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Abstract Background Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease. Aim To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease. Methods We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces. Results We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium. Conclusion The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.

Abstract Background Influenza has been associated with a heavy burden of mortality. In tropical or subtropical regions where influenza viruses circulate in the community most of the year, it is possible that there are seasonal variations in the effects of influenza on mortality, because of periodic changes in environment and host factors as well as the frequent emergence of new antigenically drifted virus strains. In this paper we explored this seasonal effect of influenza. Methods A time-varying coefficient Poisson regression model was fitted to the weekly numbers of mortality of Hong Kong from 1996 to 2002. Excess risks associated with influenza were calculated to assess the seasonal effects of influenza. Results We demonstrated that the effects of influenza were higher in winter and late spring/early summer than other seasons. The two-peak pattern of seasonal effects of influenza was found for cardio-respiratory disease and sub-categories pneumonia and influenza, chronic obstructive pulmonary disease, cerebrovascular diseases and ischemic heart disease as well as for all-cause deaths. Conclusion The results provide insight into the possibility that seasonal factors may have impact on virulence of influenza besides their effects on virus transmission. The results warrant further studies into the mechanisms behind the seasonal effect of influenza.

Abstract Background The SARS outbreak in 2003 provides a unique opportunity for the study of human responses to a novel virus. We have previously reported that dendritic cells (DCs) might be involved in the immune escape mechanisms for SARS-CoV. In this study, we focussed on the gene expression of toll-like receptors (TLRs), chemokine receptors (CCRs) and death receptor ligands in SARS-CoV infected DCs. We also compared adult and cord blood (CB) DCs to find a possible explanation for the age-dependent severity of SARS. Results Our results demonstrates that SARS-CoV did not modulate TLR-1 to TLR-10 gene expression but significantly induced the expression of CCR-1, CCR-3, and CCR-5. There was also strong induction of TNF-related apoptosis-inducing ligand (TRAIL), but not Fas ligand gene expression in SARS-CoV infected DCs. Interestingly, the expressions of most genes studied were higher in CB DCs than adult DCs. Conclusion The upregulation of chemokines and CCRs may facilitate DC migration from the infection site to the lymph nodes, whereas the increase of TRAIL may induce lymphocyte apoptosis. These findings may explain the increased lung infiltrations and lymphoid depletion in SARS patients. Further explorations of the biological significance of these findings are warranted.

Abstract Background Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of α2,3-linked and α2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SAα2,6Gal and Maackia amurensis agglutinin (MAA) for SAα2,3Gal in the respiratory tract of normal adults and children. Methods We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers. Results We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the α2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs. Conclusion The lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SAα2,3 binding viruses, such as influenza or human parainfluenza are to be made.

Abstract Background Chemokines play important roles in inflammation and antiviral action. We examined whether polymorphisms of RANTES, IP-10 and Mig affect the susceptibility to and outcome of severe acute respiratory syndrome (SARS). Methods We tested the polymorphisms of RANTES, IP-10 and Mig for their associations with SARS in 495 Hong Kong Chinese SARS patients and 578 controls. Then we tried to confirm the results in 356 Beijing Chinese SARS patients and 367 controls. Results RANTES -28 G allele was associated with SARS susceptibility in Hong Kong Chinese (P < 0.0001, OR = 2.80, 95%CI:2.11–3.71). Individuals with RANTES -28 CG and GG genotypes had a 3.28-fold (95%CI:2.32–4.64) and 3.06-fold (95%CI:1.47–6.39) increased risk of developing SARS respectively (P < 0.0001). This -28 G allele conferred risk of death in a gene-dosage dependent manner (P = 0.014) with CG and GG individuals having a 2.12-fold (95% CI: 1.11–4.06) and 4.01-fold (95% CI: 1.30–12.4) increased risk. For the replication of RANTES data in Beijing Chinese, the -28 G allele was not associated with susceptibility to SARS. However, -28 CG (OR = 4.27, 95%CI:1.64–11.1) and GG (OR = 3.34, 95%CI:0.37–30.7) were associated with admission to intensive care units or death due to SARS (P = 0.011). Conclusion RANTES -28 G allele plays a role in the pathogenesis of SARS.

Abstract Background Cytokines play important roles in antiviral action. We examined whether polymorphisms of IFN-γ,TNF-α and IL-10 affect the susceptibility to and outcome of severe acute respiratory syndrome (SARS). Methods A case-control study was carried out in 476 Chinese SARS patients and 449 healthy controls. We tested the polymorphisms of IFN-γ,TNF-α and IL-10 for their associations with SARS. Results IFN-γ +874A allele was associated with susceptibility to SARS in a dose-dependent manner (P < 0.001). Individuals with IFN-γ +874 AA and AT genotype had a 5.19-fold (95% Confidence Interval [CI], 2.78-9.68) and 2.57-fold (95% CI, 1.35-4.88) increased risk of developing SARS respectively. The polymorphisms of IL-10 and TNF-α were not associated with SARS susceptibility. Conclusion IFN-γ +874A allele was shown to be a risk factor in SARS susceptibility.

Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

Swine populations are known to be an important source of new human strains of influenza A, including those responsible for global pandemics. Yet our knowledge of the epidemiology of influenza in swine is dismayingly poor, as highlighted by the emergence of the 2009 pandemic strain and the paucity of data describing its origins. Here, we analyse a unique dataset arising from surveillance of swine influenza at a Hong Kong abattoir from 1998 to 2010. We introduce a state-space model that estimates disease exposure histories by joint inference from multiple modes of surveillance, integrating both virological and serological data. We find that an observed decrease in virus isolation rates is not due to a reduction in the regional prevalence of influenza. Instead, a more likely explanation is increased infection of swine in production farms, creating greater immunity to disease early in life. Consistent with this, we find that the weekly risk of exposure on farms equals or exceeds the exposure risk during transport to slaughter. We discuss potential causes for these patterns, including competition between influenza strains and shifts in the Chinese pork industry, and suggest opportunities to improve knowledge and reduce prevalence of influenza in the region.

SUMMARY Reliable estimates of the burden of 2009 pandemic influenza A(pH1N1) cannot be easily obtained because only a small fraction of infections were confirmed by laboratory tests in a timely manner. In this study we developed a Poisson prediction modelling approach to estimate the excess mortality associated with pH1N1 in 2009 and seasonal influenza in 1998-2008 in the subtropical city Hong Kong. The results suggested that there were 127 all-cause excess deaths associated with pH1N1, including 115 with cardiovascular and respiratory disease, and 22 with pneumonia and influenza. The excess mortality rates associated with pH1N1 were highest in the population aged 65 years. The mortality burden of influenza during the whole of 2009 was comparable to those in the preceding ten inter-pandemic years. The estimates of excess deaths were more than twofold higher than the reported fatal cases with laboratory-confirmed pH1N1 infection. [ABSTRACT FROM AUTHOR]

Like [alpha][beta] T cells, human [gamma][delta] T cells also have different subsets with distinct characteristics. Whether human V[gamma]9V[delta]2 T cells have functionally different subsets in response to influenza A (fluA) viruses remains unknown. In this study, we show for the first time that both central (CD45RA(-)CD27(+)) and effector (CD45RA(-)CD27(-)) memory V[gamma]9V[delta]2 T cells have similar levels of immediate interferon (IFN) [gamma] and cytotoxic responses to human and avian fluA virus-infected cells. In contrast, CD56(+) V[gamma]9V[delta]2 T cells have significantly higher cytotoxicity against fluA virus-infected cells compared with their CD56(-) counterparts, whereas both subsets have similar IFN-[gamma] responses. We further demonstrate that the CD16-dependent degranulation pathway, but not antibody-dependent cell-mediated cytotoxicity, contribute to the superior cytotoxicity of CD56(+) V[gamma]9V[delta]2 T cells. Our study provides further evidence for the phenotypic and functional characterization of human V[gamma]9V[delta]2 T-cell subsets during fluA virus infection and may help improve the [gamma][delta] T-cell-based immunotherapy for viral infection. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian influenza H5N1 viruses cause severe disease in humans, and dysregulation of cytokine responses is believed to contribute to the pathogenesis of human H5N1 disease. However, mechanisms leading to the increased induction of proinflammatory cytokines by H5N1 viruses are poorly understood. We show that the innate sensing receptor RIG-I is involved in interferon regulatory factor 3 (IRF3), NF-κB nuclear translocation, p38 activation, and the subsequent interferon (IFN) β, IFN-λ1, and tumor necrosis factor α induction during H5N1 infection. Soluble mediators from H5N1-infected human macrophages upregulate RIG-I, MDA5, and TLR3 to much higher levels than those from seasonal H1N1 in uninfected human macrophages and alveolar epithelial cells via paracrine IFNAR1/JAK but not IFN-λ receptor signaling. Compared with H1N1 virus-induced mediators, H5N1 mediators markedly enhance the cytokine response to PolyIC and to both seasonal and H5N1 virus infection in a RIG-I-dependent manner. Thus, sensitizing neighboring cells by upregulation of RIG-I contributes to the amplified cytokine cascades during H5N1 infection. [ABSTRACT FROM AUTHOR]

The emergence of pandemic (H1N1) 2009 virus highlighted the need for enhanced surveillance of swine influenza viruses. We used real-time reverse-transcription PCR-based genotyping and found that this rapid and simple genotyping method may identify reassortants derived from viruses of Eurasian avian-like, triple reassortant-like, and pandemic (H1N1) 2009 virus lineages. [ABSTRACT FROM AUTHOR]

