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To describe the severity, human adenovirus (HAdV) type and respiratory morbidity following adenovirus pneumonia in children. Retrospective review of children under 12 years of age, admitted with HAdV pneumonia, between January 2011 and July 2013, in a single centre in Malaysia. HAdV isolated from nasopharyngeal secretions were typed by sequencing hypervariable regions 1-6 of the hexon gene. Patients were reviewed for respiratory complications. HAdV was detected in 131 children of whom 92 fulfilled inclusion criteria. Median (range) age was 1.1 (0.1-8.0) years with 80% under 2 years. Twenty percent had severe disease with a case-fatality rate of 5.4%. Duration of admission (p = 0.02) was independently associated with severe illness. Twenty-two percent developed respiratory complications, the commonest being bronchiolitis obliterans (15.2%) and recurrent wheeze (5.4%). The predominant type shifted from HAdV1 and HAdV3 in 2011 to HAdV7 in 2013. The commonest types identified were types 7 (54.4%), 1(17.7%) and 3 (12.6%). Four out of the five patients who died were positive for HAdV7. Infection with type 7 (OR 8.90, 95% CI 1.32, 59.89), family history of asthma (OR 14.80, 95% CI 2.12-103.21) and need for invasive or non-invasive ventilation (OR 151.84, 95% CI 9.93-2.32E) were independent predictors of respiratory complications. One in five children admitted with HAdV pneumonia had severe disease and 22% developed respiratory complications. Type 7 was commonly isolated in children with severe disease. Family history of asthma need for invasive or non-invasive ventilation and HAdV 7 were independent predictors of respiratory complications.

The antibody isotype IgM appears earlier than IgG, within days of onset of symptoms, and is important during the early stages of the adaptive immune response. Little is known about the functional role of IgM during infection with chikungunya virus (CHIKV), a recently reemerging arbovirus that has caused large global outbreaks. In this study, we studied antibody responses in 102 serum samples collected during CHIKV outbreaks in Malaysia. We described the neutralizing role of IgM at different times post-infection and examined the independent contributions of IgM and IgG towards the neutralizing capacity of human immune sera during the early phase of infection, including the differences in targets of neutralizing epitopes. Neutralizing IgM starts to appear as early as day 4 of symptoms, and their appearance from day 6 is associated with a reduction in viremia. IgM acts in a complementary manner with the early IgG, but plays the main neutralizing role up to a point between days 4 and 10 which varies between individuals. After this point, total neutralizing capacity is attributable almost entirely to the robust neutralizing IgG response. IgM preferentially binds and targets epitopes on the CHIKV surface E1-E2 glycoproteins, rather than individual E1 or E2. These findings provide insight into the early antibody responses to CHIKV, and have implications for design of diagnostic serological assays.

Background/Objectives: Enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) are major causative agents of hand, foot and mouth disease (HFMD), often co-circulating and occasionally undergoing genetic recombination. While natural recombinants often involve genomic regions encoding non-structural proteins, their effects on replication and pathogenesis remain unclear. Methods: To address this, four chimera viruses (Chi-CCE, Chi-ECE, Chi-EEC, and Chi-CEC) were constructed with 5′UTR, capsid P1, and non-structural P2 and P3 genes, from CVA16 (denoted as C) or EV-A71 (denoted as E). These chimeras were tested for replication kinetics and cytopathic effects in rhabdomyosarcoma cells while in vivo virulence and protection efficacy were evaluated using a newborn BALB/c mouse model. Results: All chimeric viruses remained viable and exhibited higher replication than CVA16. In vivo, all chimeric viruses were avirulent except Chi-CCE and CVA16, which showed high virulence and viral titres in the brains and limbs of infected newborn mice. This suggests that 5′UTR and capsid P1 genes of CVA16 are critical genetic determinants of virulence. Notably, only the anti-inflammatory cytokine IL-10 was elevated, suggesting potential immune modulation during infection. Inactivated Chi-CCE immunisation conferred 100% protection against lethal CVA16 or mouse-adapted EV-A71 challenge revealing its potential as a bivalent vaccine candidate. Conclusions: Our study demonstrates that recombination between CVA16 and EV-A71 influences viral virulence and protection efficacy with implications for future development of multivalent vaccines.

