BACKGROUND
newcastle disease  is one of the most contagious and devastating diseases to poultry in the world  <cit> .
the causative agents are virulent strains of newcastle disease virus , which belong to the genus avulavirus, sub-family paramyxoviridae, family paramyxovirinae  <cit> .
the viral rna genome is approximately 15 kb in length and encodes six major virus proteins: the nucleocapsid protein , phosphoprotein , matrix protein , fusion protein , hemagglutinin–neuraminidase  and large polymerase protein .
in addition, the hn protein is an important multifunctional surface glycoprotein that consists of a cytoplasmic domain, a transmembrane region, a stalk region and a globular head, in which the receptor–binding site, the site responsible for neuraminidase activity and all of the antigenic sites reside  <cit> .
it has been confirmed that there are seven overlapping antigenic sites in the hn protein, whereas residues  <dig> to  <dig> constitute the only linear epitope identified in the hn gene, which is susceptible to immune pressure to generate antigenic variation  <cit> .
researchers have recently confirmed that the variation in the linear epitope in the hn protein would intensify the antigenic difference  <cit> .
previously, seven neutralizing epitopes positioned at residues  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> to  <dig>  and  <dig> of the f protein have been identified.
the strains of ndv can be divided into two distinct clades: class i and class ii, and based on the older classification system, both clades could be divided into  <dig> genotypes, and the class ii clade can be divided into fifteen genotypes when based on the new classification system, while the class i clade can be divided into three sub-genotypes.
to date, a strict vaccination policy has been implemented in china and the vaccine strain lasota which belonged to genotype ii has been widely used in china for over 40 years.
however, infections of genotype viid ndvs have still frequently occurred in china and other asian countries .
meanwhile, there are also some virulent genotypes  circulating among avian species in china  <cit> .
to elucidate the circulation of virulent ndvs among chickens in eastern china,  <dig> virulent ndvs isolated from chicken flocks, including broilers and layers, from  <dig> to  <dig> were phylogenetically characterized.
RESULTS
isolation and identification of ndvs from clinical samples
fifty-five ndvs from clinical samples of different chicken flocks in eastern china were isolated from  <dig> to  <dig>  identified and plaque-purified, and the details of the ndv isolates are shown in table  <dig> table  <dig> ndvs characterized in this study
phylogenetic analysis
the phylogenetic analysis of the  <dig> ndvs based on the whole f gene sequences showed that all strains were clustered into sub-genotype viid and they could be divided into two separate groups, namely viid <dig>  and viid <dig> , as previously described   <cit> .
all of the strains isolated in 2012– <dig> belonged to viid <dig>  and only three of the twenty one strains isolated in  <dig> belonged to viid <dig>  all of the ndv isolates possessed the virulent f protein cleavage site motif 112rrqkr/f <dig>  and over 70 %  of the isolates shared k78r variation on the f gene.fig.
<dig> phylogenetic tree of  <dig> sub-genotype viid ndv strains based on the whole region of the f gene
additionally, a phylogenetic analysis was also performed based on the whole hn gene .
as determined from the phylogenetic tree based on the hn gene, all of the ndv isolates showed a distribution similar to that obtained from the tree based on the f genes: three in viid <dig> and  <dig> in viid <dig>  three residues, namely t <dig>  a <dig> and t <dig>  were unique for viid <dig>  whereas i <dig>  e <dig> and m <dig> were characteristic residues of viid <dig>  all of the ndv isolates shared  <dig>  % to 100 % nucleotide identity and  <dig>  % to  <dig>  % amino acid identity.
only two isolates  shared the e347g variation.
more than  <dig>  % of the isolates in  <dig>  exhibited the e347k variation in hn, whereas  <dig>  %  of those isolated in  <dig> possessed this variation.
the latter percentage is nearly two-fold higher than that found in  <dig>  and 100 %  of the strains isolated in  <dig> possessed this variation.
in addition, the layer-origin stains isolated in  <dig>   <dig> and  <dig> shared 67 % , 75 %  and 100 %  of the e347k variation in the hn gene, respectively.
furthermore, we found that all of the strains harboring 347 k on hn also shared a g362a mutation.
however, the role of this mutation has not been well elucidated.
the  <dig> cysteine residues were conserved as described previously, and with the exception of the loss of a glycosylation site at  <dig> of js-02- <dig>  all of the other ndvs possessed six well-identified glycosylation sites.fig.
