Boon Huan Tan

2009 Influenza A(H1N1) Seroconversion Rates and Risk Factors Among Distinct Adult Cohorts in Singapore

CONTEXT Singapore experienced a single epidemic wave of 2009 influenza A(H1N1) with epidemic activity starting in late June 2009 and peaking in early August before subsiding within a month. OBJECTIVE To compare the risk and factors associated with H1N1 seroconversion in different adult cohorts. DESIGN, SETTING, AND PARTICIPANTS A study with serial serological samples from 4 distinct cohorts: general population (n = 838), military personnel (n = 1213), staff from an acute care hospital (n = 558), and staff as well as residents from long-term care facilities (n = 300) from June 22, 2009, to October 15, 2009. Hemagglutination inhibition results of serum samples taken before, during, and after the epidemic and data from symptom questionnaires are presented. MAIN OUTCOME MEASURES A 4-fold or greater increase in titer between any of the 3 serological samples was defined as evidence of H1N1 seroconversion. RESULTS Baseline titers of 40 or more were observed in 22 members (2.6%; 95% confidence interval [CI], 1.7%-3.9%) of the community, 114 military personnel (9.4%; 95% CI, 7.9%-11.2%), 37 hospital staff (6.6%; 95% CI, 4.8%-9.0%), and 20 participants from long-term care facilities (6.7%; 95% CI, 4.4%-10.1%). In participants with 1 or more follow-up serum samples, 312 military personnel (29.4%; 95% CI, 26.8%-32.2%) seroconverted compared with 98 community members (13.5%; 95% CI, 11.2%-16.2%), 35 hospital staff (6.5%; 95% CI, 4.7%-8.9%), and only 3 long-term care participants (1.2%; 95% CI, 0.4%-3.5%). Increased frequency of seroconversion was observed for community participants from households in which 1 other member seroconverted (adjusted odds ratio [OR], 3.32; 95% CI, 1.50-7.33), whereas older age was associated with reduced odds of seroconversion (adjusted OR, 0.77 per 10 years; 95% CI, 0.64-0.93). Higher baseline titers were associated with decreased frequency of seroconversion in community (adjusted OR for every doubling of baseline titer, 0.48; 95% CI, 0.27-0.85), military (adjusted OR, 0.71; 95% CI, 0.61-0.81), and hospital staff cohorts (adjusted OR, 0.50; 95% CI, 0.26-0.93). CONCLUSION Following the June-September 2009 wave of 2009 influenza A(H1N1), 13% of the community participants seroconverted, and most of the adult population likely remained susceptible.

Oseltamivir ring prophylaxis for containment of 2009 H1N1 influenza outbreaks.

BACKGROUND From June 22 through June 25, 2009, four outbreaks of infection with the pandemic influenza A (H1N1) virus occurred in Singapore military camps. We report the efficacy of ring chemoprophylaxis (geographically targeted containment by means of prophylaxis) with oseltamivir to control outbreaks of 2009 H1N1 influenza in semiclosed environments. METHODS All personnel with suspected infection were tested and clinically isolated if infection was confirmed. In addition, we administered postexposure ring chemoprophylaxis with oseltamivir and segregated the affected military units to contain the spread of the virus. All personnel were screened three times weekly both for virologic infection, by means of nasopharyngeal swabs and reverse-transcriptase-polymerase-chain-reaction assay with sequencing, and for clinical symptoms, by means of questionnaires. RESULTS A total of 1175 personnel were at risk across the four sites, with 1100 receiving oseltamivir prophylaxis. A total of 75 personnel (6.4%) were infected before the intervention, and 7 (0.6%) after the intervention. There was a significant reduction in the overall reproductive number (the number of new cases attributable to the index case), from 1.91 (95% credible interval, 1.50 to 2.36) before the intervention to 0.11 (95% credible interval, 0.05 to 0.20) after the intervention. Three of the four outbreaks showed a significant reduction in the rate of infection after the intervention. Molecular analysis revealed that all four outbreaks were derived from the New York lineage of the 2009 H1N1 virus and that cases within each outbreak were due to transmission rather than unrelated episodes of infection. Of the 816 personnel treated with oseltamivir who were surveyed, 63 (7.7%) reported mild, nonrespiratory side effects of the drug, with no severe adverse events. CONCLUSIONS Oseltamivir ring chemoprophylaxis, together with prompt identification and isolation of infected personnel, was effective in reducing the impact of outbreaks of 2009 H1N1 influenza in semiclosed settings.

Protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein 90 in virus particle assembly

In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation. Fluorescence microscopy of virus-infected cells revealed the presence of virus-induced cytoplasmic inclusion bodies and mature virus particles, the latter appearing as virus filaments. In situ electron tomography suggested that the virus filaments were complex structures that were able to package multiple copies of the virus genome. The virus particles were purified, and the protein content was analyzed by one-dimensional nano-LC MS/MS. In addition to all the major virus structural proteins, 25 cellular proteins were also detected, including proteins associated with the cortical actin network, energy pathways, and heat shock proteins (HSP70, HSC70, and HSP90). Representative actin-associated proteins, HSC70, and HSP90 were selected for further biological validation. The presence of β-actin, filamin-1, cofilin-1, HSC70, and HSP90 in the virus preparation was confirmed by immunoblotting using relevant antibodies. Immunofluorescence microscopy of infected cells stained with antibodies against relevant virus and cellular proteins confirmed the presence of these cellular proteins in the virus filaments and inclusion bodies. The relevance of HSP90 to virus infection was examined using the specific inhibitors 17-N-Allylamino-17-demethoxygeldanamycin. Although virus protein expression was largely unaffected by these drugs, we noted that the formation of virus particles was inhibited, and virus transmission was impaired, suggesting an important role for HSP90 in virus maturation. This study highlights the utility of proteomics in facilitating both our understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy.