Myxovirus resistance A (MxA) is an antiviral protein induced by interferon alpha and beta (IFN-alpha, IFN-beta) that can inhibit viral replication. The minor alleles of the -88G>T and -123C>A MxA promoter single-nucleotide polymorphisms (SNPs) are associated with increased promoter activity and altered response to IFN-alpha and IFN-beta treatment. Here, we demonstrate that the -123A minor allele provided stronger binding affinity to nuclear proteins extracted from IFN-beta-untreated cells than did the wild-type allele, whereas the -88T allele showed preferential binding after IFN-beta stimulation. Endogenous IFN-alpha and IFN-beta induction can be suppressed in severe acute respiratory syndrome (SARS) coronavirus infection. In support of our in vitro findings, a large case-control genetic-association study for SARS coronavirus infection confirmed that the -123A minor-allele carriers were significantly associated with lower risk of SARS coronavirus infection, whereas the -88T minor-allele carriers were insignificant after adjustment for confounding effects. This suggests that -123C>A plays a more important role in modulating basal MxA expression, thus contributing more significantly to innate immune response against viral infections that suppress endogenous IFN-alpha and IFN-beta induction such as SARS coronavirus. [ABSTRACT FROM AUTHOR]

Background: Few data are available about the effectiveness of nonpharmaceutical interventions for preventing influenza virus transmission.Objective: To investigate whether hand hygiene and use of facemasks prevents household transmission of influenza.Design: Cluster randomized, controlled trial. Randomization was computer generated; allocation was concealed from treating physicians and clinics and implemented by study nurses at the time of the initial household visit. Participants and personnel administering the interventions were not blinded to group assignment. (ClinicalTrials.gov registration number: NCT00425893)Setting: Households in Hong Kong.Patients: 407 people presenting to outpatient clinics with influenza-like illness who were positive for influenza A or B virus by rapid testing (index patients) and 794 household members (contacts) in 259 households.Intervention: Lifestyle education (control) (134 households), hand hygiene (136 households), or surgical facemasks plus hand hygiene (137 households) for all household members.Measurements: Influenza virus infection in contacts, as confirmed by reverse-transcription polymerase chain reaction (RT-PCR) or diagnosed clinically after 7 days.Results: Sixty (8%) contacts in the 259 households had RT-PCR-confirmed influenza virus infection in the 7 days after intervention. Hand hygiene with or without facemasks seemed to reduce influenza transmission, but the differences compared with the control group were not significant. In 154 households in which interventions were implemented within 36 hours of symptom onset in the index patient, transmission of RT-PCR-confirmed infection seemed reduced, an effect attributable to fewer infections among participants using facemasks plus hand hygiene (adjusted odds ratio, 0.33 [95% CI, 0.13 to 0.87]). Adherence to interventions varied.Limitation: The delay from index patient symptom onset to intervention and variable adherence may have mitigated intervention effectiveness.Conclusion: Hand hygiene and facemasks seemed to prevent household transmission of influenza virus when implemented within 36 hours of index patient symptom onset. These findings suggest that nonpharmaceutical interventions are important for mitigation of pandemic and interpandemic influenza.Primary Funding Source: Centers for Disease Control and Prevention. [ABSTRACT FROM AUTHOR]

To determine the origin of influenza A virus (H5N1) epizootics in Cambodia, we used maximum-likelihood and Bayesian methods to analyze the genetic sequences of subtype H5N1 strains from Cambodia and neighboring areas. Poultry movements, rather than repeated reintroduction of subtype H5N1 viruses by wild birds, appear to explain virus circulation and perpetuation. [ABSTRACT FROM AUTHOR]

Background: Influenza virus is a cause of substantial annual morbidity and mortality worldwide. The potential emergence of a new pandemic strain (eg, avian influenza virus) is a major concern. Currently available vaccines and anti-influenza drugs have limited effectiveness for influenza virus infections, especially for new pandemic strains. Therefore, there is an acute need to develop alternative strategies for influenza therapy. gammadelta T cells have potent antiviral activities against different viruses, but no data are available concerning their antiviral activity against influenza viruses.Methods: In this study, we used virus-infected primary human monocyte-derived macrophages (MDMs) to examine the antiviral activity of phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vgamma9Vdelta2 T cells against influenza viruses.Results: Vgamma9Vdelta2 T cells were selectively activated and expanded by IPP from peripheral blood mononuclear cells. IPP-expanded Vgamma9Vdelta2 T cells efficiently killed MDMs infected with human (H1N1) or avian (H9N2 or H5N1) influenza virus and significantly inhibited viral replication. The cytotoxicity of Vgamma9Vdelta2 T cells against influenza virus-infected MDMs was dependent on NKG2D activation and was mediated by Fas-Fas ligand and perforin-granzyme B pathways.Conclusion: Our findings suggest a potentially novel therapeutic approach to seasonal, zoonotic avian, and pandemic influenza-the use of phosphoantigens to activate gammadelta T cells against influenza virus infections. [ABSTRACT FROM AUTHOR]

