Wild type and mutant 2009 pandemic influenza A (H1N1) viruses cause more severe disease and higher mortality in pregnant BALB/c mice

Background: Pregnant women infected by the pandemic influenza A (H1N1) 2009 virus had more severe disease and higher mortality but its pathogenesis is still unclear. Principal Findings: We showed that higher mortality, more severe pneumonitis, higher pulmonary viral load, lower peripheral blood T lymphocytes and antibody responses, higher levels of proinflammatory cytokines and chemokines, and worse fetal development occurred in pregnant mice than non-pregnant controls infected by either wild type (clinical isolate) or mouse-adapted mutant virus with D222G substitution in hemagglutinin. These disease-associated changes and the lower respiratory tract involvement were worse in pregnant mice challenged by mutant virus. Though human placental origin JEG-3 cell line could be infected and proinflammatory cytokines or chemokines were elevated in amniotic fluid of some mice, no placental or fetal involvement by virus were detected by culture, real-time reverse transcription polymerase chain reaction or histopathological changes. Dual immunofluorescent staining of viral nucleoprotein and type II alveolar cell marker SP-C protein suggested that the majority of infected alveolar epithelial cells were type II pneumocytes. Conclusion: The adverse effect of this pandemic virus on maternal and fetal outcome is largely related to the severe pulmonary disease and the indirect effect of inflammatory cytokine spillover into the systemic circulation. © 2010 Chan et al.published_or_final_versio

A SNP in IFNGR1 promoter is correlated to the susceptibility to chronic HBV infection in Chinese population

Posters: no. P725This journal suppl. entitled: Abstracts of the 19th ECCMID (European Congress of Clinical Microbiology and Infectious Diseases). Helsinki, Finland. May 16-19, 2009.OBJECTIVES: The antiviral mechanism stimulated by interferon-gamma is found to be crucial for clearance of hepatitis B virus (HBV) in vivo. The antiviral signaling transduction is triggered by the specific binding between interferon-gamma and its receptor IFNGR1 (interferongamma receptor 1). Interferon-gamma signaling transduction pathway is directly controlled by the IFNGR1 expression level. In our study, single nucleotide polymorphisms (SNPs) in the IFNGR1 gene and the correlation between the SNPs and susceptibility to chronic HBV in Chinese were investigated. METHODS: Blood samples of 983 Chinese, including 361 chronic hepatitis B patients, 366 healthy individuals, and 256 hepatitis B spontaneously recovered patients, were collected. Seven SNPs (−611A/G, −56C/T, 40G/A, 95C/T, 130A/G, 20685A/G, 21227T/C) in IFNGR1 gene were identified by restriction fragment-length polymorphism (RFLP) assays. The transcription levels of different SNPs variants were compared by luciferase assays. RESULTS: −56C and −56T allele were found to be correlated to HBV clearance and persistence. In luciferase assays, the transcription level of IFNGR1 promoter with the −56C is significantly higher than that with −56T. CONCLUSION: −56C/T SNP in IFNGR1 promoter region is associated with susceptibility to chronic HBV in Chinese population.link_to_OA_fulltex

A regulatory polymorphism in interferon-γ receptor 1 promoter is associated with the susceptibility to chronic hepatitis B virus infection

The antiviral cascade triggered by interferon-γ (IFN-γ) represents a vital event for eradicating hepatitis B virus (HBV) in experimental animals. IFN-γ signaling is mediated through the ligand binding to IFN-γ receptor 1 (IFNGR1). Control of IFNGR1 expression level is one of the mechanisms by which cells modulate the potency of IFN-γ signaling. In this study, we comprehensively investigated the single nucleotide polymorphisms (SNPs) in IFNGR1 gene and correlated their occurrence to susceptibility to HBV infection in a Chinese population. A total of 983 participants, including 361 chronic hepatitis B patients, 256 individuals who had spontaneously recovered from HBV infection, and 366 healthy control subjects, were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphism was used to identify seven SNPs (-611A/G, -56C/T, 40G/A, 95C/T, 130A/G, 20685A/G, 21227T/C) in IFNGR1 gene. We found that -56C and -56T allele were associated with viral clearance and viral persistence, respectively (P∈=∈0.014). In a reporter-driven assay, we validated that the promoter variant with -56C exhibited a higher transcription level than that with -56T in HepG2 cells in a cell-type-specific pattern. We conclude that a functional -56C/T SNP in IFNGR1 promoter is associated with the clinical outcome of HBV infection in this Chinese population. © 2009 Springer-Verlag.link_to_subscribed_fulltex

Functional dissection of an IFN-α/β receptor 1 promoter variant that confers higher risk to chronic hepatitis B virus infection

