| Newcastle disease (ND) is one of the most contagious and devastating diseases which has caused severe economic losses all around the world. Its causative agent is Newcastle disease virus (NDV), which belongs to the genus Avulavirus, sub-family Paramyxovirinae, family Paramyxoviridae. The viral RNA genome is approximately 15.2 kb in length and encodes six major virus proteins:the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and RNA polymerase protein (L). Since the emergence in the early 1990s, sub-genotype Ⅶd NDVs have become the predominant genotype in China. Researchers have recently confirmed that the variation in and around the linear epitope of the HN protein may intensify the antigenic difference. Moreover, the circulation of the class I virus among chickens has not been well elucidated. To elucidate the circulation of the virulent and the avirulent NDVs at the live bird markets (LBMs), we characterized the NDVs isolated in Eastern China during 2008 to 2013 biologically and phylogentically. In order to elucidate the evolutionary history of sub-genotype Ⅶd NDVs, we analyzed the whole genome sequences of 25 sub-genotype Ⅶd strains isolated in Jiangsu Province during 1997-2013. We have also explored the role of HN protein in protection efficiency of ND vaccine and the effect of E347K mutation in HN on viral antigenicity and found that HN gene of the vaccine strain plays an important role in the protection efficacy by the changing of the antigenicity match with the circulating field viruses which determines the virus load of virus shedding in vaccinated birds.1. Molecular epidemiology of NDVs isolated in Eastern China during 2008-2013In this study, we characterized the NDVs isolated in Eastern China during 2008-2013 phylogenetically and biologically. Seventy-five Newcastle disease viruses (NDVs) isolated from clinical specimens of different poultry species including chickens, ducks and geese. We also obtained a total of 7106 samples by monthly collections from multiple avian species at LBMs in Eastern China and 247 NDVs were isolated and identified with a total isolation rate of 3.48%. Over 95% of the NDV isolates (236/247) were identified as class I and 4.45%(11/247) were class Ⅱ, while the 11 class II strains shared 100% identities to V4 or LaSota. No virulent strain was isolated during 2008-2010, whereas fifty-five sub-genotype Ⅶd NDVs were isolated during 2008-2013. These sub-genotype Ⅶd isolates were further divided into two subgroups, Ⅶd1 and VIId2, in which 3 isolates belonged to sub-genotype Ⅶd1 while the rest 52 isolates belonged to sub-genotype VIId2. Thirty-nine of fifty-five sub-genotype Ⅶd isolates occurred K78R variation in F protein, while most Ⅶd NDVs isolated in recent years (38 of 55) shared both E347K and G362A mutations in HN protein. The result of the vaccine protective efficacy against the challenge of variant strains indicated that the inactivated LaSota vaccine did not protect chickens from virus shedding. In this study,21 of the 34 class I NDV-positive flocks were the chicken-origin, indicating that the cross-species transmission may occur and class I NDVs have already become prevalent among the chickens. Except two strains, all the class I viruses isolated before 2010 belonged to sub-genotype 3b, while the viruses isolated in 2011 and 2012 were all sub-genotype 3c. In conclusion, the sub-genotype Ⅶd NDVs harboring 347K in HN protein have been the prevalent NDVs in Eastern China and the avirulent class I NDVs isolated at LBMs during 2008-2013 belonged to sub-genotype 3b and a newly identified cluster 3c which has already transferred from waterfowls to terrestrial birds and exist in the chicken flocks extensively.2. The evolutionary dynamics of sub-genotype Ⅶd NDVs isolated in Jiangsu ProvinceIn this study, we analyzed the whole genome sequences of 25 sub-genotype Ⅶd strains isolated in Jiangsu Province during 1997-2013. When we performed a phylogenetic analysis based on the complete genome sequences of the 25 isolates, they diverged into two subgroups designated as Ⅶd1 and Ⅶd2. Similar results were obtained when genetic trees were constructed with ORF sequences of NP, P, M, F, HN and L genes, respectively. When compared the deduced amino acid sequence of the six genes, we found that there were changes in three sites in NP gene and the 2011-2013 isolates showed T220I variation compared with the previous isolates. There were five variant sites in P gene, while the 2011-2013 isolates showed G320E and R380K variation compared with the previous isolates. Five variation sites were found in F genes, and the 2011-2013 isolates showed K78R variation compared with the previous isolates. Moreover, there were six variant sites in HN gene, and the 2011-2013 isolates showed E347K and G362A co-variation compared with the previous isolates. In addition, there were six variations