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Immunohistochemical Detection of Chikungunya Virus Antigens in Formalin-Fixed and Paraffin-Embedded Tissues

Chiam CW, Sam IC, Chan YF, Wong KT, Ong KC

Methods Mol Biol, 2016;1426:235-40.
PMID: 27233276 DOI: 10.1007/978-1-4939-3618-2_21

Immunohistochemistry is a histological technique that allows detection of one or more proteins of interest within a cell using specific antibody binding, followed by microscopic visualization of a chromogenic substrate catalyzed by peroxidase and/or alkaline phosphatase. Here, we describe a method to localize Chikungunya virus (CHIKV) antigens in formalin-fixed and paraffin-embedded infected mouse brain.
Fulltext Comparative Genetic Analyses of Human Rhinovirus C (HRV-C) Complete Genome from Malaysia

Khaw YS, Chan YF, Jafar FL, Othman N, Chee HY

Front Microbiol, 2016;7:543.
PMID: 27199901 DOI: 10.3389/fmicb.2016.00543

Human rhinovirus-C (HRV-C) has been implicated in more severe illnesses than HRV-A and HRV-B, however, the limited number of HRV-C complete genomes (complete 5' and 3' non-coding region and open reading frame sequences) has hindered the in-depth genetic study of this virus. This study aimed to sequence seven complete HRV-C genomes from Malaysia and compare their genetic characteristics with the 18 published HRV-Cs. Seven Malaysian HRV-C complete genomes were obtained with newly redesigned primers. The seven genomes were classified as HRV-C6, C12, C22, C23, C26, C42, and pat16 based on the VP4/VP2 and VP1 pairwise distance threshold classification. Five of the seven Malaysian isolates, namely, 3430-MY-10/C22, 8713-MY-10/C23, 8097-MY-11/C26, 1570-MY-10/C42, and 7383-MY-10/pat16 are the first newly sequenced complete HRV-C genomes. All seven Malaysian isolates genomes displayed nucleotide similarity of 63-81% among themselves and 63-96% with other HRV-Cs. Malaysian HRV-Cs had similar putative immunogenic sites, putative receptor utilization and potential antiviral sites as other HRV-Cs. The genomic features of Malaysian isolates were similar to those of other HRV-Cs. Negative selections were frequently detected in HRV-Cs complete coding sequences indicating that these sequences were under functional constraint. The present study showed that HRV-Cs from Malaysia have diverse genetic sequences but share conserved genomic features with other HRV-Cs. This genetic information could provide further aid in the understanding of HRV-C infection.
Fulltext The use of the amplification refractory mutation system (arms) in the detection of rare beta-thalassemia mutations in the Malays and Chinese in Malaysia

Chan YF, Tan KL, Wong YC, Wee YC, Yap SF, Tan JAMA

Southeast Asian J. Trop. Med. Public Health, 2001 Dec;32(4):872-9.
PMID: 12041567

Molecular characterization and prenatal diagnosis for beta-thalassemia can be carried out using the Amplification Refractory Mutation System (ARMS). The ARMS is a rapid and direct molecular technique in which beta-thalassemia mutations are visualized immediately after DNA amplification by gel electrophoresis. In the University of Malaya Medical Center, molecular characterization and prenatal diagnosis for beta-thalassemia is carried out using ARMS for about 96% of the Chinese and 84.6% of the Malay patients. The remaining 4% and 15.4% of the uncharacterized mutations in the Chinese and Malay patients respectively are detected using DNA sequencing. DNA sequencing is an accurate technique but it is more time-consuming and expensive compared with the ARMS. The ARMS for the rare Chinese beta-mutations at position -29 (A-->G) and the ATG-->AGG base substitution at the initiator codon for translation in the beta-gene was developed. In the Malays, ARMS was optimized for the beta-mutations at codon 8/9 (+G), Cap (+1) (A-->C) and the AATAAA-->AATAGA base substitution in the polyadenylation region of the beta-gene. The ARMS protocols were developed by optimization of the parameters for DNA amplification to ensure sensitivity, specificity and reproducibility. ARMS primers (sequences and concentration), magnesium chloride concentration, Taq DNA polymerase and PCR cycling parameters were optimized for the specific amplification of each rare beta-thalassemia mutation. The newly-developed ARMS for the 5 rare beta-thalassemia mutations in the Chinese and Malays in Malaysia will allow for more rapid and cost-effective molecular characterization and prenatal diagnosis for beta-thalassemia in Malaysia.
Immune responses against enterovirus A71 infection: Implications for vaccine success

