Genetic characterisation of influenza C viruses detected in Singapore in 2006.

In an earlier study on respiratory infections in Singapore military recruits, four influenza C virus (FLUCV) infections were detected out of the 1354 samples collected. All four isolates were detected in 2006, and their whole genome was completely sequenced and analysed. Phylogenetic analysis of the hemagglutinin esterase fusion (HEF) gene revealed that all four Singapore isolates belonged to the C/Japan-Kanagawa/1/76-related lineage. However, the genes of the four FLUCV isolates had origins from several different lineages, and the genome composition resembles that of the C/Japan-Miyagi/9/96-like strains that had been circulating in Japan between 1996 and 2000.

Surveillance and clinical characterization of influenza in a university cohort in Singapore.

BACKGROUND: Southeast Asia is a potential locus for the emergence of novel influenza strains. However, information on influenza within the region is limited. OBJECTIVES: This study was to determine the proportion of influenza-like illness (ILI) caused by influenza A and B viruses in a university cohort in Singapore, identify important distinctive clinical features of influenza infection and potential factors associated with influenza infection compared with other causes of ILI. METHODOLOGY: A surveillance study was conducted from 2007 to 2009, at the University Health and Wellness Centre, National University of Singapore (NUS). Basic demographic information and nasopharyngeal swabs were collected from consenting students and staff with ILI, with Influenza A and B identified by both culture and molecular methods. RESULTS: Proportions of influenza A and B virus infections in subjects with ILI were 153/500 (30.6%) and 11/500 (2.2%) respectively. The predominant subtype was A/H1N1, including both the seasonal strain (20/153) and the pandemic strain (72/153). The clinical symptom of fever was more common in subjects with laboratory confirmed influenza than other ILIs. On-campus hostel residence and being a student (compared with staff) were associated with increased risk of laboratory confirmed influenza A/H1N1 2009 infection. CONCLUSIONS: This study provides a baseline prevalence of influenza infection within young adults in Singapore in a university setting. Potential risk factors, such as hostel residence, were identified, allowing for more targeted infection control measures in the event of a future influenza pandemic.

Prospective surveillance and molecular characterization of seasonal influenza in a university cohort in Singapore.

BACKGROUND: Southeast Asia is believed to be a potential locus for the emergence of novel influenza strains, and therefore accurate sentinel surveillance in the region is critical. Limited information exists on sentinel surveillance of influenza-like illness (ILI) in young adults in Singapore in a University campus setting. The objective of the present study was to determine the proportion of ILI caused by influenza A and B viruses in a university cohort in Singapore. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a prospective surveillance study from May through October 2007, at the National University of Singapore (NUS). Basic demographic information and nasopharyngeal swabs were collected from students and staff with ILI. Reverse-transcriptase PCR (RT-PCR) and viral isolation were employed to detect influenza viruses. Sequencing of hemagglutinin (HA) and neuraminidase (NA) genes of some representative isolates was also performed. Overall proportions of influenza A and B virus infections were 47/266 (18%) and 9/266 (3%) respectively. The predominant subtype was A/H3N2 (55%) and the rest were A/H1N1 (45%). The overall sensitivity difference for detection of influenza A viruses using RT-PCR and viral isolation was 53%. Phylogenetic analyses of HA and NA gene sequences of Singapore strains showed identities higher than 98% within both the genes. The strains were more similar to strains included in the WHO vaccine recommendation for the following year (2008). Genetic markers of oseltamivir resistance were not detected in any of the sequenced Singapore isolates. CONCLUSIONS/SIGNIFICANCE: HA and NA gene sequences of Singapore strains were similar to vaccine strains for the upcoming influenza season. No drug resistance was found. Sentinel surveillance on university campuses should make use of molecular methods to better detect emerging and re-emerging influenza viral threats.

A highly divergent Encephalomyocarditis virus isolated from nonhuman primates in Singapore.

BACKGROUND: In 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. METHODS: Of 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. RESULTS: Electron 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. CONCLUSIONS: The 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.

Viral agents responsible for febrile respiratory illnesses among military recruits training in tropical Singapore.

BACKGROUND: Military personnel are highly susceptible to febrile respiratory illnesses (FRI), likely due to crowding, stress and other risk factors present in the military environment. OBJECTIVE: Our objective was to investigate the viral etiological agents responsible for FRI among military recruits training in a tropical climate in Singapore. STUDY DESIGN: From March 2006 through April 2007, a total of 1354 oropharyngeal (throat) swabs were collected from military recruits who reported sick with an oral temperature of > or =38 degrees C and a cough and/or sore throat. Real-time polymerase chain reaction (PCR) was used to assay for the presence of influenza A and B viruses and adenoviruses (H-AdV), and conventional PCR used for the remaining respiratory viruses in all specimens. RESULTS: Influenza A virus was the dominant infection with a laboratory-confirmed incidence of 24% (326/1354) and a predominance of the H3N2 subtype. The temporal pattern for influenza A virus infections coincided with the nation-wide pattern in the civilian community. Detection rates of 12% (159/1354) and 2.7% (5/1354) were obtained for influenza B virus and other respiratory viruses, respectively. CONCLUSIONS: 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.

Molecular characterization of low pathogenic avian influenza viruses, isolated from food products imported into Singapore.

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.

The incidence of human bocavirus infection among children admitted to hospital in Singapore.

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.

Detection of porcine circovirus type 2 in pigs imported from Indonesia.

We have detected the presence of porcine circovirus (PCV) type 2 in Indonesian pigs imported to Singapore for food consumption. A total of three viral isolates were identified, and to genetically characterise them further, their full genomes were sequenced. Each genome showed a typical organization of PCV type 2, with the three isolates sharing similar genome lengths of 1767 nucleotide (nt) at high nt identities of 99.8-100%, further indicating that the viral isolates were quite homogeneous. Sequence analysis further revealed that the ORF2 genes contain the nt sequence CCCCGC (from nt position 262 to 267) that was previously reported to be associated with PCV type 2, group 1C. The phylogenetic tree was constructed for the ORF2 genes, and the PCV type 2 isolates distributed into two distinctive groups. The Indonesian PCV type 2 clustered tightly with one China isolate, accession number AY035820, as a sub-cluster in group 1C. The sequence and phylogenetic analyses both confirmed that the three Indonesian PCV type 2 isolates belong to group 1C, and that the genetic changes for the three Indonesian isolates were very stable, possibly due to the low-scale evolution.

Diagnostic value of real-time capillary thermal cycler in virus detection.

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.