Vaccine efficacy against SARS-CoV-2 could be compromised by the emergence of SARS-CoV-2 variants and it is important to study how it impacts the booster vaccination regime. We investigated the humoral and T cell responses longitudinally in vaccinated uninfected (n = 25) and post-COVID-19 individuals (n = 8), and those who had received a BNT162b2 booster following complete two-doses regimes of either BNT162b2 (homologous) (n = 14) or ChAdOx1-S (heterologous) (n = 15) vaccines, by means of a SARS-CoV-2 pseudovirus neutralization test and QuantiFERON SARS-CoV-2 assay. Vaccinated post-COVID-19 individuals showed higher neutralizing antibodies with longer durability against SARS-CoV-2 wild type (WT) and Omicron spikes, but demonstrated similar declining T cell responses compared to the uninfected vaccinated. Two doses of BNT162b2 induced higher neutralizing antibodies against WT and T cell responses than ChAdOx1-S for six months. The BNT162b2 booster confers a greater humoral response against WT, but a similar cross-neutralizing antibody against Omicron and T cell responses in the homologous booster group compared to the heterologous booster group. Breakthrough infection in the homologous booster group (n = 11) significantly increased the neutralizing antibody, but T cell responses remained low. Our data may impact government public health policy regarding the administration of mix-and-match vaccines, where both vaccination regimes can be employed should there be shortages of certain vaccines.

Background Methicillin-resistant Staphylococcus aureus (MRSA) is an established pathogen that causes hospital- and community-acquired infections worldwide. The prevalence rate of MRSA infections were reported to be the highest in Asia. As there is limited epidemiological study being done in Malaysia, this study aimed to determine the prevalence of MRSA infection and the molecular characteristics of MRSA bacteraemia. Methods Two hundred and nine MRSA strains from year 2011 to 2012 were collected from a tertiary teaching hospital in Malaysia. The strains were characterized by antimicrobial susceptibility testing, staphylococcal cassette chromosome mec (SCC mec ) typing, detection of Panton-Valentine leukocidin (PVL) gene, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Patient’s demographic and clinical data were collected and correlated with molecular data by statistical analysis. Results Male gender and patient >50 years of age ( p  < 0.0001) were significantly associated with the increased risk of MRSA acquisition. Fifty-nine percent of MRSA strains were HA-MRSA that carried SCC mec type II, III, IV and V while 31% were CA-MRSA strains with SCC mec III, IV and V. The prevalence of PVL gene among 2011 MRSA strains was 5.3% and no PVL gene was detected in 2012 MRSA strains. All of the strains were sensitive to vancomycin. However, vancomycin MIC creep phenomenon was demonstrated by the increased number of MRSA strains with MIC ≥1.5 μg/mL ( p  = 0.008) between 2011 and 2012. Skin disease ( p  = 0.034) and SCC mec type III ( p  = 0.0001) were found to be significantly associated with high vancomycin MIC. Forty-four percent of MRSA strains from blood, were further subtyped by MLST and PFGE. Most of the bacteraemia cases were primary bacteraemia and the common comorbidities were diabetes, hypertension and chronic kidney disease. The predominant pulsotype was pulsotype C exhibited by SCC mec III-ST239. This is a first study in Malaysia that reported the occurrence of MRSA clones such as SCC mec V-ST5, untypeable-ST508, SCC mec IV-ST1 and SCC mec IV-ST1137. Conclusions SCC mec type III remained predominant among the MRSA strains in this hospital. The occurrence of SCC mec IV and V among hospital strains and the presence of SCC mec III in CA-MRSA strains are increasing. MRSA strains causing bacteraemia over the two-year study period were found to be genetically diverse.