Aw-Yong KL, NikNadia NMN, Tan CW, Sam IC, Chan YF

Rev Med Virol, 2019 09;29(5):e2073.
PMID: 31369184 DOI: 10.1002/rmv.2073

Enterovirus A71 (EV-A71) from the Picornaviridae family is an important emerging pathogen causing hand, foot, and mouth disease (HFMD) outbreaks worldwide. EV-A71 also caused fatal neurological complications in young children especially in Asia. On the basis of seroepidemiological studies from many Asian countries, EV-A71 infection is very common. Children of very young age are particularly vulnerable. Large-scale epidemics that occur every 3 to 4 years are associated with accumulation of an immunologically naive younger population. Capsid proteins especially VP1 with the presence of major B- and T-cell epitopes are the most antigenic proteins. The nonstructural proteins mainly contribute to T-cell epitopes that induce cross-reactive immune responses against other enteroviruses. Dominant epitopes and their neutralization magnitudes differ in mice, rabbits, and humans. Neutralizing antibody is sufficient for immune protection, but poorer cellular immunity may lead to severe neurological complications and deaths. Some chemokines/cytokines are consistently found in severely ill patients, for example, IL-6, IL-10, IL-17A, MCP-1, IL-8, MIG, IP-10, IFN-γ, and G-CSF. An increase in white cell counts is a risk factor for severe HFMD. Recent clinical trials on EV-A71 inactivated vaccine showed >90% efficacy and a robust neutralization response that was protective, indicating neutralizing antibody correlates for protection. No protection against other enteroviruses was observed. A comprehensive understanding of the immune responses to EV-A71 infection will benefit the development of diagnostic tools, potential therapeutics, and subunit vaccine candidates. Future development of a multivalent enterovirus vaccine will require knowledge of correlates of protection, understanding of cross-protection and memory T-cell responses among enteroviruses.
Low seroprevalence rates of Zika virus in Kuala Lumpur, Malaysia

Sam IC, Montoya M, Chua CL, Chan YF, Pastor A, Harris E

Trans R Soc Trop Med Hyg, 2019 11 01;113(11):678-684.
PMID: 31294807 DOI: 10.1093/trstmh/trz056

BACKGROUND: Zika virus (ZIKV) is believed to be endemic in Southeast Asia. However, there have been few Zika cases reported to date in Malaysia, which could be due to high pre-existing levels of population immunity.
METHODS: To determine Zika virus (ZIKV) seroprevalence in Kuala Lumpur, Malaysia, 1085 serum samples from 2012, 2014-2015 and 2017 were screened for anti-ZIKV antibodies using a ZIKV NS1 blockade-of-binding assay. Reactive samples were confirmed using neutralization assays against ZIKV and the four dengue virus (DENV) serotypes. A sample was possible ZIKV seropositive with a ZIKV 50% neutralization (NT50) titre ≥20. A sample was probable ZIKV seropositive if, in addition, all DENV NT50 titres were <20 or the ZIKV NT50 titre was >4-fold greater than the highest DENV NT50 titre.
RESULTS: We found low rates of possible ZIKV seropositivity (3.3% [95% confidence interval {CI} 2.4 to 4.6]) and probable ZIKV seropositivity (0.6% [95% CI 0.3 to 1.4]). Possible ZIKV seropositivity was independently associated with increasing age (odds ratio [OR] 1.04 [95% CI 1.02 to 1.06], p<0.0001) and male gender (OR 3.5 [95% CI 1.5 to 8.6], p=0.005).
CONCLUSIONS: The low ZIKV seroprevalence rate, a proxy for population immunity, does not explain the low incidence of Zika in dengue-hyperendemic Kuala Lumpur. Other factors, such as the possible protective effects of pre-existing flavivirus antibodies or reduced transmission by local mosquito vectors, should be explored. Kuala Lumpur is at high risk of a large-scale Zika epidemic.
Evaluation of rapid influenza diagnostic tests for influenza A and B in the tropics

