| Influenza virus, one of the most important respiratory viruses which can infect humans and result into deseases, presents globally seasonal epidemics every year. The data from world healthy organization showed that global attack rate of seasonal influenza estimated at5-10%in adults and20-30%in children including3-5million cases of severe illness and up to300000-500000deaths per year. In spring2009, a previously undescribed A (H1N1) influenza virus emerged to infect humans in Mexico and North America in priority. The virus transmitted and speaded in dozens of countries in several weeks, and induced a global pandemic in a short time.,The pandemic still resulted into280000human death although the2009influenza A (H1N1) pandemic is mild on humans on the whole. And most fatal infection majored in young adults. The question is what the reasons was own to fatal infection. Combined the analysis of clinical data of2009pdmHIN1fatal cases, immunopathology and molecular pathology studies on lung tissue of fatal cases with experimental studies bosed on in vitro infection models, the object of this study is trying to expound the characteristics of the immune response of innate immune system in2009pdmH1N1infection and the possible effects of antiviral drugs for treatment on severe infection, to explore the pathogenesis of severe infection, and to provide a possible evidence for the clinical treatment policy on severe patients.The study included two parts as following.Part I:The study on the role of the innate immune response and it’s pathogenesis associated in2009pdm H1N1fatal infection.We investigated viral load, the immune response and apoptosis in lung tissues from50fatal cases with2009pdmH1N1virus infection. The results suggested that seven out of the27cytokines/chemokines showed remarkably high expression including IL-1RA, IL-6, TNF-a, IL-8, MCP-1, MIP-1β and IP-10in lung tissues of2009pdmH1N1fatal cases. Viral load, which showed the highest level on day7of illness onset and persisted until day17of illness, was positively correlated with mRNA levels of IL-1RA, MCP-1, MIP-1β, IP-10and RANTES. IHC staining showed that IL-6, IP-10and IL-8with similar distribution with influenza A virus NP staining were detected in pneumocytes and submucosal glands of lung tissues of fatal cases. Apoptosis was evident in lung tissues stained by the TUNEL assay, and cleaved caspase-3with similar location in lung tissues of fatal cases was frequently seen in pneumocytes, submucosal glands and lymphoid tissues. In addition, apoptosis associated genes Fas-L present remarkably increased expression in lung tissues of fatal cases but TRAIL didn’t present significant difference in lung tissues between fatal cases and normal control. Another finding was that decreased Fas and elevated Fas-L mRNA levels were present in lung tissues by quantitative RT-PCR assay. The pathogenesis of the2009pdmH1N1virus infection is associated with viral replication and a host-imposed immune response. Fas-L and caspase-3are involved in the pathway of2009pdmH1N1virus-induced apoptosis in lung tissues, and the disequilibrium between the Fas and Fas-L level in lung tissues could contribute to delayed clearance of the virus and subsequent pathological damages.Part II:The preliminary study of the possible affect of Ostamivir treatmenton2009pdmH1N1fatal patient and its mechanismoseltamivir has been shown to be effective for the treatment and chemoprophylaxis of seasonal as well as2009pdmH1N1influenza infections. However, the safety consideration on oseltamivir could never be neglected since the adverse events have been reported frequently after the treatment of oseltamivir for influenza patients. This study analyzed the clinical data of the2009pdmH1N1fatal cases studied in part I. Twenty-nine cases with clear records of antiviral drug treatment were devived into two groups of oseltamivir treat (No.=24) and no antiviral treatment (No.=5). The relationship was analyzed between oseltamivir treatment ant the viral load, host immune response or apoptosis in lung tissures. The results showed that the median admission duriation was5days (rang of1-12days) after illness onset in oseltamivir treatment group, median time of intial using oseltamivir was5days (rang of1-12days) after illness onset, and median time of death was9days (range of 3-17days) after illness onset. Two out of5cases without antiviral treatment did not hospitalize. The other3cases hospitalized at3,4or8days fater illness onset respectively. The median time of death was4days (range of2-8days) after illness onset in no antivial treatment group. Viral load tests showed no significant difference (p=0.39) in lung tissues between two groups although viral load level was slightly higher in oseltamivir treatment group that in no viral treatment group. The cytokines levels of IL-1RA, IL-6, TNF-a, IL-8, MCP-1, MIP-1β, IP-10and RANTES in lung tissues was no significant difference (p>0.05) between the two groups. The apoptosis-related gene FASL mRNA expression levels in the oseltamivir treatment group was significantly higher than no antivial treatment group (p=0.01),but TRAIL mRNA expression levels did not differ between the two groups in (p>0.05).In addition, the apoptosis and viral replication kenetics were tested on the infected cells after seasonal or2009pandemic influenza A (H1N1) viruses infected A-549or THP-1cells with or without oseltamivir-treatment were tested. Apoptotic levels of infected THP-1or A-549cells were determined by FCM with annexin V/P1stain or TUNEL assay. The viral kenetics were demonstrated by viral titration or quantitative RT-PCR. The cytokines response was tested in supernatant of infected THP-1cells by Cytometric beads assay. The results showed that the apoptosis of infected cells was increased at24h post of infection (POI) in influenza infected A A-549or THP-1cells with oseltamivir treatment compared with non-treatment. To be consistent, the viral loads in infected cells were increased at6h or24h POI in oseltaviral treatment group although viral loads were dicreased at Oh POI.The results suggested that oseltamivir may inhibit both viral entry and release but it may enhance apoptosis of infected cells. The present clinical treatment policy of neraminidase inhibitor oseltamivir may not disease the viral load in lung tissues of severe infection. The delayed using of oseltamivir in severe influenza patients may enhanced the pathological damage of local infected-tissues, and may delay viral clearance in vivo. |
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