Malaysia had 10,219 confirmed cases of COVID-19 as of September 20, 2020. About 33% were associated with a Tablighi Jamaat religious mass gathering held in Kuala Lumpur between February 27 and March 3, 2020, which drove community transmission during Malaysia’s second wave. We analysed genome sequences of SARS-CoV-2 from Malaysia to better understand the molecular epidemiology and spread. We obtained 58 SARS-CoV-2 whole genome sequences from patients in Kuala Lumpur and performed phylogenetic analyses on these and a further 57 Malaysian sequences available in the GISAID database. Nine different SARS-CoV-2 lineages (A, B, B.1, B.1.1, B.1.1.1, B.1.36, B.2, B.3 and B.6) were detected in Malaysia. The B.6 lineage was first reported a week after the Tablighi mass gathering and became predominant (65.2%) despite being relatively rare (1.4%) globally. Direct epidemiological links between lineage B.6 viruses and the mass gathering were identified. Increases in reported total cases, Tablighi-associated cases, and community-acquired B.6 lineage strains were temporally linked. Non-B.6 lineages were mainly travel-associated and showed limited onward transmission. There were also temporally correlated increases in B.6 sequences in other Southeast Asian countries, India and Australia, linked to participants returning from this event. Over 95% of global B.6 sequences originated from Asia Pacific. We also report a nsp3-C6310A substitution found in 47.3% of global B.6 sequences which was associated with reduced sensitivity using a commercial diagnostic real-time PCR assay. Lineage B.6 became the predominant cause of community transmission in Malaysia after likely introduction during a religious mass gathering. This event also contributed to spikes of lineage B.6 in other countries in the Asia-Pacific. Mass gatherings can be significant causes of local and global spread of COVID-19. Shared genomic surveillance can be used to identify SARS-CoV-2 transmission chains to aid prevention and control, and to monitor diagnostic molecular assays. Clinical Trial Registration : COVID-19 paper.

Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly understood. In mice, adaptation and virulence determinants have been mapped to mutations at VP2-149, VP1-145 and VP1-244. We investigate how these amino acids alter heparin-binding phenotype and shapes EV-A71 virulence in one-day old mice. We constructed six viruses with varying residues at VP1-98, VP1-145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 149K/98E/145Q, termed KEQ) to generate KKQ, KKE, KEE, IEE and IEQ variants. We demonstrated that the weak heparin-binder IEE was highly lethal in mice. The initially strong heparin-binding IEQ variant acquired an additional mutation VP1-K244E, which confers weak heparin-binding phenotype resulting in elevated viremia and increased virus antigens in mice brain, with subsequent high virulence. IEE and IEQ-244E variants inoculated into mice disseminated efficiently and displayed high viremia. Increasing polymerase fidelity and impairing recombination of IEQ attenuated the virulence, suggesting the importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico docking and deep sequencing approaches, we inferred that virus population diversity is shaped by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, which ultimately results in increased neurovirulence. The dynamic switching between heparin-binding and weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71.

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.

In 2008-2010, Malaysia experienced a nationwide chikungunya virus (CHIKV) outbreak caused by the Indian Ocean lineage E1-226V (valine) variant, adapted to Aedes albopictus. In 2017-2022, transition to an E1-226A (alanine) variant occurred. Ae. albopictus prevails in rural areas, where most cases occurred during the E1-226V outbreak, while Ae. aegypti dominates urban areas. The shift in circulating CHIKV variants from E1-226V to E1-226A (2009-2022) was hypothesized to result in a transition from rural to urban CHIKV distribution, driven by differences in Ae. aegypti vector competence for the two variants. This study aimed to: (1) map the spatiotemporal spread of CHIKV cases in Malaysia between 2009-2022; and (2) compare replication of E1-226A and E1-226V variants in the midguts and head/thoraxes of Ae. aegypti. Spatiotemporal analysis of national notified CHIKV case addresses was performed. Between 2009-2022, 12,446 CHIKV cases were reported, with peaks in 2009 and 2020, and a significant shift from predominantly rural cases in 2009-2011 (85.1% rural), to urban areas in 2017-2022 (86.1% urban; p<0.0001). Two Ae. aegypti strains, field-collected MC1 and laboratory Kuala Lumpur (KL) strains, were fed infectious blood containing constructed CHIKV clones, pCMV-p2020A (E1-226A) and pCMV-p2020V (E1-226V) to measure CHIKV replication by real-time PCR and/or virus titration. The pCMV-p2020A clone replicated better in Ae. aegypti cell line Aag2 and showed higher replication, infection and dissemination efficiency in both Ae. aegypti strains, compared to pCMV-p2020V. This study revealed that a change in circulating CHIKV variants can be associated with changes in vector competence and outbreak epidemiology. Continued genomic surveillance of arboviruses is important.