Chong YM, Tan XH, Hooi PS, Lee LM, Sam IC, Chan YF

J Med Virol, 2019 08;91(8):1562-1565.
PMID: 31032971 DOI: 10.1002/jmv.25495

Rapid diagnosis of influenza is important for early treatment and institution of control measures. In developing tropical countries such as Malaysia, influenza occurs all year round, but molecular assays and conventional techniques (such as immunofluorescence and culture) for diagnosis are not widely available. Rapid influenza diagnostic tests (RIDTs) may be useful in this setting. A total of 552 fresh respiratory specimens were assessed from patients with respiratory symptoms at a teaching hospital in Kuala Lumpur, Malaysia from November 2017 to March 2018. Two digital immunoassays (DIAs), STANDARD F Influenza A/B Fluorescence Immunoassay (STANDARD F) and Sofia Influenza A + B Fluorescence Immunoassay (Sofia) and one conventional RIDT (immunochromatographic assay), SD Bioline Influenza Ag A/B/A(H1N1) Pandemic rapid test kit (SD Bioline) were evaluated in comparison with a WHO-recommended reverse transcription quantitative PCR (RT-qPCR). Of the 552 samples, influenza A virus was detected in 47 (8.5%) and influenza B virus in 7 (1.3%). The digital immunoassays STANDARD F and Sofia had significantly higher overall sensitivity rates (71.7% and 70.6%, respectively) than the conventional RIDT SD Bioline and immunofluorescence/viral culture (55.8% and 52.8%, respectively). Sensitivity rates were higher for influenza A than influenza B, and specificity rates were uniformly high, ranging from 98% to 100%. Digital readout RIDTs can be used in tropical settings with year-round influenza if PCR is unavailable.
Fulltext An Overview of the Genetic Variations of the SARS-CoV-2 Genomes Isolated in Southeast Asian Countries

Yap PSX, Tan TS, Chan YF, Tee KK, Kamarulzaman A, Teh CSJ

J Microbiol Biotechnol, 2020 Jul 28;30(7):962-966.
PMID: 32627759 DOI: 10.4014/jmb.2006.06009

Monitoring the mutation dynamics of human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical in understanding its infectivity, virulence and pathogenicity for development of a vaccine. In an "age of mobility," the pandemic highlights the importance and vulnerability of regionalization and labor market interdependence in Southeast Asia. We intend to characterize the genetic variability of viral populations within the region to provide preliminary information for regional surveillance in the future. By analyzing 142 complete genomes from South East Asian (SEA) countries, we identified three central variants distinguished by nucleotide and amino acid changes.
Updates on chikungunya epidemiology, clinical disease, and diagnostics

Sam IC, Kümmerer BM, Chan YF, Roques P, Drosten C, AbuBakar S

Vector Borne Zoonotic Dis, 2015 Apr;15(4):223-30.
PMID: 25897809 DOI: 10.1089/vbz.2014.1680