<dig> phylogenetic tree of  <dig> sub-genotype viid ndv strains based on the complete hn gene
pathogenicity tests
as is shown in table  <dig>  all the ten isolates were highly virulent, which were consistent with the tyipical virulent motif 112rrqkrf <dig> at the f cleavage sites.table  <dig> coefficients of antigenic similarity  between ndv isolates and lasota strain
r valuec
achicken embryo cross-neutralization test
bcross-hemagglutination inhibition test
c <dig>  ≤ r ≤  <dig> , indicates no significant antigenic difference between the two viruses
<dig>  ≤ r ≤  <dig>  indicates a minor difference between the two viruses
r <  <dig>  indicates a major difference between the two virus strains
cross hemagglutination inhibition  test and virus neutralization test
the hi titers of the anti-lasota serum for the ten isolates were 4-8-fold lower than that for the homologous strain lasota, displaying the significance of the antigenic difference between the ndv isolates and the vaccine strain lasota.
as determined from the cross hi test, the r values between the predominant strains and lasota were all lower than  <dig> , indicating the existence of a significant antigenic difference between the strains.
meanwhile the r values between the e347k-variant strains and lasota were lower than that found for the 347e/g strains, which indicate that the e/g347k variation would expand the antigenic difference with the vaccine strain.
and the significant antigenic difference was also confirmed by the r value obtained from cross-neutralization test between lasota and the predominant strains .table  <dig> pathogenicity tests of the isolates
amdt = mean death time
bicpi = intracerebral pathogenicity index
test of the protective efficacy of the lasota strain against the variant strains
as is shown in table  <dig>  none of the birds vaccinated with the lasota vaccine showed clinical signs post challenge with js-22-11-ch or js-14-12-ch.
in contrast, 100 % of the unvaccinated birds challenged with js-22-11-ch or js-14-12-ch died within five days post challenge.
all of the unchallenged birds remained normal throughout the experiment.table  <dig> protection of vaccinated spf chickens with inactivated oil-emulsion vaccines against challenge with the predominant variant ndvs
vaccine-challengea
ala-js <dig> and control-js22 = challenge with  <dig> eid <dig> js-22-11-ch; la-js <dig> and control-js14 = challenge with  <dig> eid <dig> js-14-12-ch
as is shown in table  <dig>  tracheal and cloacal swabs were collected from the birds at days  <dig>   <dig>  and  <dig> post challenge and subjected to virus isolation tests.
the virus isolation rate from the tracheal swabs was 100 %  at day  <dig> post challenge in both the control-js <dig> and control-js <dig> groups, whereas the isolation rates for the la-js <dig> and la-js <dig> groups were 70 %  and 100 % , respectively.
and the virus isolation rate from the tracheal swabs was 50 %  and 70 %  at day  <dig> post challenge in la-js <dig> and la-js <dig> groups, whereas the isolation rates at day  <dig> post challenge were 10 %  and 30 % , respectively.table  <dig> frequency of isolation of challenge virus from spf chickens
–the birds were all dead within the day 5pc
os = oral swabs; cs = cloacal swabs
discussion
the genotype vii ndvs have become the most prevalent strains in china since the 1990s, and the sub-genotype viid ndvs are mainly responsible for the present nd epizootic in china  <cit> .
in this study, virulent ndvs from the vaccinated chicken flocks were characterized as sub-genotype viid and could be clustered into sub-genotype viid <dig> and viid <dig> based on the phylogenetic trees based on both f and hn genes  <cit> .
