Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia

Thirteen enterovirus 71 (EV71) isolates were obtained from both fatal and non-fatal infections of patients seen in Peninsula Malaysia and in Sarawak during an outbreak of hand, foot and mouth disease (HFMD) in Malaysia in 1997, with incidences of fatal brainstem encephalomyelitis. The isolates were identified using immunofluorescence staining, neutralization assays, and partial sequencing of the 5' untranslated regions (UTR). Assessment of the potential genetic relationships of the isolates using the partial 5'UTR sequences suggested clustering of the isolates into at least two main clusters. Isolates from Peninsula Malaysia were found in both clusters whereas Sarawak-derived isolates clustered only in cluster II. Isolates derived from fatal infections, however, occurred in both clusters and no distinctive nucleotide sequences could be attributed to the fatal isolates. Examination of the nucleotide sequences revealed at least 13 nucleotide positions in all the isolates which differ completely from the previously reported EV71 5'UTR sequences. In addition, at least 11 nucleotide position differences within the 5'UTR were noted which differentiated cluster I from cluster II. Predicted secondary RNA structures drawn using the nucleotide sequences also suggested differences between isolates from the two clusters. These findings suggest the presence of at least two potentially virulent EV71 co-circulating in Malaysia during the 1997 HFMD outbreak.     

Induction of chromosome aberrations and mitotic arrest by cytomegalovirus in human cells

Human cytomegalovirus (CMV) is potentially an effective but often overlooked genotoxic agent in humans. We report here evidence that indicates that infection by CMV can induce chromosome alterations and mitotic inhibition. The frequency of chromosome aberrations induced was dependent on the input multiplicity of infection (m.o.i.) for human lung fibroblasts (LU), but not for human peripheral blood lymphocytes (PBLs) when both cell types were infected at the GO phase of the cell cycle. The aberrations induced by CMV were mostly chromatid breaks and chromosome pulverizations that resembled prematurely condensed S-phase chromatin. Pulverized chromosomes were not observed in LU cells infected with virus stocks that had been rendered nonlytic by UV-irradiation at 24,000 ergs/mm2 or from infection of human lymphocytes. In LU cells infected with UV-irradiated CMV, the frequency of aberrations induced was inversely dependent on the extent of the exposure of the CMV stock to the UV-light. In permissive CMV infection of proliferating LU cells at 24 hr after subculture, a high percentage (greater than 40%) of the metaphase cells were arrested at their first metaphase and displayed severely condensed chromosomes when harvested 48 hr later. A significant increase (p less than 0.05) in the chromosome aberration frequency was also observed. Our study shows that CMV infection is genotoxic to host cells. The types and extent of damage are dependent on the viral genome expression and on the cell cycle stage of the cells at the time of infection. The possible mechanisms for induction of chromosome damage by CMV are discussed.     

Differences in Perceived Severity of Zika Virus Infection and Dengue Fever and its Influence on Mosquito Control Practices in Malaysia

The aims of this study were to explore the differences in (1) the perception of severity towards ZIKV infection and dengue fever, and (2) mosquito control practices before and after the ZIKV outbreak were declared a Public Health Emergency of International Concern (PHEIC). Data were collected between Feb to May 2016 using a computer-assisted telephone interviewing system. The median scale score for perceived severity of ZIKV was 3 (interquartile range [IQR] 1–5) versus 4 (IQR 3–5) for dengue (P?<?0.001). The scores for mosquito control practices before and after ZIKV was declared a PHEIC were similar, at 4 (IQR 3–5). Multivariate analysis revealed that participants with a higher score for perception of severity of ZIKV were more likely to report greater mosquito control practices after the declaration of the PHEIC (OR 1.822 [95% CI 1.107–2.998]). The emerging ZIKV pandemic requires concerted efforts to enhance mosquito control practices among the Malaysian public. Efforts to improve public mosquito control practices should focus on enhancing the perception of the severity of ZIKV.     

