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In China, H9N2 subtype avian influenza outbreak is firstly reported in Guangdong province in 1992. Subsequently, the disease spreads into vast majority regions nationwide and has currently become endemic there. Over vicennial genetic evolution, the viral pathogenicity and transmissibility have showed an increasing trend as year goes by, posing serious threat to poultry industry. In addition, H9N2 has demonstrated significance to public health as it could not only directly infect mankind, but also donate partial or even whole cassette of internal genes to generate novel human-lethal reassortants like H5N1, H7N9, H10N8 and H5N6 viruses. In this review, we mainly focused on the epidemiological dynamics, biological characteristics, molecular phylogeny and vaccine strategy of H9N2 subtype avian influenza virus in China to present an overview of the situation of H9N2 in China.

We generated and characterized site-directed HA mutants on the genetic backbone of H5N1 clade 2.3.4 virus preferentially binding to α-2,3 receptors in order to identify the key determinants in hemagglutinin rendering the dual affinity to both α-2,3 (avian-type) and α-2,6 (human-type) linked sialic acid receptors of the current clade 2.3.4.4 H5NX subtype avian influenza reassortants. The results show that the T160A substitution resulted in the loss of a glycosylation site at 158N and led not only to enhanced binding specificity for human-type receptors but also transmissibility among guinea pigs, which could be considered as an important molecular marker for assessing pandemic potential of H5 subtype avian influenza isolates.

Background: Newcastle disease (ND) is one of the most contagious and devastating diseases to poultry in the world. The causative agents are virulent strains of Newcastle disease virus (NDV), 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., Results: The results showed that all of the virulent strains belonged sub-genotype VIId shared the same cleavage site (112)RRQKR/F(117) in the fusion protein. At least 69 % (38 of 55) 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., Conclusions: 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.

Background: The strains of the genotype VIId Newcastle disease virus (NDV) induce more severe tissue damage in lymphoid organs than other virulent strains. The underlying molecular mechanisms are poorly understood., Methods: Genotype IV NDV Herts/33 and genotype VIId NDV JS5/05 have a distinctive pathological profile in the spleen. These two strains of viruses were selected as parental viruses to generate a panel of chimeric viruses by replacing the M, F and HN genes of Herts/33 individually or in combination with the corresponding genes of JS5/05 using reverse genetic. Virulence and in vitro characteristics of the recombinant viruses were assessed. In addition, pathological changes, virus load, and transcriptional cytokine response in the spleen of chickens infected with these recombinant viruses were also analyzed., Results: Pathogenicity test showed that all chimeric viruses are virulent. In vitro characterization revealed that gene replacement did not change growth kinetics and HN expression on cell surface of the recombinant viruses. However, replacement of the M, F and HN genes resulted in apparent changes in the fusion activity. Moreover, pathological studies revealed that only inclusion of the homologous M, F and HN genes of JS5/05 in Herts/33 backbone resulted in severe pathological changes characterized by extensive necrosis in the spleen, similar to that induced by JS5/05. In addition, this gene replacement significantly increased virus replication and the levels of transcriptional cytokine response, compared to Herts/33. Conversely, inclusion of the M, F and HN genes of Herts/33 into JS5/05 backbone resulted in Herts/33-specific pathological changes and significantly decreased virus load and the expression levels of cytokine genes, compared to JS5/05., Conclusions: The M, F and HN genes are related to the severe pathological changes in the spleen of chickens infected with genotype VIId NDV.

The emerging H5 clade 2.3.4.6 viruses of different NA subtypes have been detected in different domestic poultry in China. We evaluated the receptor binding property and transmissibility of four novel H5 clade 2.3.4.6 subtype highly pathogenic avian influenza viruses. The results show that these viruses bound to both avian-type (α-2,3) and human-type (α-2,6) receptors. Furthermore, we found that one of these viruses, GS/EC/1112/11, not only replicated but also transmitted efficiently in guinea pigs. Therefore, such novel H5 subtype viruses have the potential of a pandemic threat.

Background: The strains of Newcastle disease virus (NDV) can be divided into two distinct clades: class I and class II. At present, limited molecular epidemiological data are available for the class I virus at live bird markets (LBMs). Knowing the genomic and antigenic characteristics of class I NDVs might provide important insights into the evolution dynamics of these viruses. In this study class I NDVs isolated from LBMs in Eastern China between 2008 and 2012 were characterized., Results: We characterized 34 class I NDVs genetically and 15 of the 34 NDVs pathologically which originated from geese, chickens and ducks at live bird markets. Based on the older classification system, twelve of fourteen strains isolated from 2008 to 2010 belonged to sub-genotype 3b. However, the rest 22 strains formed a separate novel cluster in genotype 3, which was designated as sub-genotype 3c. When based on the new classification system, sub-genotype 3b was classified into sub-genotype 1a and the sub-genotype 3c was classified into sub-genotype 1b. Over 62% (21/34) of the viruses were chicken-origin and only 13 isolates were waterfowl-origin. The Cross-neutralization reactions between CK/JS/05/11, CK/JS/06/12 and the vaccine strain LaSota showed significant antigenic differences between them., Conclusions: Currently, sub-genotype 3c (or 1b) NDVs are the most frequently isolated classI strains at LBMs in Eastern China., and the class I NDVs has transferred from waterfowls to chickens and circulated in chicken flocks extensively.

