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. Twenty-six 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 influenza 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 112E/G-R-Q-E/G-R-L117 (unlisted).
The terrestrial birds and sub-genotype 3c have become the popular host and genotype, respectively, and a new sub-genotype was confirmed
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 sub-genotype 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 classification 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 sub-genotype 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 classified into a unified 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.