in M gene and eight in L gene. The evolutionary rates of the six genes in order of NP-P-M-F-HN-L were 1.18×10-3,8.47×10-4, 1.00×10-3,1.25×10-3,1.24×10-3 and 1.16×10-3 substitution/site/year, respectively. The evolutionary rate for the concatenated genomes was 1.06×10-3 substitution/site/year. BSP analyses based on the concatenated genomes demonstrated that sub-genotype Ⅶd NDVs emerged before 1991 in Jiangsu Province. Results in this study revealed the unique evolutionary history of sub-genotype Ⅶd NDVs in Jiangsu Province.3. Role of variant HN protein in protection efficiency of ND vaccineThe vaccination of ND vaccines is an important strategy for the prevention and control of ND in poultry. F and HN are the two important membrane glycoproteins of NDV which directly determine the virus antigenicity. Researchers recently confirmed that Newcastle disease virus fusion protein is the major contributor to protective immunity. We constructed a vaccine strain designated as replacing the HN gene of the vaccine strain NDV/AI4 from more recent Ⅶd variant NDV strain JS-16-12-Ch with HN gene bearing both E347K and G362A mutations. To investigate the effects of HN gene in vaccine strain on the protection efficacy against the predominant NDVs, we compared the protection efficiency difference between LaSota, A14 and O/AI4 at the same level of induced antibody when challenged with the strain JS-16-12-Ch. The vaccine strain LaSota showed significant antigenic difference with the challenge strain, and they were also distinct in the phylogenic tree. In addition, the NDV/AI4 strain showed significant antigenic difference with the challenge strain, but they were closer in the phylogenic tree. And the strain O/AI4 showed no antigenic difference with the challenge strain. From our results of vaccination studies, we found that O/AI4 caused a decrease in the number of birds shedding, and significantly reduced the amount of virus shedding in tracheal swabs when compared to the LaSota and NDV/AI4. O/AI4 provided improved protection as determined by a significant decrease in the number of birds shedding and the amount of virus shedding from infected birds. On the other hand, the O/AI4 group provided improved protection in terms of the significant decrease in viral load in visceral organs while the AI4 only decreased the viral load in part of the tested organs. In addition, histopathology showed that more severe histological changes were observed in LaSota vaccinated group than in the two sub-genotype VIId vaccine groups. In summary, HN gene of the vaccine strain plays an important role in the protection efficacy by the changing of the antigenicity match with the circulating field viruses which determines the virus load of virus shedding in vaccinated birds.4. The effects of the E347K and G362A variation in HN gene on antigenicityIt has been estimated that sub-genotype Vlld NDVs harboring 347K on HN gene are the prevalent NDVs in Eastern China. Researchers more recently confirmed that the HN gene of vaccine strain plays an important role in protection efficacy of ND vaccines by the changes of antigenicity match with the challenge virus. A cDNA clone of NDV strain 14 was used to introduce individual amino acid substitution in the HN protein. Three mutant strains E347K (347/14), G362A (362/14) and E347K+G362A (347+362/14) co-mutant strain were successfully rescued. Results of cross-neutralization tests revealed that there were significant antigenic differences between 347/14,347+362/14 and 14, respectively, and there were a minor difference between 362/14 and 14. We constructed the rFPV-HN expressing the HN gene of 14, and inoculated SPF chickens with it. Twenty-one days after immunization, we challenged the vaccinated groups with the four strains 347/14,362/14,347+362/14 and 14, respectively. We found that 347/14,362/14 and 347+362/14 all caused an increase in the number of birds shedding, and significantly increase in the amount of virus shedding in tracheal swabs when compared to the 14 challenge group. On the other hand, the 347/14 and 347+362/14 challenge group caused a significant increase in viral load in visceral organs than the 14 challenge group while the 362/14 challenge group showed no significant difference with 14. These results of virus load and sheding are consistant with histopathology findings that the 347+362/14 challenge group showed more severe changes than the 347/14 challenge group, while the 362/14 challenge group and the 14 challenge group showed mild histological changes. In summary, E347K mutation in HN protein significantly intensifies the antigenic difference; G362A variation plays a minor role in this respect, while the E347K, G362A co-variation further expands the antigenic difference, although it was not significantly higher than E347K variation. |
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