Background/Aims: We previously demonstrated that two linked single nucleotide polymorphisms (SNPs) at -408 and -3 of type I interferon receptor 1 (IFNAR1) promoter are associated with susceptibility to chronic HBV infection. We aimed to elucidate the mechanism by which -3 and/or -408 C/T SNPs had such profound effects. Methods: A functional SNP in IFNAR1 promoter was defined by reporter gene assay, mutational analysis, flow cytometry analysis and gel shift assay. The nuclear protein binding to the essential polymorphic site was identified and its effect on transcriptional regulation of IFNAR1 was further demonstrated in a series of ex vivo and in vivo experiments. Results: We found C > T change at the -3 locus reduced the transcriptional activity of IFNAR1 promoter. High mobility group B protein 1 (HMGB1) and PARP-1 were co-recruited to the IFNAR1 promoter to regulate its transcription. We demonstrated HMGB1-binding affinity to IFNAR1 promoter was reduced in the -3T variant. Additionally, PARP-1, a cofactor for IFNAR1 transcription activation, was significantly suppressed by HBV. Conclusion: Upon HBV infection, decreased binding affinity of HMGB1 to the IFNAR1 promoter -3T variant is aggravated by the suppressed PARP-1 expression caused by HBV, resulting in a further attenuated IFNAR1 expression. This compromises the antiviral and immuno-regulatory effects of IFN-α/β, which may in turn affect the clinical outcome of HBV infection. © 2009 European Association for the Study of the Liver.link_to_subscribed_fulltex

A critical role of IL-17 in modulating the B-cell response during H5N1 influenza virus infection

Interleukin-17 (IL-17), a member of the IL-17 cytokine family, plays a crucial role in mediating the immune response against extracellular bacteria and fungi in the lung. Although there is increasing evidence that IL-17 is involved in protective immunity against H1 and H3 influenza virus infections, little is known about the role of IL-17 in the highly pathogenic H5N1 influenza virus infection. In this study, we show that H5N1-infected IL-17 knockout (KO) mice exhibit markedly increased weight loss, more pronounced lung immunopathology and significantly reduced survival rates as compared with infected wild-type controls. Moreover, the frequency of B cells in the lung were substantially decreased in IL-17 KO mice after virus infection, which correlated with reduced CXCR5 expression in B cells and decreased CXCL13 production in the lung tissue of IL-17 KO mice. Consistent with this observation, B cells from IL-17 KO mice exhibited a significant reduction in chemokine-mediated migration in culture. Taken together, these findings demonstrate a critical role for IL-17 in mediating the recruitment of B cells to the site of pulmonary influenza virus infection in mice. © 2011 CSI and USTC. All rights reserved.link_to_subscribed_fulltex

Translationally controlled tumor protein induces mitotic defects and chromosome missegregation in hepatocellular carcinoma development

Emerging evidence implicates the chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) as a specific oncogene in human hepatocellular carcinoma (HCC). To better understand the molecular mechanisms underlying HCC cases carrying CHD1L amplification (>50% HCCs), we identified a CHD1L target, translationally controlled tumor protein (TCTP), and investigated its role in HCC progression. Here, we report that CHD1L protein directly binds to the promoter region (nt -733 to -1,027) of TCTP and activates TCTP transcription. Overexpression of TCTP was detected in 40.7% of human HCC samples analyzed and positively correlated with CHD1L overexpression. Clinically, overexpression of TCTP was significantly associated with the advanced tumor stage (P = 0.037) and overall survival time of HCC patients (P = 0.034). In multivariate analyses, TCTP was determined to be an independent marker associated with poor prognostic outcomes. In vitro and in vivo functional studies in mice showed that TCTP has tumorigenic abilities, and overexpression of TCTP induced by CHD1L contributed to the mitotic defects of tumor cells. Further mechanistic studies demonstrated that TCTP promoted the ubiquitin-proteasome degradation of Cdc25C during mitotic progression, which caused the failure in the dephosphorylation of Cdk1 on Tyr15 and decreased Cdk1 activity. As a consequence, the sudden drop of Cdk1 activity in mitosis induced a faster mitotic exit and chromosome missegregation, which led to chromosomal instability. The depletion experiment proved that the tumorigenicity of TCTP was linked to its role in mitotic defects. Conclusion: Collectively, we reveal a novel molecular pathway (CHD1L/TCTP/Cdc25C/Cdk1), which causes the malignant transformation of hepatocytes with the phenotypes of accelerated mitotic progression and the production of aneuploidy. © 2011 American Association for the Study of Liver Diseases.link_to_OA_fulltex

D225G mutation in hemagglutinin of pandemic influenza H1N1 (2009) virus enhances virulence in mice