Background: Genotype VIId Newcastle disease virus (NDV) isolates induce more severe damage to lymphoid tissues, especially to the spleen, when compared to virulent viruses of other genotypes. However, the biological basis of the unusual pathological changes remains largely unknown., Methods: Virus replication, cytokine gene expression profile and cell death response in chicken splenocytes infected with two genotype VIId NDV strains (JS5/05 and JS3/05), genotype IX NDV strain F48E8 and genotype IV NDV strain Herts/33 were evaluated. Statistical significance of differences between experimental groups was determined using the Independent-Samples T test., Results: JS5/05 and JS3/05 caused hyperinduction of type I interferons (IFNs) (IFN-α and -β) during detection period compared to F48E8 and Herts/33. JS5/05 increased expression level of IFN-γ gene at 6 h post-inoculation (pi) and JS3/05 initiated sustained activation of IFN-γ within 24 h pi, whereas transcriptional levels of IFN-γ remained unchanged at any of the time points during infection of F48E8 and Herts/33. In addition, compared to F48E8 and Herts/33, JS3/05 and JS5/05 significantly increased the amount of free nucleosomal DNA in splenocytes at 6 and 24 h pi respectively. Annexin-V and Proidium iodid (PI) double staining of infected cells showed that cell death induced by JS3/05 and JS5/05 was characterized by marked necrosis compared to F48E8 and Herts/33 at 24 h pi. These results indicate that genotype VIId NDV strains JS3/05 and JS5/05 elicited stronger innate immune and cell death responses in chicken splenocytes than F48E8 and Herts/33. JS5/05 replicated at a significantly higher efficiency in splenocytes than F48E8 and Herts/33. Early excessive cell death induced by JS3/05 infection partially impaired virus replication., Conclusions: Viral dysregulaiton of host response may be relevant to the severe pathological manifestation in the spleen following genotype VIId NDV infection.

Background: The goose is usually considered to be resistant even to strains of Newcastle disease virus (NDV) that are markedly virulent for chickens. However, ND outbreaks have been frequently reported in goose flocks in China since the late 1990s with the concurrent emergence of genotype VIId NDV in chickens. Although the NDVs isolated from both chickens and geese in the past 15 years have been predominantly VIId viruses, published data comparing goose- and chicken-originated ND viruses are scarce and controversial., Results: In this paper, we compared genotype VIId NDVs originated from geese and chickens genetically and pathologically. Ten entire genomic sequences and 329 complete coding sequences of individual genes from genotype VIId NDVs of both goose- and chicken-origin were analyzed. We then randomly selected two goose-originated and two chicken-originated VIId NDVs and compared their pathobiology in both geese and chickens in vivo and in vitro with genotype IV virus Herts/33 as a reference. The results showed that all the VIId NDVs either from geese or from chickens shared high sequence homology and characteristic amino acid substitutions and clustered together in phylogenetic trees. In addition, geese and chickens infected by goose or chicken VIId viruses manifested very similar pathological features distinct from those of birds infected with Herts/33., Conclusions: There is no genetic or phenotypic difference between genotype VIId NDVs originated from geese and chickens. Therefore, no species-preference exists for either goose or chicken viruses and more attention should be paid to the trans-species transmission of VIId NDVs between geese and chickens for the control and eradication of ND.

Background: Although extensive data demonstrates that the majority of H6 duck isolates belonged to a single H6N2 virus lineage with a single gene constellation in southern China from 2000 to 2005, the prevalence of H6N2 virus in poultry in Eastern China is largely unknown., Results: Epidemiology revealed that H6N2 viruses were the most frequently detected influenza subtypes in live bird markets from 2002 to 2008 in Eastern China, but from 2009 onwards, they were replaced with novel H6N6 viruses. We phylogenetically and antigenically analyzed 42 H6 viruses isolated mainly in domestic ducks from 2002 to 2010 in Eastern China. Surprisingly, none of these isolates grouped with the previously described H6N2 viruses which belonged to a single H6N2 virus lineage with a single gene constellation in domestic ducks in southern China from 2000 to 2005. Two distinct hemagglutinin lineages were identified and they all underwent frequent reassortment with multiple virus subtypes from the natural gene pool, but few reassortants were persistent or prevalent., Conclusions: Five subtypes of H6 influenza viruses (H6N1, H6N2, H6N5, H6N6 and H6N8) cocirculated in Eastern China, which form a significant part of the natural influenza virus reservoir in domestic ducks, and significant viral reassortment is still ongoing in this species.