| H9N2avian influenza virus (AIV) has been circulating worldwide in many avian speciesand resulted in great economic losses to poultry industries. Although its pathogenicity wastrivial, it could cause high morbidity and lethality to poultry when co-infected withother pathogens. More importantly, human cases of avian H9N2virus infection have beenreported in Hong Kong and mainland China since1999. H9N2influenza viruses of chickenorigin cause only mild symptoms in humans, but they have the pandemic and cross-speciespotential for the high level of genetic plasticity and ressortment with other subtypes. H9N2isalso regarded by the World Health Organization (WHO) as the next potential avian influenzasubtype that may have high prevalence in the future, and which may bring huge damagesupon global economies and public health. So far, data on seroprevalence of H9N2virusesamong humans and their infection in mammals are limited. This literature gap is veryunfavorable for the effective prevention and control of human H9N2infection.Based on this, firstly, we investigated the seroprevalence of H9N2viruses amongpoultry workers with occupational exposure to domestic poultry in Shandong, China. Ninesubjects (2.3%) were positive for H9N2virus infection in the poultry workers in the currentstudy, which indicates that H9N2may have previously infected commercial laying poultryworker in Shandong, China. Therefore, in our study, avian-origin H9N2Shandong isolateswere used to investigate their infectivity and pathological changes in mammals. Then, thelungs were used as the target organ as their obvious lesions based on the above study. Theexpressions of cytokines in the lungs of H9N2-infected mice were examined by real-timepolymerase chain reaction(Real-time PCR), enzyme-linked immunosorbent assay (ELISA)and western blot. At the same time, proteomic analysis by using2DE on the lung tissues ofmice were also performed. This study will provide theoretical basis to the mechanisms of lung injury in mammals and the development of anti-influenza virus drug by providing drugtargets.There are four parts of this study.1Seroprevalence of avian influenza H9N2among poultry workersThis study was carried out to identify the seroprevalence of H9N2AIV among poultryworkers in Shandong, China. During December2011to February2012, a total of482subjectstook part in this study, including382poultry workers and100healthy residents withoutoccupational poultry exposure. Serum samples were collected and tested for the presence ofantibodies against H9N2AIV by hemagglutination inhibition (HI) and microneutralization(MN) assays. Nine subjects (9/382=2.3%) were positive for antibodies against H9N2AIVamong the poultry workers by either HI or MN assays using≥40cut-off, while none of the100healthy residents were seropositive. In conclusion, our study identified the H9N2avianinfluenza infections among poultry workers in Shandong, China, and continuous surveillanceof H9N2AIV infection in humans should be carried out to evaluate the threat to the publichealth.2Gene analysis of H9N2AIV of Shandong isolates and their infectivity in mammalsThe study was conducted to understand the evolution of H9N2subtype AIV isolatedfrom Shandong province and their pathogenicity to mammals. We analyzed the sequences of8segments of five H9N2AIVs isolated from Shandong province, and evaluated theirpathogenicity to mammals of the viruses by using BABL/c mice and guinea pigs model. TheHA and NA results indicated that all five strains belonged to CK/Beijing lineage. Two of thestrains possessed human-like receptor binding specificity (Leu234), while other threepossessed avain-like receptor binding specificity (Gln234). The HA cleavage site showed thatall strains were low pathogenic. Potential glycosylation sites varied among the viruses. Twoviruses could replicate in mice’s lung, caused interstitial pneumonia, and could be detected inbronchial epithelial cells by immunohistochemistry. The virus titers in mice lung were5.3±0.12log10TCID50/g and4.75±0.2log10TCID50/g.CK/SD/W3and CK/SD/ch could replicatein turbinate, trachea and lungs in the guinea pigs, the average virus titer of turbinate ranging3.4±0.55log10TCID50/g to4.2±0.42log10TCID50/g. The transmission experiments showedthat CK/SD/W3can spread by direct contact. Our study showed that H9N2Shandong isolatescan infect mice and guinea pigs without adaptation. It is essential to monitor the epidemicstrain in order to evaluate the public health threat to humans. 3Response profiles of cytokines and chemokines against avian H9N2influenza viruswithin the mouse lungSpecificity primers were designed according the gene sequences of cytokines andchemokines from the GenBank for real-time RT-PCR method. The results showed that thereal-time RT-PCR possessed high specificity and sensitivity. To obtain insight into the hostimmune responses to the avian H9N2virus, the expressions of11cytokines and chemokinesin the lungs of infected mice were examined by Real-time PCR, ELISA and Western. Afterinfection, there was a two-to20-fold mRNA induction of IFN-γ, IL-6, IP-10, CCL-5andMIP-1α; the concentrations of CCL-5and IFN-γ were, respectively, increased up to over15-fold at2dpi and20-fold at6dpi. Protein expressions of all or part of the five correspondingcytokines and chemokines (IFN-γ, IL-6, IP-10, CCL-5, and MIP-1α) were confirmed byELISA and western. We found that interferon gamma (IFN-γ) was the dominant antiviralcomponent, and interferon gamma-induced protein10kDa (IP-10), interleukin6(IL-6),chemokine (C-C motif) ligand5(CCL-5) and macrophage inflammatory protein-1alpha(MIP-1α) all played a role in pro-inflammatory responses to H9N2viruses. In conclusion, thisresearch can make us further understand the infection characteristics of H9N2virus inmammalian host by providing the data on mice lung immune responses to the avian H9N2virus.4Proteomic analysis of the lungs of mice infected with H9N2AIVTo investigate the host response against H9N2infections, we performed proteomicanalysis by using2DE on the lung tissues of mice collected on12h and72h postinoculationwith SD/CK/W3or PBS as a control. Thirty-one differentially expressed (DE) proteins, ofwhich there are four different proteins at12h and27different proteins at72h.These proteins are related to virus-host interactions and the functions includecytoskeletal proteins, stress response, antioxidant response, transcription or protein expressionregulation. The results indicated that cytoskeletal proteins are mainly regulated microtubule-associated protein CAP-Gly domain-containing linker protein1, Rab GTPase-activatingprotein1-like and annexin A4, which exchange material and transfer information through acomplex transport vesicles and microtubules. Stress response proteins HSP27and threeantioxidant protein Chloride Intracellular Channel Protein1(CLIC1), peroxiredoxin-6(Prx6)and peroxiredoxin-2(Prx2) were significantly increased, indicating that the influenza virusinfection may induce host cell oxidative stress. Ubc protein, ubiquitin-B and ISG15proteinare related to ubiquitin-proteasome pathway (UPP). Our data provide important informationregarding the interaction between influenza virus and host, which is useful to better understand the infection mechanisms of H9N2in mammals and development anti-influenzavirus drug targets. |
PDF Full Text Request