t <dig>  a <dig>  and t <dig> of the hn gene were unique residue substitutions found in the viid <dig> ndvs, and i <dig>  e <dig>  and m <dig> of the hn gene were characteristic residues of the viid <dig> ndvs.
in this study, we found that sub-genotype viid <dig> remains the predominant genotype vii ndvs from  <dig> to  <dig>  and all of the ndvs isolated from  <dig> to  <dig> were located in this clade.
the hn protein of ndv plays an important role in immune protection against virus infection, and the variation of the antigenic epitope would expand the antigenic difference.
and analysis of the role of site  <dig> in antigenicity has confirmed that the e347k variation would intensify the antigenic difference and may increase the risk of vaccine breakdown  <cit> .
the isolation rate of strains with the e347k variation is increasing yearly, as previous reported.
more than 36 % of the virus strains  were isolated in commercial layer flocks, which have resulted in losses in egg production.
cho et al.
<cit>  found that the korean variation strains have also caused losses in egg production.
moreover, more than 75 %  of the ndv strains isolated from layer flocks were the e347k-variation strains.
the high isolation rate indicates that the e347k-variation strains can infect chickens under immune pressure because the layers were all well-vaccinated in china.
both site  <dig> and site  <dig> locate around the receptor-binding pocket, and we found of e347k, g362a co-variant strains, whereas cho et al.
also identified two strains with the e347k and g362r co-variant ones.
however, the role of the co-variation remains unknown  <cit> .
at present, lasota is widely and frequently used to prevent ndv infection in the poultry industries in china; however, virulent ndv infections continue to occur in the well-vaccinated chicken flocks  <cit> .
moreover, both cross hi and cross-neutralization tests were performed, and the r values of all of the strains were lower than  <dig> .
however, the r values between the e347k-variant strains and lasota were lower than that found for the 347e/g strains, which indicate that the e347k variation would expand the antigenic difference with the vaccine strain.
therefore, the results of the cross-neutralization and cross hi experiments may also be consistent with the higher isolation rate of the e347k-variant strains, which demonstrated significant antigenic differences between the vaccine strain and the predominant variant strains should be responsible for the outbreaks of nd in china.
in addition, the protection efficiency of lasota against the e347k variation strains in china is not well-elucidated.
in this study, the vaccinated spf chickens were fully protected against morbidity and mortality, but virus shedding was not stopped.
our data suggest that the lasota inactivated vaccine cannot protect chickens from virus shedding when infected with the variant ndvs, and the variant strain challenged group showed higher virus isoation rate which may be one of the reasons for the circulation of sub-genotype viid variation strains in vaccinated chicken flocks.
in conclusion, our findings indicate that the sub-genotype viid ndvs with e347k variation in the hn protein are predominant in eastern china and that this variation would intensify the antigenic difference with the vaccine strains.
moreover, the vaccine strain lasota has been used for more than 40 years and can hardly protect chickens effectively from virus shedding when infected with the circulation strains.
therefore, new ndv vaccines closely related to the prevalent genotype viid viruses should be developed to control nd in china.
CONCLUSIONS
currently, sub-genotype viid ndvs are the prevalent virulent strains circulating among vaccinated chicken flocks in eastern china.
our findings indicate that the e347k variation in hn protein would expand the antigenic difference with lasota, which may be responsible for the increasing isolation rate of these strains from vaccinated chickens.

