Jingyu Wang

Deterioration of eggshell quality in laying hens experimentally infected with H9N2 avian influenza virus

This study aimed to determine the mechanism by which H9N2 avian influenza virus (AIV) affects eggshell quality. Thirty-week-old specific pathogen free egg-laying hens were inoculated with the chicken-origin H9N2 AIV strain (A/Chicken/shaanxi/01/2011) or with inoculating media without virus by combined intraocular and intranasal routes. The time course for the appearance of viral antigen and tissue lesions in the oviduct was coincident with the adverse changes in egg production in the infected hens. The viral loads of AIV have a close correlation with the changes in the uterus CaBP-D28k mRNA expression as well as the Ca concentrations in the eggshells in the infected hens from 1 to 7xa0days post inoculation (dpi). Ultrastructural examination of eggshells showed significantly decreased shell thickness in the infected hens from 1 to 5xa0dpi (Pxa0<xa00.05). Furthermore, obvious changes in the structure of the external shell surface and shell membrane were detected in the infected hens from 1 to 5xa0dpi as compared with the control hens. In conclusion, this study confirmed that H9N2 AIV strain (A/Chicken/shaanxi/01/2011) infection is associated with severe lesions of the uterus and abnormal expression of CaBP-D28k mRNA in the uteri of the infected hens. The change of CaBP-D28k mRNA expression may contribute to the deterioration of the eggshell quality of the laying hens infected with AIV. It is noteworthy that the pathogenicity of H9N2 AIV strains may vary depending on the virus strain and host preference.

Apoptosis induction and release of inflammatory cytokines in the oviduct of egg-laying hens experimentally infected with H9N2 avian influenza virus

The H9N2 subtype avian influenza virus (AIV) can cause serious damage to the reproductive tract of egg-laying hens, leading to severe egg-drop and poor egg shell quality. However, previous studies in relation to the oviductal-dysfunction resulted from this agent have not clearly been elucidated. In this study, apoptosis and pathologic changes in the oviducts of egg-laying hens caused by H9N2 AIV were evaluated. To understand the immune response in the pathogenic processes, 30-week old specific pathogen free (SPF) egg-laying hens inoculated with H9N2 subtype of AIV through combined intra-ocular and intra-nasal routes. H9N2 AIV infection resulted in oviductal lesions, triggered apoptosis and expression of immune related genes accompanied with infiltration of CD3(+)CD4(+) and CD3(+)CD8α(+) cells. Significant tissue damage and apoptosis were observed in the five oviductal parts (infundibulum, magnum, isthmus, uterus and vagina) at 5 days post-inoculation (dpi). Furthermore, immune-related genes, including chicken TLR3 (7, 21), MDA5, IL-2, IFN-β, CXCLi1, CXCLi2, XCL1, XCR1 and CCR5 showed variation in the egg-laying hens infected with H9N2 AIV. Notably, mRNA expression of IFN-α was suppressed during the infection. These results show distinct expression patterns of inflammatory cytokines and chemokines amongst segments of the oviduct. Differential gene expression of inflammatory cytokines and lymphocytes aggregation occurring in oviducts may initiate the infected tissue in response to virus replication which may eventually lead to excessive cellular apoptosis and tissue damage.

The distribution of sialic acid receptors of avian influenza virus in the reproductive tract of laying hens.

The susceptibility of the host to influenza virus is determined by the distribution of the sialic acid (SA) receptors on host cell membrane. Avian influenza virus (AIV) preferentially binds to SA α-2,3-galactose (SA α2,3-gal) linked receptors, while human strains bind to sialic acid α2,6-galactose (SA α2,6-gal) linked receptors. Here, we describe the SA patterns and distributions in the reproductive tract of hens by employing two specific lectins, Maackia amurensis agglutinin (MAA) for SA α2,3-gal and sambucus nigra agglutinin (SNA) for SA α 2,6-gal receptors. Our results revealed that both SA α2,3-gal and SA α2,6-gal receptors exist in the reproductive tract of hens, including magnum, isthmus, uterus and vagina except for infundibulum. The distribution of SAα-2,3-gal receptor was more abundantly in the columnar epithelium cells of magnum, isthmus and uterus. Only minimal positive results for SA α-2,6-gal receptors were detected in the columnar epithelium cells of magnum, isthmus, uterus and vagina. Furthermore, AIV in tissues of the reproductive tract tissues of laying hens were detected by SYBR green-based reverse transcription and polymerase chain reaction (RT-PCR). Results showed that both viral loads and pathological changes in different parts of the reproductive tract were positively correlated with the expression of both receptors. Our results revealed that the reproductive tract of hens may provide an environment for the replication of both avian and human influenza viruses.