Chikungunya virus (CHIKV) is an Aedes-borne alphavirus, historically found in Africa and Asia, where it caused sporadic outbreaks. In 2004, CHIKV reemerged in East Africa and spread globally to cause epidemics, including, for the first time, autochthonous transmission in Europe, the Middle East, and Oceania. The epidemic strains were of the East/Central/South African genotype. Strains of the Asian genotype of CHIKV continued to cause outbreaks in Asia and spread to Oceania and, in 2013, to the Americas. Acute disease, mainly comprising fever, rash, and arthralgia, was previously regarded as self-limiting; however, there is growing evidence of severe but rare manifestations, such as neurological disease. Furthermore, CHIKV appears to cause a significant burden of long-term morbidity due to persistent arthralgia. Diagnostic assays have advanced greatly in recent years, although there remains a need for simple, accurate, and affordable tests for the developing countries where CHIKV is most prevalent. This review focuses on recent important work on the epidemiology, clinical disease and diagnostics of CHIKV.
Vaccine candidates generated by codon and codon pair deoptimization of enterovirus A71 protect against lethal challenge in mice

Lee MHP, Tan CW, Tee HK, Ong KC, Sam IC, Chan YF

Vaccine, 2021 03 19;39(12):1708-1720.
PMID: 33640144 DOI: 10.1016/j.vaccine.2021.02.024

Enterovirus A71 (EV-A71) causes hand, foot and mouth disease (HFMD) in young children. It is associated with severe neurological complications and death. This study aims to develop a live-attenuated vaccine by codon deoptimization (CD) and codon-pair deoptimization (CPD) of EV-A71. CD is generated by introducing the least preferred codons for amino acids while CPD increases the presence of underrepresented codon pairs in the specific genes. CD and CPD chimeras were generated by synonymous mutations at the VP2, VP3, VP1 and 2A gene regions, designated as XYZ. All twelve deoptimized viruses were viable with similar replication kinetics, but the plaque sizes were inversely proportional to the level of deoptimization. All the deoptimized viruses showed attenuated growth in vitro with reduced viral protein expression at 48 h and lower viral RNA at 39 °C. Six-week-old ICR mice were immunized intraperitoneally with selected CD and CPD X and XY vaccine candidates covering the VP2-VP3 and VP2-VP3-VP1 genes, respectively. All vaccine candidates elicited high anti-EV-A71 IgG levels similar to wild-type (WT) EV-A71. The CD X and CPD X vaccines produced robust neutralizing antibodies but not the CD XY and CPD XY. On lethal challenge, offspring of mice immunized with WT, CD X and CPD X were fully protected, but the CD XY- and CPD XY-vaccinated mice had delayed symptoms and eventually died. Similarly, active immunization of 1-day-old suckling mice with CD X, CPD X and CD XY vaccine candidates provided complete immune protection but CPD XY only protected 40% of the challenged mice. Histology of the muscles from CD X- and CPD X-vaccinated mice showed minimal pathology compared to extensive inflammation in the post-challenged mock-vaccinated mice. Overall, we demonstrated that the CD X and CPD X elicited good neutralizing antibodies, conferred immune protection and are promising live-attenuated vaccine candidates for EV-A71.
Socioeconomic costs of children <5 years hospitalised with acute respiratory infections in Kuala Lumpur, Malaysia

Sam IC, Ahmad Jaafar N, Wong LP, Nathan AM, de Bruyne JA, Chan YF

Vaccine, 2021 05 21;39(22):2983-2988.
PMID: 33931252 DOI: 10.1016/j.vaccine.2021.04.010

BACKGROUND: Acute respiratory infections (ARI) are a major cause of morbidity and mortality in Malaysian children
Fulltext Chikungunya virus-associated death in Malaysia

Sam IC, Kamarulzaman A, Ong GS, Veriah RS, Ponnampalavanar S, Chan YF, et al.

