced, we still chose the classical classi cation method because the published F gene sequences were concentrated in region 47-420 nt. From the host perspective, both terrestrial birds and water birds occupied a place at the early phase, but terrestrial birds became the dominant host of class I NDVs after 2014. From the genotype perspective, the sub-genotype 3c has been gradually becoming the predominant genotype since 2010 and is the current epidemic genotype at LBMs [22,27]. Meanwhile, we found that the occurrences of popular genotypes were not random, and the epidemic genotype would change with the replacement of the host and time.
To further study the sub-genotype 3c, we performed a systematic analysis of sub-genotype 3c NDVs isolated from China. The results showed that terrestrial birds have currently become the dominant host in 3c, and the sub-genotype 3c has constantly been evolving. By plotting the phylogenetic tree of class I NDVs, we were delighted to nd two isolates belonging to a new branch. Although these two isolates have been reported to be genotype 3 in NCBI, there is no further sub-genotype classi cation. Based on the genetic distance of the new sub-genotype classi cation, the two isolates met the sub-genotype classi cation condition and were named as the sub-genotype 3d. Furthermore, we analyzed the subgenotype 3c NDVs during 2009-2018 to con rm whether the 3c underwent amino acid chemotaxis during evolution. The result revealed that the trend changes of the sub-genotype 3c NDVs existed in the signal peptide of the F protein. The sequence of the signal peptide could guide the membrane fusion of the virus and in uence the NDVs [32, 33], which might contribute to the prevalence of the sub-genotype 3c NDVs. It also indicated that the sub-genotype 3c NDV has been evolving to adapt to the environment.
Taken the data together, it is necessary to monitor the spread of class I NDVs strictly to prevent the ND outbreak.

Conclusion
Through long-time epidemiological surveillance at LBMs and commercial farms in Eastern China, we gathered data emphasizing that the traditional feeding model was the main cause of class I NDVs circulating at LBMs. The study also indicated the replacement of the epidemic genotype and host in China during 2002-2018, and links were found among the host, genotype, and time. Interestingly, a new sub-genotype of class I was con rmed based on genetic evolution analysis. Furthermore, the epidemic sub-genotype 3c is under ongoing evolution, which might be an adaptive variation to the environment.

Declarations
Availability of data and materials The datasets generated and analyzed during the current study are included in this article and the additional le.
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