 newcastle disease is one of the most contagious and devastating diseases to poultry in the world. the causative agents are virulent strains of newcastle disease virus, which belong to the genus avulavirus, sub-family paramyxoviridae, family paramyxovirinae. the viral rna genome is approximately 15 kb in length and encodes six major virus proteins: the nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin–neuraminidase and large polymerase protein. in addition, the hn protein is an important multifunctional surface glycoprotein that consists of a cytoplasmic domain, a transmembrane region, a stalk region and a globular head, in which the receptor–binding site, the site responsible for neuraminidase activity and all of the antigenic sites reside. it has been confirmed that there are seven overlapping antigenic sites in the hn protein, whereas residues to constitute the only linear epitope identified in the hn gene, which is susceptible to immune pressure to generate antigenic variation. researchers have recently confirmed that the variation in the linear epitope in the hn protein would intensify the antigenic difference. previously, seven neutralizing epitopes positioned at residues to and of the f protein have been identified. the strains of ndv can be divided into two distinct clades: class i and class ii, and based on the older classification system, both clades could be divided into genotypes, and the class ii clade can be divided into fifteen genotypes when based on the new classification system, while the class i clade can be divided into three sub-genotypes. to date, a strict vaccination policy has been implemented in china and the vaccine strain lasota which belonged to genotype ii has been widely used in china for over 40 years. however, infections of genotype viid ndvs have still frequently occurred in china and other asian countries. meanwhile, there are also some virulent genotypes circulating among avian species in china. to elucidate the circulation of virulent ndvs among chickens in eastern china, virulent ndvs isolated from chicken flocks, including broilers and layers, from to were phylogenetically characterized.  isolation and identification of ndvs from clinical samples fifty-five ndvs from clinical samples of different chicken flocks in eastern china were isolated from to identified and plaque-purified, and the details of the ndv isolates are shown in table  table ndvs characterized in this study phylogenetic analysis the phylogenetic analysis of the ndvs based on the whole f gene sequences showed that all strains were clustered into sub-genotype viid and they could be divided into two separate groups, namely viid and viid, as previously described. all of the strains isolated in 2012– belonged to viid and only three of the twenty one strains isolated in belonged to viid all of the ndv isolates possessed the virulent f protein cleavage site motif 112rrqkr/f and over 70 % of the isolates shared k78r variation on the f gene.fig. phylogenetic tree of sub-genotype viid ndv strains based on the whole region of the f gene additionally, a phylogenetic analysis was also performed based on the whole hn gene. as determined from the phylogenetic tree based on the hn gene, all of the ndv isolates showed a distribution similar to that obtained from the tree based on the f genes: three in viid and in viid three residues, namely t a and t were unique for viid whereas i e and m were characteristic residues of viid all of the ndv isolates shared  % to 100 % nucleotide identity and  % to  % amino acid identity. only two isolates shared the e347g variation. more than  % of the isolates in exhibited the e347k variation in hn, whereas  % of those isolated in possessed this variation. the latter percentage is nearly two-fold higher than that found in and 100 % of the strains isolated in possessed this variation. in addition, the layer-origin stains isolated in and shared 67 %, 75 % and 100 % of the e347k variation in the hn gene, respectively. furthermore, we found that all of the strains harboring 347 k on hn also shared a g362a mutation. however, the role of this mutation has not been well elucidated. the cysteine residues were conserved as described previously, and with the exception of the loss of a glycosylation site at of js-02- all of the other ndvs possessed six well-identified glycosylation sites.fig. phylogenetic tree of sub-genotype viid ndv strains based on the complete hn gene pathogenicity tests as is shown in table  all the ten isolates were highly virulent, which were consistent with the tyipical virulent motif 112rrqkrf at the f cleavage sites.table coefficients of antigenic similarity between ndv isolates and lasota strain r valuec achicken embryo cross-neutralization test bcross-hemagglutination inhibition test c  ≤ r ≤ , indicates no significant antigenic difference between the two viruses  ≤ r ≤  indicates a minor difference between the two viruses r <  indicates a major difference between the two virus strains cross hemagglutination inhibition test and virus neutralization test the hi titers of the anti-lasota serum for the ten isolates were 4-8-fold lower than that for the homologous strain lasota, displaying the significance of the antigenic difference between the ndv isolates and the vaccine strain lasota. as determined from the cross hi test, the r values between the predominant strains and lasota were all lower than, indicating the existence of a significant antigenic difference between the strains. meanwhile the r values between the e347k-variant strains and lasota were lower than that found for the 347e/g strains, which indicate that the e/g347k variation would expand the antigenic difference with the vaccine strain. and the significant antigenic difference was also confirmed by the r value obtained from