Trop Biomed, 2010 Aug;27(2):343-7.
PMID: 20962735

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus which causes fever, rash, and arthralgia. In the past, life-threatening complications were very rarely reported. However, during the recent worldwide outbreaks, there have been several reports of unusually severe complications and deaths. Malaysia is experiencing a nationwide outbreak of CHIKV, with over 10 000 patients affected since April 2008. We report the first case of culture-confirmed CHIKV-associated death in Malaysia, in a patient with fever, rash, acute exacerbation of pre-existing heart failure, rhabdomyolysis, and multiple organ failure. CHIKV infections may cause atypical, severe or fatal presentations.
The effect of non-native and non-regional speech testing on a multi-lingual population

Quar TK, Soli SD, Chan YF, Ishak WS, Abdul Wahat NH

Int J Audiol, 2017 02;56(2):92-98.
PMID: 27686009 DOI: 10.1080/14992027.2016.1210828

OBJECTIVE: This study was conducted to evaluate the speech perception of Malaysian Chinese adults using the Taiwanese Mandarin HINT (MHINT-T) and the Malay HINT (MyHINT).
DESIGN: The MHINT-T and the MyHINT were presented in quiet and noise (front, right and left) conditions under headphones. Results for the two tests were compared with each other and with the norms for each test.
STUDY SAMPLE: Malaysian Chinese native speakers of Mandarin (N = 58), 18-31 years of age with normal hearing.
RESULTS: On average, subjects demonstrated poorer speech perception ability than the normative samples for these tests. Repeated measures ANOVA showed that speech reception thresholds (SRTs) were slightly poorer on the MHINT-T than on the MyHINT for all test conditions. However, normalized SRTs were poorer by 0.6 standard deviations for MyHINT as compared with MHINT-T.
CONCLUSIONS: MyHINT and MHINT-T can be used as norm-referenced speech perception measures for Mandarin-speaking Chinese in Malaysia.
Fulltext New Paradigms for Virus Detection, Surveillance and Control of Zika Virus Vectors in the Settings of Southeast Asia

Vythilingam I, Sam JI, Chan YF, Khaw LT, Sulaiman WY

Front Microbiol, 2016;7:1452.
PMID: 27679623 DOI: 10.3389/fmicb.2016.01452

Zika virus (ZIKV) has now become a global public health concern. The vectors for ZIKV are Aedes aegypti and A. albopictus. Both these mosquitoes are predominant in Southeast Asia and are also responsible for the spread of other arboviral diseases like dengue virus and chikungunya virus. The incidence of dengue has been increasing over the years and this is of concern to public health workers. Simple laboratory tools for the detection of ZIKV is also lacking. In the absence of drugs and vaccine for these arboviral diseases, vector control is the main option for surveillance and control. Aedes larval surveys have been the hallmark of dengue control along with larviciding and fogging when cases are reported. However, we need new paradigms and options for control of these vectors. The current situation in Southeast Asia clearly proves that effective strategies for vector control need to be proactive and not reactive. This will be the way forward to control epidemics of these diseases inclusive of ZIKV until a vaccine becomes available.
Fulltext Enterovirus A71 DNA-Launched Infectious Clone as a Robust Reverse Genetic Tool

