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Complete sequence analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in Singapore (2000)

Singh S, Poh CL, Chow VT

Microbiol. Immunol., 2002;46(11):801-8.
PMID: 12516778

Enterovirus 71 (EV71) is a major aetiological agent of hand, foot and mouth disease (HFMD). In recent years, several outbreaks in East Asia were associated with neurological complications and numerous deaths. An outbreak in Singapore in October 2000 afflicted thousands of children, resulting in four fatal cases from three of whom EV71 was isolated. The genomes of two representative EV71 strains isolated from a fatal case and a surviving patient were completely sequenced, and their nucleotide and amino acid sequences compared with known EV71 strains. The two outbreak strains were classified under genogroup B, together with those previously isolated in Singapore, Malaysia and Japan. Comparative sequence analysis of the two Singapore strains revealed 99% nucleotide similarity, while their deduced amino acid sequences were almost identical except for residue 1506 in the 3A non-structural region. Given that the outbreak involved closely related genetic variants of EV71, the broad spectrum of disease severity may be attributed to critical factors such as varying viral inoculation doses or differing host immune responses following infection, but is less likely to be due to the emergence of EV71 strains with heightened virulence.
Comparative analysis of NS3 sequences of temporally separated dengue 3 virus strains isolated from southeast Asia

Chow VT, Seah CL, Chan YC

Intervirology, 1994;37(5):252-8.
PMID: 7698880

By a combination of PCR and direct-cycle sequencing using consensus primers, we analyzed approximately 400-bp fragments within the NS3 genes of twenty-one dengue virus type 3 strains isolated from five neighboring Southeast Asian countries at different time intervals from 1956 to 1992. The majority of base disparities were silent mutations, with few predicted amino acid substitutions, thus emphasizing the strict conservation of the NS3 gene. Phylogenetic trees constructed on the basis of these nucleotide differences revealed distinct but related clusters of strains from the Philippines, Indonesia, and strains from Singapore and Malaysia of the 1970s and early 1980s, while the Thai cluster was relatively more distant. This genetic relationship was compatible with that proposed by other workers who have studied other dengue 3 virus genes such as E, M and prM. However, we observed that the more recent, epidemic-associated dengue 3 strains from Singapore and Malaysia of the late 1980s and early 1990s were more closely related to the Thai cluster, implying their evolution from the latter, and emphasizing the importance of viral spread via increasing travel within the Southeast Asian area and elsewhere. Nucleotide sequence analysis of the NS3 genes of dengue viruses can serve to advance the understanding of the epidemiology and evolution of these viruses.
Diagnosis of nipah virus encephalitis by electron microscopy of cerebrospinal fluid

Chow VT, Tambyah PA, Yeo WM, Phoon MC, Howe J

J Clin Virol, 2000 Dec;19(3):143-7.
PMID: 11090749

BACKGROUND: between 1998 and 1999, an outbreak of potentially fatal viral encephalitis erupted among pig farm workers in West Malaysia, and later spread to Singapore where abattoir workers were afflicted. Although Japanese encephalitis virus was initially suspected, the predominant aetiologic agent was subsequently confirmed to be Nipah virus, a novel paramyxovirus related to but distinct from Hendra virus.
OBJECTIVE: to describe a case of Nipah virus encephalitis in a pig farm worker from Malaysia.
STUDY DESIGN: the clinical, laboratory and radiological findings of this patient were scrutinized. Special emphasis was placed on the electron microscopic analysis of the cerebrospinal fluid (CSF) specimen from this patient.
RESULTS: the neurological deficits indicative of cerebellar involvement were supported by the magnetic resonance imaging that showed prominent cerebellar and brainstem lesions. CSF examination provided further evidence of viral encephalitis. Complement fixation and/or RT-PCR assays were negative for Japanese encephalitis, herpes simplex, measles and mumps viruses. ELISA for detecting IgM and IgG antibodies against Hendra viral antigens were equivocal for the CSF specimen, and tested initially negative for the first serum sample but subsequently positive for the repeat serum sample. Transmission electron microscopy of negatively-stained preparations of CSF revealed enveloped virus-like structures fringed with surface projections as well as nucleocapsids with distinctive helical and herringbone patterns, features consistent with those of other paramyxoviruses, including Hendra virus.
CONCLUSION: this case report reiterates the relevant and feasible role of diagnostic electron microscopy for identifying and/or classifying novel or emerging viral pathogens for which sufficiently specific and sensitive tests are lacking.
RT-PCR, nucleotide, amino acid and phylogenetic analyses of enterovirus type 71 strains from Asia