Characterization of emerging Newcastle disease virus isolates in China

BackgroundNewcastle disease (ND) is a devastating worldwide disease of poultry characterized by increased respiration, circulatory disturbances, hemorrhagic enteritis, and nervous signs. Sequence analysis shows several amino acid residue substitutions at neutralizing epitopes on the F and HN proteins of recent Shaanxi strains. Both Cross protection and cross serum neutralization tests revealed that the traditional vaccine strains were unable to provide full protection for the flocks.MethodsTo better understand the epidemiology of Newcastle disease outbreak, a portion of the F gene and the full-length HN gene were amplified from Shaanxi isolates by reverse transcription-polymerase chain reaction (RT-PCR) and then conducted sequence and phylogenetic analyzes. In pathogenicity analysis, both high intra-cerebral pathogenicity index (ICPI) and mean death time (MDT) tests of chicken embryo were carried out. Furthermore, a cross-protection experiment in which specific-pathogen-free chickens vaccinated with a LaSota vaccine strain were challenged by the recent Shaanxi strain was also performed.ResultsNine Newcastle disease (ND) virus (NDV) isolates which were recovered from ND outbreaks in chicken flocks in China were genotypically and pathotypically characterized. Amino acid sequence analysis revealed that all the recent Shaanxi-isolated NDVs have 112R-R-Q-K-R-F117 for the C-terminus of the F2 protein and exhibit high ICPI and MDT of chicken embryos, suggesting that they were all classified as velogenic type of NDVs. Phylogenetic analysis of these isolates showed that they belong to subgenotype VIId that have been implicated in the recent outbreaks in northwestern China. The percentage of amino acid sequence identity of F protein between recent Shaanxi stains and five vaccine strains was in the range of 81.9xa0%–88.1xa0%, while the percentage of amino acid sequence identity of HN protein between recent Shaanxi strains and vaccine strains was in the range of 87.4xa0%–91.2xa0%. Furthermore, a number of amino acid residue substitutions at neutralizing epitopes on the F and HN proteins of these isolates were observed, which may lead to the change of antibody recognition and neutralization capacity. A cross-protection experiment indicated that specific-pathogen-free chickens vaccinated with a LaSota vaccine strain was not capable of providing full protection for the flocks that were challenged by the recent Shaanxi strain.ConclusionsTaken together, our findings reveal that recent Shannxi NDVstrains exhibit antigenic variations that could be responsible for recent outbreaks of NDVs in northwestern China.

Complete Genome Sequence of a New H9N2 Avian Influenza Virus Isolated in China.

ABSTRACT The complete genomic sequence of a new H9N2 avian influenza virus (AIV), isolated in northwestern China, was determined. Sequence and phylogenetic analyses based on the sequences of eight genomic segments revealed that the isolate is phylogenetically related to the Y280-like sublineage.

Complete Genome Sequence of a Newly Emerging Newcastle Disease Virus

ABSTRACT The complete genome sequence of a newly emerging Newcastle disease virus, isolated in China, was determined. A phylogenetic analysis based on the F gene revealed that the isolate is phylogenetically related to Newcastle disease virus genotype VIId. Sequence analysis indicated that amino acid residue substitutions occur at neutralizing epitopes on the hemagglutinin-neuraminidase (HN) protein.