Although the majority of infections by the pandemic influenza H1N1 (2009) virus is mild, a higher mortality occurs in young adults with no risk factors for complications. Some of these severe cases were infected by the virus with an aspartate to glycine substitution at 225 position (D225G, H3 numbering) in the hemagglutinin (HA). Previous studies with the highly virulent 1918 pandemic H1N1 virus suggested that such substitution was associated with a dual binding specificity of the virus for both α2,3- and α2,6-linked sialic acid receptors on host cells. Thus, the D225G mutant may cause more severe disease with its increased predilection for the lower respiratory tract, where the α2,3 sialic acid receptor is more prevalent, but this hypothesis has not been investigated. We obtained a mutant virus after four sequential passages in lungs of BALB/c mice with a wild-type pandemic influenza A H1N1 (2009) virus. One plaque purified mutant virus had a single non-synonymous D225G mutation in the HA gene. This mutant was more lethal to chick embryo and produced a viral load of about two log higher than that of the wild-type parental virus during the first 24 h. A pathogenicity test showed that the 50% lethal dose in mice (LD50) was reduced from over 2 × 10 6 plaque-forming units (PFU) with the parental virus to just 150 PFU with the mutant virus. The survival of mice challenged with the mutant virus was significantly decreased when compared with the parental virus (P < 0.0001). Significantly higher viral titers and elevated proinflammatory cytokines in lung homogenates of mice infected with the mutant virus were found, which were compatible with severe histopathological changes of pneumonitis. The only consistent mutation in the genomes of viral clones obtained from dying mice was D225G substitution. Copyright © 2010 by the Society for Experimental Biology and Medicine.link_to_subscribed_fulltex

Active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: Implications for pathogenesis

Middle East respiratory syndrome coronavirus (MERS-CoV) infection caused severe pneumonia and multiorgan dysfunction and had a higher crude fatality rate (around 50% vs. 10%) than SARS coronavirus (SARS-CoV) infection. To understand the pathogenesis, we studied viral replication, cytokine/chemokine response, and antigen presentation in MERS-CoV-infected human monocyte-derived macrophages (MDMs) versus SARS-CoV-infected MDMs. Only MERS-CoV can replicate in MDMs. Both viruses were unable to significantly stimulate the expression of antiviral cytokines (interferon alpha [IFN-alpha] and IFN-beta) but induced comparable levels of tumor necrosis factor alpha and interleukin 6. Notably, MERS-CoV induced significantly higher expression levels of interleukin 12, IFN-gamma, and chemokines (IP-10/CXCL-10, MCP-1/CCL-2, MIP-1alpha/CCL-3, RANTES/CCL-5, and interleukin 8) than SARS-CoV. The expression of major histocompatibility complex class I and costimulatory molecules were significantly higher in MERS-CoV-infected MDMs than in SARS-CoV-infected cells. MERS-CoV replication was validated by immunostaining of infected MDMs and ex vivo lung tissue. We conclusively showed that MERS-CoV can establish a productive infection in human macrophages. The aberrant induction of inflammatory cytokines/chemokines could be important in the disease pathogenesis.link_to_subscribed_fulltex

Improved detection of Zika virus RNA in human and animal specimens by a novel, highly sensitive and specific real-time RT-PCR assay targeting the 5'-untranslated region of Zika virus

Poster presentationBackground: Highly sensitive and specific laboratory diagnostics are especially important for controlling the rapidly expanding Zika virus (ZIKV) epidemic, as the clinical features of ZIKV infection may be mild or indistinguishable from other arbovirus infections. Non-vector-borne transmissions may occur due to persistent virus shedding in ZIKV-infected patients’ urogenital tract, blood, and/or bodily fluids. Some existing RT-PCR assays utilizing primers/probes designed before the present epidemic emerged might misdiagnose up to 20-80% of ZIKV infected patients because of limited sensitivity and nucleotide mismatches. Methods: We developed and evaluated five novel real-time RT-PCR assays targeting conserved regions in the 5’ untranslated region (5’UTR), envelope (E’), non-structural protein 2A (NS2A), NS5, and 3’-UTR of the ZIKV genome. Results: The ZIKV-5’-UTR assay exhibited the lowest in-vitro limit of detection (5-10 RNA copies/reaction and 3.0 x 10-1 plaque-forming units/ml). Compared to the modified version of a widely adopted RT-PCR assay targeting the ZIKV-E gene, the ZIKV-5’UTR assay showed better sensitivity in human clinical specimens, and representative mouse specimens, including many organs which are known to be involved in human ZIKV infection but difficult to obtain in clinical settings. The ZIKV-5’UTR assay detected ZIKV RNA in all 84/84 (100.0%) ZIKV-E’-positive and an additional 30/296 (10.1%, P<0.01) ZIKV-E’-negative mouse specimens. The higher sensitivity of the ZIKV-5’UTR assay was most significant in kidney and testis/epididymis specimens (P<0.01). No in-vitro or in-vivo cross-reactivity was found between the ZIKV-5’UTR assay and other common flaviviruses/arboviruses. Conclusions: The highly sensitive and specific ZIKV-5’UTR assay may help to improve the laboratory diagnosis of ZIKV infection