Chikungunya infection in Malaysia: Comparison with dengue infection in adults and predictors of persistent arthralgia

BackgroundChikungunya virus (CHIKV) and dengue virus (DENV) co-circulate in areas endemic with the Aedes mosquito vectors. Both viruses cause similar illnesses which may be difficult to distinguish clinically. CHIKV is also associated with persistent arthralgia.ObjectivesTo compare and describe factors which differentiate between DENV and CHIKV infections on presentation; and to describe predictors of persistent arthralgia in CHIKV patients.Study designPatients aged >14 years diagnosed with acute CHIKV and DENV infections in Kuala Lumpur, Malaysia were retrospectively identified. Clinical and laboratory data were obtained from medical records, and compared. CHIKV patients were telephoned 15–24 months later and interviewed about persistent symptoms. Logistic regression analysis was performed.ResultsA total of 53 CHIKV and 113 DENV patients were included. CHIKV patients were older and more likely to be female. CHIKV was independently associated with arthralgia and rash, while DENV was associated with myalgia, raised aspartate transaminase, and leucopaenia. Forty CHIKV patients were followed up, with a median duration of self-reported arthralgia of 3 months (range, 0–24 months). Eighteen (45%) had persistent arthralgia beyond 4 months, for which age >40 years was an independent predictor. At 1 year, 9 (22.5%) patients had arthralgia.ConclusionIn Kuala Lumpur, selected clinical and laboratory predictors help to distinguish between DENV and CHIKV infections. Persistent arthralgia was a frequent sequel of CHIKV infection in this cohort.     

Indirect effects of cigarette butt waste on the dengue vector Aedes aegypti (Diptera: Culicidae)

Despite major insecticide-based vector control programs, dengue continues to be a major threat to public health in urban areas. The reasons for this failure include the emergence of insecticide resistance and the narrowing of the spectrum of efficient products. Cigarette butts (CBs), the most commonly discarded piece of waste, also represent a major health hazard to human and animal life. CBs are impregnated with thousands of chemical compounds, many of which are highly toxic and none of which has history of resistance in mosquitoes. This study was performed to examine whether exposure to CB alters various biological parameters of parents and their progeny. We examined whether the mosquito changes its ovipositional behaviors, egg hatching, reproductive capacity, longevity and fecundity in response to CB exposure at three different concentrations. Females tended to prefer microcosms containing CBs for egg deposition than those with water only. There were equivalent rates of eclosion success among larvae from eggs that matured in CB and water environments. We also observed decreased life span among adults that survived CB exposure. Extracts of CB waste have detrimental effects on the fecundity and longevity of its offspring, while being attractive to its gravid females. These results altogether indicate that CB waste indirectly affect key adult life traits of Aedes aegypti and could conceivably be developed as a novel dengue vector control strategy, referring to previously documented direct toxicity on the larval stage. But this will require further research on CB waste effects on non-target organisms including humans.     

Senescence Affects Endothelial Cells Susceptibility to Dengue Virus Infection

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.     

Enterovirus 71 infection induces apoptosis in Vero cells

The effects of Enterovirus 71 (HEV71) infection on African green monkey kidney cells (Vero) were investigated. It was found that the infected cells showed progressive cellular morphological changes characteristic in apoptotic cells within 10 hours post-infection. The number of apoptotic cells correlated significantly with the number of HEV71 antigen positive cells when cells were labeled using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and stained for HEV71 antigen. Approximately 11, 26, 45 and 50% of the infected cells were apoptotic at 12, 24, 48 and 72 hours post-infection, respectively. Internucleosomal DNA fragmentation, characteristic in the late stage of apoptosis was noted beginning on day 2 post-infection. The DNA fragmentation, however, was absent in cells treated with the heat- and ultraviolet light-inactivated virus inocula. These results demonstrate the capacity of HEV71 to induce apoptosis in the infected cells. The induction, however, requires high level of HEV71 infectivity and the presence of live virus particles, suggesting the need for the presence of specific viral proteins for apoptosis to occur.     

Imipenem Treatment Induces Expression of Important Genes and Phenotypes in a Resistant Acinetobacter baumannii Isolate

Acinetobacter baumannii has emerged as a notorious multidrug-resistant pathogen, and development of novel control measures is of the utmost importance. Understanding the factors that play a role in drug resistance may contribute to the identification of novel therapeutic targets. Pili are essential for A. baumannii adherence to and biofilm formation on abiotic surfaces as well as virulence. In the present study, we found that biofilm formation was significantly induced in an imipenem-resistant (Imp^r) strain treated with a subinhibitory concentration of antibiotic compared to that in an untreated control and an imipenem-susceptible (Imp^s) isolate. Using microarray and quantitative PCR analyses, we observed that several genes responsible for the synthesis of type IV pili were significantly upregulated in the Imp^r but not in the Imp^s isolate. Notably, this finding is corroborated by an increase in the motility of the Imp^r strain. Our results suggest that the ability to overproduce colonization factors in response to imipenem treatment confers biological advantage to A. baumannii and may contribute to clinical success.