The laboratory findings identified influenza A virus as the primary causative viral agent for FRI in the Singapore military, in strong contrast to findings from temperate countries and countries where recruits are often vaccinated for influenza. Our results suggest that influenza vaccination should be considered as a requirement to reduce the incidence of influenza infections. This is the first report describing respiratory infections in a tropical military setting, in a developed country in Asia.

BackgroundIn 2001 and 2002, fatal myocarditis resulted in the sudden deaths of four, two adult and two juvenile, orang utans out of a cohort of 26 in the Singapore Zoological Gardens.MethodsOf the four orang utans that underwent post-mortem examination, virus isolation was performed from the tissue homogenates of the heart and lung obtained from the two juvenile orang utans in Vero cell cultures. The tissue culture fluid was examined using electron microscopy. Reverse transcription and polymerase chain reaction with Encephalomyocarditis virus (EMCV)-specific primers targeting the gene regions of VP3/VP1 and 3D polymerase (3Dpol) confirmed the virus genus and species. The two EMCV isolates were sequenced and phylogenetic analyses of the virus genes performed. Serological testing on other animal species in the Singapore Zoological Gardens was also conducted.ResultsElectron microscopy of the two EMCV isolates, designated Sing-M100-02 and Sing-M105-02, revealed spherical viral particles of about 20 to 30 nm, consistent with the size and morphology of members belonging to the family Picornaviridae. In addition, infected-Vero cells showed positive immunoflorescence staining with antiserum to EMCV. Sequencing of the viral genome showed that the two EMCV isolates were 99.9% identical at the nucleotide level, indicating a similar source of origin. When compared with existing EMCV sequences in the VP1 and 3Dpol gene regions, the nucleotide divergence were at a maximum of 38.8% and 23.6% respectively, while the amino acid divergence were at a maximum of 33.9% and 11.3% respectively. Phylogenetic analyses of VP1 and 3Dpol genes further grouped the Sing-M100-02 and Sing-M105-02 isolates to themselves, away from existing EMCV lineages. This strongly suggested that Sing-M100-02 and Sing-M105-02 isolates are highly divergent variants of EMCV. Apart from the two deceased orang utans, a serological survey conducted among other zoo animals showed that a number of other animal species had neutralizing antibodies to Sing-M105-02 isolate, indicating that the EMCV variant has a relatively wide host range.ConclusionsThe etiological agent responsible for the fatal myocarditis cases among two of the four orang utans in the Singapore Zoological Gardens was a highly divergent variant of EMCV. This is the first report of an EMCV infection in Singapore and South East Asia.

IntroductionInfluenza infections present with wide-ranging clinical features. We aim to compare the differences in presentation between influenza and non-influenza cases among those with febrile respiratory illness (FRI) to determine predictors of influenza infection.MethodsPersonnel with FRI (defined as fever≥37.5°C, with cough or sore throat) were recruited from the sentinel surveillance system in the Singapore military. Nasal washes were collected, and tested using the Resplex II and additional PCR assays for etiological determination. Interviewer-administered questionnaires collected information on patient demographics and clinical features. Univariate comparison of the various parameters was conducted, with statistically significant parameters entered into a multivariate logistic regression model. The final multivariate model for influenza versus non-influenza cases was used to build a predictive probability clinical diagnostic model.Results821 out of 2858 subjects recruited from 11 May 2009 to 25 Jun 2010 had influenza, of which 434 (52.9%) had 2009 influenza A (H1N1), 58 (7.1%) seasonal influenza A (H3N2) and 269 (32.8%) influenza B. Influenza-positive cases were significantly more likely to present with running nose, chills and rigors, ocular symptoms and higher temperature, and less likely with sore throat, photophobia, injected pharynx, and nausea/vomiting. Our clinical diagnostic model had a sensitivity of 65% (95% CI: 58%, 72%), specificity of 69% (95% CI: 62%, 75%), and overall accuracy of 68% (95% CI: 64%, 71%), performing significantly better than conventional influenza-like illness (ILI) criteria.ConclusionsUse of a clinical diagnostic model may help predict influenza better than the conventional ILI definition among young adults with FRI.

Human bocavirus (HBoV) is a parvovirus, belonging to the genus Bocavirus. The virus was identified recently in Sweden, and has now been detected in several different countries. Although it is associated with lower respiratory tract infections in pediatric patients, the incidence of HBoV infection in a developed country in South East Asia, has not been examined. The objective of this study was to determine the importance of HBoV as a cause of lower respiratory tract infections among children admitted to hospital in Singapore. Five hundred nasopharyngeal swabs were collected from anonymized pediatric patients admitted to the Kandang Kerbau Women's and Children's Hospital for acute respiratory infections. The specimens were tested for the presence of HBoV using polymerase chain reactions. HBoV was detected in 8.0% of the patients tested, and a majority of these HBoV patients exhibited lower respiratory tract infections. A significant level of coinfection with respiratory syncytial viruses and rhinoviruses was also observed in these HBoV patients. The data suggest that HBoV is an important cause of lower respiratory tract infections among children admitted to hospital in Singapore, and is the first study examining the incidence of HBoV infection in a developed country in South East Asia. J. Med. Virol. 81:82–89, 2009. © 2008 Wiley‐Liss, Inc.

An outbreak of acute hemorrhagic conjunctivitis (AHC) was reported in Singapore military camps in the year 2005. A total of 103 conjunctival swab specimens were collected from military personnel diagnosed clinically with AHC. PCR testing on these conjunctival specimens revealed the presence of an enterovirus, and this was confirmed by virus isolation. Molecular typing using a partial VP1 gene confirmed a variant of coxsackievirus A24 (CA24v) as the most likely etiological agent for the outbreak. Full-length genome sequencing was carried out on 2 selected virus strains, DSO-26SIN05 and DSO-52SIN05. Sequence comparison and phylogenetic analyses of the VP4, VP1 and 3Cpro gene regions were performed, clustering the Singapore CA24v strains with viruses originating from Asia in the post-2000 era. In addition, we report evolution rates of 4.2 x 10(-3) and 1.0 x 10(-3) nucleotide/year, respectively, for the VP4 capsid and 3Cpro gene regions. Our result shows a focal evolutionary point around 1965-1966, suggesting that the CA24v virus has been evolving constantly since its emergence in Singapore, nearly 40 years ago.

We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia.

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Molecular-based detection methods such as PCR techniques have had a significant impact on the diagnosis of viral infections because of their superior sensitivity and rapid turnaround time. This review describes the use of real-time PCR on the capillary thermal cycler, the Roche LightCycler trade mark, for early disease detection in diagnostic virology. The advantages of using the LightCycler, the detection processes using SYBR Green I and different hybridization strategies will be discussed in detail, with specific examples drawn from our in-house viral assays. The use of the LightCycler for the investigation of two recent viral outbreaks in Singapore will also be briefly described.