Outbreak of Febrile Respiratory Illness Associated with Adenovirus 11a Infection in a Singapore Military Training Camp

ABSTRACT Outbreak cases of acute respiratory disease (ARD) associated with subspecies B2 human adenovirus 11a (HAdV-11a) infection were detected during 2005 in a military basic training camp in Singapore. The Singapore HAdV-11a strain is highly similar to other Asian strains of HAdV-11, including strain QS-DLL, which is responsible for the recently described 2006 outbreak of ARD in China.

Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses.

The host response to the low pathogenic avian influenza (LPAI) H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN) expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG) expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture.

Serological Response in RT-PCR Confirmed H1N1-2009 Influenza A by Hemagglutination Inhibition and Virus Neutralization Assays: An Observational Study

Background We describe the serological response following H1N1-2009 influenza A infections confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Methodology and Principal Findings The study included patients admitted to hospital, subjects of a seroepidemiologic cohort study, and participants identified from outbreak studies in Singapore. Baseline (first available blood sample) and follow-up blood samples were analyzed for antibody titers to H1N1-2009 and recently circulating seasonal influenza A virus strains by hemagglutination inhibition (HI) and virus micro-neutralization (VM) assays. 267 samples from 118 cases of H1N1-2009 were analyzed. Geometric mean titers by HI peaked at 123 (95% confidence interval, CI 43-356) between days 30 to 39. The chance of observing seroconversion (four-fold or greater increase of antibodies) was maximized when restricting analysis to 45 participants with baseline sera collected within 5 days of onset and follow-up sera collected 15 or more days after onset; for these participants, 82% and 89% seroconverted to A/California/7/2009 H1N1 by HI and VM respectively. A four-fold or greater increase in cross-reactive antibody titers to seasonal A/Brisbane/59/2007 H1N1, A/Brisbane/10/2007 H3N2 and A/Wisconsin/15/2009 H3N2 occurred in 20%, 18% and 16% of participants respectively. Conclusions and Significance Appropriately timed paired serology detects 80–90% RT-PCR confirmed H1N1-2009; Antibodies from infection with H1N1-2009 cross-reacted with seasonal influenza viruses.

Human metapneumovirus in children, Singapore.

Four hundred specimens were collected from pediatric patients hospitalized in Singapore; 21 of these specimens tested positive for human metapneumovirus (HMPV), with the A2 genotype predominating. A 5% infection rate was estimated, suggesting that HMPV is a significant cause of morbidity among the pediatric population of Singapore.

Evidence that selective changes in the lipid composition of raft-membranes occur during respiratory syncytial virus infection.

We examined the structure of lipid-raft membranes in respiratory syncytial virus infected cells. Cholesterol depletion studies using methyl-beta-cyclodextrin suggested that membrane cholesterol was required for virus filament formation, but not inclusion bodies. In addition, virus filament formation coincided with elevated 3-hydroxy-3-methylglutaryl-coenzyme A reductase expression, suggesting an increase in requirement for endogenous cholesterol synthesis during virus assembly. Lipid raft membranes were examined by mass spectrometry, which suggested that virus infection induced subtle changes in the lipid composition of these membrane structures. This analysis revealed increased levels of raft-associated phosphatidylinositol (PI) and phosphorylated PI during RSV infection, which correlated with the appearance of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate (PIP(3)) within virus inclusion bodies, and inhibiting the synthesis of PIP(3) impaired the formation of progeny virus. Collectively, our analysis suggests that RSV infection induces specific changes in the composition of raft-associated lipids, and that these changes play an important role in virus maturation.

A systems-based approach to analyse the host response in murine lung macrophages challenged with respiratory syncytial virus

BackgroundRespiratory syncytial virus (RSV) is an important cause of lower respiratory tract infection in young children. The degree of disease severity is determined by the host response to infection. Lung macrophages play an important early role in the host response to infection and we have used a systems-based approach to examine the host response in RSV-infected lung-derived macrophage cells.ResultsLung macrophage cells could be efficiently infected (>95%) with RSV in vitro, and the expression of several virus structural proteins could be detected. Although we failed to detect significant levels of virus particle production, virus antigen could be detected up until 96 hours post-infection (hpi). Microarray analysis indicated that 20,086 annotated genes were expressed in the macrophage cells, and RSV infection induced an 8.9% and 11.3% change in the global gene transcriptome at 4 hpi and 24 hpi respectively. Genes showing up-regulated expression were more numerous and exhibited higher changes in expression compared to genes showing down-regulated expression. Based on gene ontology, genes with cytokine, antiviral, cell death, and signal transduction functions showed the highest increases in expression, while signalling transduction, RNA binding and protein kinase genes showed the greatest reduction in expression levels. Analysis of the global gene expression profile using pathway enrichment analysis confirmed that up-regulated expression of pathways related to pathogen recognition, interferon signalling and antigen presentation occurred in the lung macrophage cells challenged with RSV.ConclusionOur data provided a comprehensive analysis of RSV-induced gene expression changes in lung macrophages. Although virus gene expression was detected, our data was consistent with an abortive infection and this correlated with the activation of several antivirus signalling pathways such as interferon type I signalling and cell death signalling. RSV infection induced a relatively large increase in pro-inflammatory cytokine expression, however the maintenance of this pro-inflammatory response was not dependent on the production of infectious virus particles. The sustained pro-inflammatory response even in the absence of a productive infection suggests that drugs that control the pro-inflammatory response may be useful in the treatment of patients with severe RSV infection.

Risk factors for pandemic (H1N1) 2009 virus seroconversion among hospital staff, Singapore.

TOC Summary: Infection was associated with occupational and nonoccupational risk factors.