Tan CW, Tee HK, Lee MH, Sam IC, Chan YF

PLoS One, 2016;11(9):e0162771.
PMID: 27617744 DOI: 10.1371/journal.pone.0162771

Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot and mouth disease, and is occasionally associated with neurological complications and death in children. Reverse genetics is widely used in the field of virology for functional study of viral genes. For EV-A71, such tools are limited to clones that are transcriptionally controlled by T7/SP6 bacteriophage promoter. This is often time-consuming and expensive. Here, we describe the development of infectious plasmid DNA-based EV-A71 clones, for which EV-A71 genome expression is under transcriptional control by the CMV-intermediate early promoter and SV40 transcriptional-termination signal. Transfection of this EV-A71 infectious DNA produces good virus yield similar to in vitro-transcribed EV-A71 infectious RNA, 6.4 and 5.8 log10PFU/ml, respectively. Infectious plasmid with enhanced green fluorescence protein and Nano luciferase reporter genes also produced good virus titers, with 4.3 and 5.0 log10 PFU/ml, respectively. Another infectious plasmid with both CMV and T7 promoters was also developed for easy manipulation of in vitro transcription or direct plasmid transfection. Transfection with either dual-promoter infectious plasmid DNA or infectious RNA derived from this dual-promoter clone produced infectious viral particles. Incorporation of hepatitis delta virus ribozyme, which yields precise 3' ends of the DNA-launched EV-A71 genomic transcripts, increased infectious viral production. In contrast, the incorporation of hammerhead ribozyme in the DNA-launched EV-A71 resulted in lower virus yield, but improved the virus titers for T7 promoter-derived infectious RNA. This study describes rapid and robust reverse genetic tools for EV-A71.
Polysulfonate suramin inhibits Zika virus infection

Tan CW, Sam IC, Chong WL, Lee VS, Chan YF

Antiviral Res, 2017 07;143:186-194.
PMID: 28457855 DOI: 10.1016/j.antiviral.2017.04.017

Zika virus (ZIKV) is an arthropod-borne flavivirus that causes newborn microcephaly and Guillian-Barré syndrome in adults. No therapeutics are available to treat ZIKV infection or other flaviviruses. In this study, we explored the inhibitory effect of glycosaminoglycans and analogues against ZIKV infection. Highly sulfated heparin, dextran sulfate and suramin significantly inhibited ZIKV infection in Vero cells. De-sulfated heparin analogues lose inhibitory effect, implying that sulfonate groups are critical for viral inhibition. Suramin, an FDA-approved anti-parasitic drug, inhibits ZIKV infection with 3-5 log10 PFU viral reduction with IC50value of ∼2.5-5 μg/ml (1.93 μM-3.85 μM). A time-of-drug-addition study revealed that suramin remains potent even when administrated at 1-24 hpi. Suramin inhibits ZIKV infection by preventing viral adsorption, entry and replication. Molecular dynamics simulation revealed stronger interaction of suramin with ZIKV NS3 helicase than with the envelope protein. Suramin warrants further investigation as a potential antiviral candidate for ZIKV infection. Heparan sulfate (HS) is a cellular attachment receptor for multiple flaviviruses. However, no direct ZIKV-heparin interaction was observed in heparin-binding analysis, and downregulate or removal of cellular HS with sodium chlorate or heparinase I/III did not inhibit ZIKV infection. This indicates that cell surface HS is not utilized by ZIKV as an attachment receptor.
VP1 residues around the five-fold axis of enterovirus A71 mediate heparan sulfate interaction

Tan CW, Sam IC, Lee VS, Wong HV, Chan YF

Virology, 2017 01 15;501:79-87.
PMID: 27875780 DOI: 10.1016/j.virol.2016.11.009

Enterovirus A71 (EV-A71) is a neurotropic enterovirus that uses heparan sulfate as an attachment receptor. The molecular determinants of EV-A71-heparan sulfate interaction are unknown. With In silico heparin docking and mutagenesis of all possible lysine residues in VP1, we identified that K162, K242 and K244 are responsible for heparin interaction and inhibition. EV-A71 mutants with K242A and K244A rapidly acquired compensatory mutations, T100K or E98A, and Q145R-T237N respectively, which restored the heparin-binding phenotype. Both VP1-98 and VP1-145 modulates heparin binding. Heparin-binding phenotype was completely abolished with VP1-E98-E145, but was restored by an E98K or E145Q substitution. During cell culture adaptation, EV-A71 rapidly acquired K98 or Q/G145 to restore the heparin-binding phenotype. Together with next-generation sequencing analysis, our results implied that EV-A71 has high genetic plasticity by modulating positively-charged residues at the five-fold axis during in vitro heparin adaptation. Our finding has impact on EV-A71 vaccine production, evolutionary studies and pathogenesis.
Fulltext 2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation

Lai JKF, Sam IC, Verlhac P, Baguet J, Eskelinen EL, Faure M, et al.