Singh S, Chow VT, Chan KP, Ling AE, Poh CL

J Virol Methods, 2000 Aug;88(2):193-204.
PMID: 10960707

A specific and sensitive method based on RT-PCR was developed to detect enterovirus 71 (EV71) from patients with hand, foot and mouth disease, myocarditis, aseptic meningitis and acute flaccid paralysis. RT-PCR primers from conserved parts of the VP1 capsid gene were designed on the basis of good correlation with sequences of EV71 strains. These primers successfully amplified 44 strains of EV71 including 34 strains isolated from Singapore in 1997 and 1998, eight strains from Malaysia isolated in 1997 and 1998, one Japanese strain and the neurovirulent strain EV71/7423/MS/87. RT-PCR of 30 strains of other enteroviruses including coxsackievirus A and B, and echoviruses failed to give any positive amplicons. Hence, RT-PCR with these primers showed 100% correlation with serotyping. Direct sequencing of the RT-PCR products of 20 EV71 strains revealed a distinct cluster with two major subgroups, thus enabling genetic typing of the viruses. The genetic heterogeneity of these strains culminated in amino acid substitutions within the VP1, VP2 and VP3 regions. The sequencing of a 2.9 kb fragment comprising the capsid region and the major part of 5' UTR of two Singapore strains revealed that they belonged to a group distinct from the prototype EV71/BrCr strain and the EV71/7423/MS/87 strain. The dendrogram generated from 341 bp fragments within the VP1 region revealed that the strains of Singapore, Malaysia and Taiwan belong to two entirely different EV71 genogroups, distinct from the three genogroups identified in another recent study.
Genital human papillomavirus infection among women from major ethnic groups in Singapore

Chow VT, Tham KM, Lim-Tan SK, Sng IT, Bernard HU

Asia Oceania J Obstet Gynaecol, 1990 Dec;16(4):373-7.
PMID: 1966000

The close epidemiological relationships between specific genital human papillomavirus (HPV) types and neoplasia of the cervix uteri have been extensively documented worldwide, including Singapore. Cervical cancer incidence rates in Singapore show variations between the major ethnic groups. To ascertain the corresponding HPV infection rates among the various races in Singapore, we analysed the cervical smears of 225 women by filter in situ DNA hybridization, and compared the data with a previous similar study. Fourteen (6.2%) individuals were HPV-positive, with HPV 16 and HPV 31 being the commonest types. No significant difference between HPV positivity rates in Chinese (5.0%) and in Malays (6.7%) was found, even though Chinese have a higher cervical cancer incidence than Malays. Furthermore, the cervical HPV carriage rate among women with normal cytology was 5.9%. In the light of reports of high genital HPV prevalence rates detected by DNA amplification, these data support the notion that HPV infection is commonly latent and requires the cooperation of other factors for cervical carcinogenesis.
Chlamydia pneumoniae IgG seropositivity and clinical history of ischemic heart disease in Singapore

Koh WP, Taylor MB, Chew SK, Phoon MC, Kang KL, Chow VT

J Microbiol Immunol Infect, 2003 Sep;36(3):169-74.
PMID: 14582560

There is still substantial uncertainty concerning the association between Chlamydia pneumoniae and ischemic heart disease. This may partly be explained by the adjustment for potential confounders in different population studies. This is the first study in Singapore to look at the association of C. pneumoniae seropositivity with ischemic heart disease in a multivariate analysis. A random sample of 714 persons aged between 35 and 69 years was selected from the participants of the Singapore National Health Survey conducted in 1998. Data on clinical measurements and conditions were collected using biochemical tests and interviewer-based questionnaires. Ischemic heart disease was defined by the Rose questionnaire and included history suggestive of angina and/or myocardial infarction. Immunoglobulin G antibodies for C. pneumoniae were detected using an indirect microimmunofluorescence test, and seropositivity was defined as IgG titers > or = 1:16. There were no statistically significant differences in the prevalence rates of seropositivity to C. pneumoniae among the three ethnic groups, that is, Chinese (80.4%), Malays (74.0%), and Asian Indians (73.2%). There was no association between seropositivity and ischemic heart disease after adjustment for age alone (OR 1.00, 95% CI 0.54-1.83) or for age, sex, and other risk factors of atherosclerosis (OR 0.99, 95% CI 0.53-1.84). C. pneumoniae Immunoglobulin G seropositivity was not associated with an increased risk of ischemic heart disease as defined by the Rose angina questionnaire in Singapore.
Seroprevalence of IgG antibodies against Chlamydia pneumoniae in Chinese, Malays and Asian Indians in Singapore

Koh WP, Taylor MB, Hughes K, Chew SK, Fong CW, Phoon MC, et al.