Severe human disease associated with influenza A H5N1 virus was first detected in Hong Kong in 1997. Its recent reemergence in Asia and high associated mortality highlight the need to understand its pathogenesis. We investigated the roles of death receptor ligands (DRLs) in H5N1 infection. Significant up-regulation of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-alpha, but not Fas ligand (FasL) mRNA, was detected in human monocyte-derived macrophages (MDMs) infected with avian influenza viruses A/Hong Kong/483/97 (H5N1/97) or its precursor, A/Quail/Hong Kong/G1/97. H5N1/97-infected MDMs exhibited the strongest induction of apoptosis in Jurkat T cells, and it could be reduced by TRAIL-receptor 2 blocking antibody. Furthermore, influenza virus infection enhanced the sensitivity of Jurkat T cells to apoptosis induced by TNF-alpha, TRAIL, and FasL. Our data suggested that functional TRAIL produced by influenza virus-infected MDMs was related to their cytotoxicity and that the enhanced sensitization to DRL-induced apoptosis detected in avian influenza may contribute to disease pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Cytokines play important roles in antiviral action. We examined whether polymorphisms of IFN-γ,TNF-α and IL-10 affect the susceptibility to and outcome of severe acute respiratory syndrome (SARS). Methods: A case-control study was carried out in 476 Chinese SARS patients and 449 healthy controls. We tested the polymorphisms of IFN-γ,TNF-α and IL-10 for their associations with SARS. Results: IFN-γ +874A allele was associated with susceptibility to SARS in a dose-dependent manner (P < 0.001). Individuals with IFN-γ +874 AA and AT genotype had a 5.19-fold (95% Confidence Interval [CI], 2.78-9.68) and 2.57-fold (95% CI, 1.35-4.88) increased risk of developing SARS respectively. The polymorphisms of IL-10 and TNF-α were not associated with SARS susceptibility. Conclusion: IFN-γ +874A allele was shown to be a risk factor in SARS susceptibility. [ABSTRACT FROM AUTHOR]

Abstract Background No country is fully prepared for a 1918-like pandemic influenza. Averting a pandemic of H5N1 influenza virus depends on the successful control of its endemicity, outbreaks in poultry and occasional spillage into human which carries a case-fatality rate of over 50%. The use of perimetric depopulation and vaccination has failed to halt the spread of the epidemic. Blanket vaccination for all poultry over a large geographical area is difficult. A combination of moratorium, segregation of water fowls from chickens and vaccination have been proved to be effective in the Hong Kong Special Administrative Region (HKSAR) since 2002 despite endemicity and outbreaks in neighbouring regions. Systematic surveillance in southern China showed that ducks and geese are the primary reservoirs which transmit the virus to chickens, minor poultry and even migratory birds. Presentation of the hypothesis We hypothesize that this combination of moratorium, poultry segregation and targeted vaccination if successfully adapted to an affected district or province in any geographical region with high endemicity would set an example for the control in other regions. Testing the hypothesis A planned one-off moratorium of 3 weeks at the hottest month of the year should decrease the environmental burden as a source of re-infection. Backyard farms will then be re-populated by hatchlings from virus-free chickens and minor poultry only. Targeted immunization of the ducks and geese present only in the industrial farms and also the chickens would be strictly implemented as blanket immunization of all backyard poultry is almost impossible. Freely grazing ducks and geese would not be allowed until neutralizing antibodies of H5 subtype virus is achieved. As a proof of concept, a simple mathematical model with susceptible-infected-recovered (SIR) structure of coupled epidemics between aquatic birds (mainly ducks and geese) and chickens was used to estimate transmissibility within and between these two poultry populations. In the field the hypothesis is tested by prospective surveillance of poultry and immunocompetent patients hospitalized for severe pneumonia for the virus before and after the institution of these measures. Implications of the Hypothesis A combination of targeted immunization with the correct vaccine, segregation of poultry species and moratorium of poultry in addition to the present surveillance, biosecurity and hygienic measures at the farm, market and personal levels could be important in the successful control of the H5N1 virus in poultry and human for an extensive geographical region with continuing outbreaks. Alternatively a lesser scale of intervention at the district level can be considered if there is virus detection without evidence of excess poultry deaths since asymptomatic shedding is common in waterfowls.