Modulation of the innate immune-related genes expression in H9N2 avian influenza virus-infected chicken macrophage-like cells (HD11) in response to Escherichia coli LPS stimulation

Macrophages play important roles in mediating virus-induced innate immune responses and are thought to be involved in the pathogenesis of bacterial superinfections. The innate immune response initiated by both low pathogenicity AIV and bacterial superinfection in their avian host is not fully understood. We therefore determine the transcripts of innate immune-related genes following avian H9N2 AIV virus infection and E. coli LPS co-stimulation of avian macrophage-like cell line HD11 cells. More pronounced expression of pro-inflammatory cytokines (IL-6 and IL-1β) as well as the inflammatory chemokines (CXCLi1 and CXCLi2) was observed in virus infected plus LPS treated HD11 cells compared to H9N2 virus solely infected control. For two superinfection groups, the levels of genes examined in a prior H9N2 virus infection before secondary LPS treatment group were significantly higher as compared with simultaneous virus infection plus LPS stimulation group. Interestingly, similar high levels of IL-6 gene were observed between LPS sole stimulation group and two superinfection groups. Moreover, IL-10 and TGF-β3 mRNA levels in both superinfection groups were moderately upregulated compared to sole LPS stimulation group or virus alone infection group. Although TLR4 and MDA5 levels in virus alone infection group were significantly lower compared to that in both superinfection groups, TLR4 upregulation respond more rapid to virus sole infection compared to LPS plus virus superinfection. Collectively, innate immune-related genes respond more pronounced in LPS stimulation plus H9N2 virus infection HD11 cells compared to sole virus infection or LPS alone stimulation control cells.

The role of hexon in egg drop syndrome virus (EDSV) inducing apoptosis in duck embryo fibroblast cells

Although extensive efforts have been made to understand adenovirus infection in human cells, little is known for egg drop syndrome virus (EDSV) infection in the avian-derived cells. In this study, the effects of EDSV infection as well as the possible role hexon protein, the main building block of the EDSV capsid, on apoptosis induction in duck embryo fibroblast (DEF) cells was examined. Flow cytometry analysis and TUNEL assay revealed that EDSV infection induced significant apoptosis in DEF cells compared with mock infected cells. Interestingly, the increase of the apoptosis rate detected in EDSV infected DEF cells were accompanied by an increased virus load in cells in a time-dependent manner. Furthermore, a time-dependent decrease in hexon protein expression levels in hexon transfected DEF cells in parallel with a gradual decrease in TUNEL-labeling cells was also observed in the current study. In addition, caspase activity detection and western blot analysis indicates that either EDSV infection or EDSV hexon transfection both induced apoptosis of DEF cells via activating both the exogenous and the mitochondrial pathway.

Strong inflammatory responses and apoptosis in the oviducts of egg-laying hens caused by genotype VIId Newcastle disease virus.

BackgroundNewcastle disease virus (NDV) can cause serious damage to the reproductive tracts of egg-laying hens and leads to egg production and quality reduction. However, the mechanism of severe pathological damage in the oviducts of egg-laying hens after NDV infection has not been fully elucidated. In this study, the correlation between the primary pathological lesions and viral load in the oviducts of egg-laying hens infected with the velogenic genotype VIId NDV strain was evaluated by pathological observation and virus detection. Subsequently, apoptosis, the expression of immune-related genes and lymphocyte infiltration into the infected oviducts were determined to explore the potential causes of the pathological changes.ResultsA higher viral load and severe tissue lesions and apoptotic bodies were observed in the oviduct of NDV-infected hens compared with the control. Immune-related genes, including TLR3/7/21, MDA5, IL-2/6/1β, IFN-β, CXCLi1/2, and CCR5, were significantly upregulated in the magnum and uterus. IL-2 presented the highest mRNA level change (137-fold) at 5xa0days post infection (dpi) in the magnum. Infection led to CD3+CD4+ and CD3+CD8α+ lymphocyte infiltration into the magnum of the oviduct. A higher viral load was found to be associated with pathological changes and the elevated expression of proinflammatory cytokines in the NDV-infected hens.ConclusionsOur results indicate that the severe lesions and apoptosis in the oviducts of egg-laying hens caused by genotype VIId NDV strains are associated with the excessive release of inflammatory cytokines, chemokines and lymphocyte infiltration, which contribute to the dysfunction of the oviducts and the decrease of egg production in hens.

Complete Genome Sequences of Six Avian-Like H1N1 Swine Influenza Viruses from Northwestern China

ABSTRACT Very little is known about swine influenza in northwestern China. Here, we report the complete genomic sequences of six avian-like H1N1 swine influenza viruses (SIVs) isolated in pigs in northwestern China. Phylogenetic analyses of the sequences of eight genomic segments demonstrated that they are avian-like H1N1 SIVs.