Int J Epidemiol, 2002 Oct;31(5):1001-7.
PMID: 12435775 DOI: 10.1093/ije/31.5.1001

BACKGROUND: Chlamydia pneumoniae, a bacterium that causes respiratory infections, is probably under-diagnosed. There is also interest in its possible role in the aetiology of coronary heart disease. This is the first population-based seroprevalence survey of C. pneumoniae infection in Singapore.

METHODS: A random sample of 1,068 people aged 18-69 years was selected from the participants of the Singapore National Health Survey conducted in 1998. Sera and data on certain clinical measurements and conditions had been collected. IgG antibodies for C. pneumoniae were detected using an indirect microimmunofluorescence test and positivity graded. Seropositivity was defined as IgG titre >/=1:16.

RESULTS: There were no statistically significant differences in the prevalence rates of seropositivity to C. pneumoniae for age group 18-69 years among the three ethnic groups, i.e. Chinese (males 76.7%, females 68.3%), Malays (males 75.4%, females 59.1%), and Asian Indians (males 74.6%, females 59.4%). The seropositivity rate for people aged 18-69 years in Singapore was 75.0% for males and 65.5% for females (difference of 9.5%, P < 0.001). In both genders combined, seropositivity increased from 46.5% in the age group 18-29 to reach a plateau of 78.9% in the age group 40-49, which remained stable to 60-69 years. There was no association of seropositivity with smoking, diabetes mellitus, hypertension or body mass index after adjustment for age and gender.

CONCLUSION: The high prevalence rates in our study population and the higher rate in males compared to females are consistent with studies from other parts of the world. No significant difference in prevalence rates was observed among Chinese, Malays and Indians. The pattern of rising and levelling off of seropositivity with age suggests that C. pneumoniae infection occurs early in life, and in older ages the high level of seropositivity is probably maintained by re-infections or chronic infections. Chlamydia pneumoniae infection was not found to be associated with the cardiovascular risk factors examined.
Fulltext Comparative Transcriptomic and Molecular Pathway Analyses of HL-CZ Human Pro-Monocytic Cells Expressing SARS-CoV-2 Spike S1, S2, NP, NSP15 and NSP16 Genes

Sharma A, Ong JW, Loke MF, Chua EG, Lee JJ, Choi HW, et al.

Microorganisms, 2021 May 31;9(6).
PMID: 34073047 DOI: 10.3390/microorganisms9061193

The ongoing COVID-19 pandemic is a clear and present threat to global public health. Research into how the causative SARS-CoV-2 virus together with its individual constituent genes and proteins interact with target host cells can facilitate the development of improved strategies to manage the acute and long-term complications of COVID-19. In this study, to better understand the biological roles of critical SARS-CoV-2 proteins, we determined and compared the host transcriptomic responses of the HL-CZ human pro-monocytic cell line upon transfection with key viral genes encoding the spike S1 subunit, S2 subunit, nucleocapsid protein (NP), NSP15 (endoribonuclease), and NSP16 (2'-O-ribose-methyltransferase). RNA sequencing followed by gene set enrichment analysis and other bioinformatics tools revealed that host genes associated with topologically incorrect protein, virus receptor activity, heat shock protein binding, endoplasmic reticulum stress, antigen processing and presentation were up-regulated in the presence of viral spike S1 expression. With spike S2 expression, pro-monocytic genes associated with the interferon-gamma-mediated signaling pathway, regulation of phosphatidylinositol 3-kinase activity, adipocytokine signaling pathway, and insulin signaling pathway were down-regulated, whereas those associated with cytokine-mediated signaling were up-regulated. The expression of NSP15 induced the up-regulation of genes associated with neutrophil degranulation, neutrophil-mediated immunity, oxidative phosphorylation, prion disease, and pathways of neurodegeneration. The expression of NSP16 resulted in the down-regulation of genes associated with S-adenosylmethionine-dependent methyltransferase activity. The expression of NP down-regulated genes associated with positive regulation of neurogenesis, nervous system development, and heart development. Taken together, the complex transcriptomic alterations arising from these viral-host gene interactions offer useful insights into host genes and their pathways that potentially contribute to SARS-CoV-2 pathogenesis.