Viruses, 2017 07 04;9(7).
PMID: 28677644 DOI: 10.3390/v9070169

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with aN-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.
Seroepidemiology of enterovirus D68 infection in Kuala Lumpur, Malaysia between 2013-2015

Chan YF, Sam IC, Nayan E, Tan XH, Yogarajah T

J Med Virol, 2021 Oct 07.
PMID: 34617599 DOI: 10.1002/jmv.27381

Enterovirus D68 (EV-D68) is an emerging respiratory pathogen since the 2014 outbreak in USA. A low level of virus circulation has been reported in Kuala Lumpur, Malaysia in the past. However, the extent of the infection in Malaysia is not known. In the present study, we determine the seroepidemiology of EV-D68 in Kuala Lumpur, Malaysia before and after the USA outbreak in Aug 2014. A luciferase-based seroneutralization test was developed using a clone-derived prototype Fermon strain carrying a nanoluciferase marker. We screened the neutralization capacity of 450 serum samples from children and adults (1-89 years old) collected between 2013 to 2015. EV-D68 seropositivity increased with age, with children aged 1-3 showing significantly lower seroprevalence compared to adults. Multivariate analysis showed that older age groups 13-49 years (odds ratio [OR] 4.78 [95% CI 2.69-8.49], p<0.0001) and >50 years (OR 3.83 [95% CI 2.19-6.68], p<0.0001) were more likely to be EV-D68 seropositive than children <13 years. Sampling post-Sept 2014 compared to pre-Sept 2014 also predicted seropositivity (OR 1.66 [95% CI 1.04-2.65]). Presence of neutralizing antibodies against EV-D68 in the study population suggests that EV-D68 was circulating prior to 2014. A higher seropositivity post-Sept 2014 suggests that Malaysia also experienced an upsurge in EV-D68 infections after the USA outbreaks in Aug 2014. A low seropositivity rate observed in children, especially those aged 1-3 years old, suggests that they are at risk and should be prioritized for future vaccination. This article is protected by copyright. All rights reserved.
Fulltext Isolation and Identification of Inter-Species Enterovirus Recombinant Genomes

Bentley K, Tee HK, Pearson A, Lowry K, Waugh S, Jones S, et al.

Viruses, 2021 11 29;13(12).
PMID: 34960659 DOI: 10.3390/v13122390

Positive-strand RNA virus evolution is partly attributed to the process of recombination. Although common between closely genetically related viruses, such as within species of the Enterovirus genus of the Picornaviridae family, inter-species recombination is rarely observed in nature. Recent studies have shown recombination is a ubiquitous process, resulting in a wide range of recombinant genomes and progeny viruses. While not all recombinant genomes yield infectious progeny virus, their existence and continued evolution during replication have critical implications for the evolution of the virus population. In this study, we utilised an in vitro recombination assay to demonstrate inter-species recombination events between viruses from four enterovirus species, A-D. We show that inter-species recombinant genomes are generated in vitro with polymerase template-switching events occurring within the virus polyprotein coding region. However, these genomes did not yield infectious progeny virus. Analysis and attempted recovery of a constructed recombinant cDNA revealed a restriction in positive-strand but not negative-strand RNA synthesis, indicating a significant block in replication. This study demonstrates the propensity for inter-species recombination at the genome level but suggests that significant sequence plasticity would be required in order to overcome blocks in the virus life cycle and allow for the production of infectious viruses.
Chikungunya encephalopathy and pneumonia in a young infant presenting with septic shock

Chee YS, Ahamad Fouzi N, Chong YM, Sam IC, Chan YF, Chua CL, et al.

J Paediatr Child Health, 2022 Feb 17.
PMID: 35175651 DOI: 10.1111/jpc.15858

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