cross-neutralization test between lasota and the predominant strains.table pathogenicity tests of the isolates amdt = mean death time bicpi = intracerebral pathogenicity index test of the protective efficacy of the lasota strain against the variant strains as is shown in table  none of the birds vaccinated with the lasota vaccine showed clinical signs post challenge with js-22-11-ch or js-14-12-ch. in contrast, 100 % of the unvaccinated birds challenged with js-22-11-ch or js-14-12-ch died within five days post challenge. all of the unchallenged birds remained normal throughout the experiment.table protection of vaccinated spf chickens with inactivated oil-emulsion vaccines against challenge with the predominant variant ndvs vaccine-challengea ala-js and control-js22 = challenge with eid js-22-11-ch; la-js and control-js14 = challenge with eid js-14-12-ch as is shown in table  tracheal and cloacal swabs were collected from the birds at days and post challenge and subjected to virus isolation tests. the virus isolation rate from the tracheal swabs was 100 % at day post challenge in both the control-js and control-js groups, whereas the isolation rates for the la-js and la-js groups were 70 % and 100 %, respectively. and the virus isolation rate from the tracheal swabs was 50 % and 70 % at day post challenge in la-js and la-js groups, whereas the isolation rates at day post challenge were 10 % and 30 %, respectively.table frequency of isolation of challenge virus from spf chickens –the birds were all dead within the day 5pc os = oral swabs; cs = cloacal swabs discussion the genotype vii ndvs have become the most prevalent strains in china since the 1990s, and the sub-genotype viid ndvs are mainly responsible for the present nd epizootic in china. in this study, virulent ndvs from the vaccinated chicken flocks were characterized as sub-genotype viid and could be clustered into sub-genotype viid and viid based on the phylogenetic trees based on both f and hn genes. t a and t of the hn gene were unique residue substitutions found in the viid ndvs, and i e and m of the hn gene were characteristic residues of the viid ndvs. in this study, we found that sub-genotype viid remains the predominant genotype vii ndvs from to and all of the ndvs isolated from to were located in this clade. the hn protein of ndv plays an important role in immune protection against virus infection, and the variation of the antigenic epitope would expand the antigenic difference. and analysis of the role of site in antigenicity has confirmed that the e347k variation would intensify the antigenic difference and may increase the risk of vaccine breakdown. the isolation rate of strains with the e347k variation is increasing yearly, as previous reported. more than 36 % of the virus strains were isolated in commercial layer flocks, which have resulted in losses in egg production. cho et al. found that the korean variation strains have also caused losses in egg production. moreover, more than 75 % of the ndv strains isolated from layer flocks were the e347k-variation strains. the high isolation rate indicates that the e347k-variation strains can infect chickens under immune pressure because the layers were all well-vaccinated in china. both site and site locate around the receptor-binding pocket, and we found of e347k, g362a co-variant strains, whereas cho et al. also identified two strains with the e347k and g362r co-variant ones. however, the role of the co-variation remains unknown. at present, lasota is widely and frequently used to prevent ndv infection in the poultry industries in china; however, virulent ndv infections continue to occur in the well-vaccinated chicken flocks. moreover, both cross hi and cross-neutralization tests were performed, and the r values of all of the strains were lower than. however, the r values between the e347k-variant strains and lasota were lower than that found for the 347e/g strains, which indicate that the e347k variation would expand the antigenic difference with the vaccine strain. therefore, the results of the cross-neutralization and cross hi experiments may also be consistent with the higher isolation rate of the e347k-variant strains, which demonstrated significant antigenic differences between the vaccine strain and the predominant variant strains should be responsible for the outbreaks of nd in china. in addition, the protection efficiency of lasota against the e347k variation strains in china is not well-elucidated. in this study, the vaccinated spf chickens were fully protected against morbidity and mortality, but virus shedding was not stopped. our data suggest that the lasota inactivated vaccine cannot protect chickens from virus shedding when infected with the variant ndvs, and the variant strain challenged group showed higher virus isoation rate which may be one of the reasons for the circulation of sub-genotype viid variation strains in vaccinated chicken flocks. in conclusion, our findings indicate that the sub-genotype viid ndvs with e347k variation in the hn protein are predominant in eastern china and that this variation would intensify the antigenic difference with the vaccine strains. moreover, the vaccine strain lasota has been used for more than 40 years and can hardly protect chickens effectively from virus shedding when infected with the circulation strains. therefore, new ndv vaccines closely related to the prevalent genotype viid viruses should be developed to control nd in china.  currently, sub-genotype viid ndvs are the prevalent virulent strains circulating among vaccinated chicken flocks in eastern china. our findings indicate that the e347k variation in hn protein would expand the antigenic difference with lasota, which may be responsible for the increasing isolation rate of these strains from vaccinated chickens. 
the cross-neutralization tests confirmed that the strains harboring 347 k showed lower antigenic relatedness with lasota. currently, sub-genotype viid ndvs are the prevalent virulent strains circulating among vaccinated chicken flocks in eastern china. the causative agents are virulent strains of newcastle disease virus, which belong to the genus avulavirus, sub-family paramyxoviridae, family paramyxovirinae. the results showed that all of the virulent strains belonged sub-genotype viid shared the same cleavage site 112rrqkr/f in the fusion protein. virulent newcastle disease virussub-genotype viide347kvariationantigenic differencechinese special fund for agro-scientific research in the public interest 201003012liu xiufan earmarked fund for modern agro-industry technology research systemnycytx-41-g07liu xiufan national natural science foundation of china31172338liu xiufan priority academic program development of jiangsu higher education institutions issue-copyright-statement© the author 2016 our findings indicated that the e347k variation in hn gene would expand the antigenic difference with lasota, which may be responsible for the increasing isolation rate of these strains from vaccinated chickens. at least 69 % of the ndv strains possessed e347k variation in the hemagglutinin-neuraminidase protein. newcastle disease is one of the most contagious and devastating diseases to poultry in the world. knowing the genomic and antigenic characteristics of virulent ndvs might contribute to nd control in china.
 newcastle disease is one of the most contagious and devastating diseases to poultry in the world. the causative agents are virulent strains of newcastle disease virus, which belong to the genus avulavirus, sub-family paramyxoviridae, family paramyxovirinae. knowing the genomic and antigenic characteristics of virulent ndvs might contribute to nd control in china.  the results showed that all of the virulent strains belonged sub-genotype viid shared the same cleavage site 112rrqkr/f in the fusion protein. at least 69 % of the ndv strains possessed e347k variation in the hemagglutinin-neuraminidase protein. the cross-neutralization tests confirmed that the strains harboring 347 k showed lower antigenic relatedness with lasota. furthermore, the immune-challenge experiment indicated that lasota could not provide complete protection against infection with the e347k variant ndvs as the vaccinated birds were still able to be infected and shed virulent challenge viruses.  currently, sub-genotype viid ndvs are the prevalent virulent strains circulating among vaccinated chicken flocks in eastern china. our findings indicated that the e347k variation in hn gene would expand the antigenic difference with lasota, which may be responsible for the increasing isolation rate of these strains from vaccinated chickens. keywords virulent newcastle disease virussub-genotype viide347kvariationantigenic differencechinese special fund for agro-scientific research in the public interest 201003012liu xiufan earmarked fund for modern agro-industry technology research systemnycytx-41-g07liu xiufan national natural science foundation of china31172338liu xiufan priority academic program development of jiangsu higher education institutions issue-copyright-statement© the author 2016 