Intracellular calcium ions facilitate dengue virus entry into endothelial cells and compromise endothelial barrier integrity

Objective: To investigate the involvement of Ca2+ in dengue virus (DENV)-infected human umbilical vein endothelial cells (HUVECs) and the disruption of endothelial integrity. Methods: HUVECs were infected with DENV-2 in the presence of intracellular Ca2+ or endoplasmic reticulum Ca2+ chelators. Virus infectivity was measured by focus-forming assay and quantitative RT-PCR. Intracellular Ca2+ was measured using Fluo-4-AM dye. VE-cadherin and focal adhesion kinase (FAK) expressions were investigated by immunofluorescence and immunoblotting assays, respectively. Results: DENV infection increased intracellular cytosolic Ca2+ levels and caused disassembly of the adherens junction protein, VEcadherin as evidenced by decreased VE-cadherin expression at the periphery of DENV-2 infected HUVECs. Depletion of intracellular Ca2+ stores, particularly those of the endoplasmic reticulum Ca2+, significantly decreased DENV yield in HUVECs. Decreased virus yield following the depletion of intracellular Ca2+ was caused by the inhibition of viral entry into HUVECs and not the inhibition of viral binding or attachment. DENV-2 infection also resulted in Ca2+- dependent activation of FAK. Conclusions: Intracellular Ca2+ is required for the early phases of DENV infection in endothelial cells. Increased cytosolic Ca2+ levels in endothelial cells during DENV infection activated FAK, disrupted adherens junctions and compromised barrier integrity. Thus, Ca2+ plays an important role in DENV infection in endothelial cells.

Microbiological analysis of raw milk unveiled the presence of a dairy contaminant, Corynebacterium lipophiloflavum

Dairy farming occupied a distinct position in agriculture since milk can be harvested every day, providing a regularsource of income to the farmers. Development of the Malaysian dairy farming industry was marred by poor farmhygiene practices, leading to the proliferation of dairy-spoilage bacteria, affecting milk quality. In this study, wereport the isolation and characterization of a rare Corynebacterium species from raw milk after the implementationof improved farm hygiene practices. All milking equipment, farm worker’s hands and the cow’s udders and teatswere washed with detergent and wiped dry with clean towels before milk sample collection. Collected foremilksamples from mastitis-free cows were inoculated onto Petrifilm™ and cultured colonies were plated onto nutrientagar. Biochemical and molecular tests were performed for the identification of peculiar bacterial isolates. A uniqueyellow-pigmented bacteria isolate was recovered from the milk of a healthy cow after the adoption of improvedfarm hygiene practices. Phenotypic and genotypic characterization confirmed the milk isolate as Corynebacteriumlipophiloflavum. This is the first description of C. lipophiloflavum in cow’s milk and could possibly imply theinfluence of bovine flora in dairy contamination. The findings highlight the increasing spectrum of Corynebacteriumspecies with potential adverse impact to the dairy industry. It is recommended to screen for C. lipophiloflavum in allmilk processing facility to ensure that milk is safe for consumption and its products prepared to the highest qualityand safety standards.

External quality assessment of dengue and chikungunya diagnostics in Asia Pacific region, 2015

OBJECTIVE: To conduct an external quality assessment (EQA) of dengue and chikungunya diagnostics among national-level public health laboratories in the Asia Pacific region following the first round of EQA for dengue diagnostics in 2013. METHODS: Twenty-four national-level public health laboratories performed routine diagnostic assays on a proficiency testing panel consisting of two modules. Module A contained serum samples spiked with cultured dengue virus (DENV) or chikungunya virus (CHIKV) for the detection of nucleic acid and DENV non-structural protein 1 (NS1) antigen. Module B contained human serum samples for the detection of anti-DENV antibodies. RESULTS: Among 20 laboratories testing Module A, 17 (85%) correctly detected DENV RNA by reverse transcription polymerase chain reaction (RT-PCR), 18 (90%) correctly determined serotype and 19 (95%) correctly identified CHIKV by RT-PCR. Ten of 15 (66.7%) laboratories performing NS1 antigen assays obtained the correct results. In Module B, 18/23 (78.3%) and 20/20 (100%) of laboratories correctly detected anti-DENV IgM and IgG, respectively. Detection of acute/recent DENV infection by both molecular (RT-PCR) and serological methods (IgM) was available in 19/24 (79.2%) participating laboratories. DISCUSSION: Accurate laboratory testing is a critical component of dengue and chikungunya surveillance and control. This second round of EQA reveals good proficiency in molecular and serological diagnostics of these diseases in the Asia Pacific region. Further comprehensive diagnostic testing, including testing for Zika virus, should comprise future iterations of the EQA.

Enterovirus 71 in Malaysia: A decade later

In the last decade, Malaysia has experienced several hand, foot and mouth disease (HFMD) epidemics, complicated by fatalities due to severe neurological involvement. Enterovirus 71 (EV-71) has been implicated as the major causative agent for these epidemics. EV-71 infection is a global public health problem with pandemic potential. In many parts of Asia-Pacifi c, the virus has emerged as one of the most deadly virus infections amongst young children. The virus is highly transmissible through faecaloral route and respiratory droplets. A recent rise in neurological complications and deaths suggests that the viruses currently circulating may be more virulent. The major risk factor associated with more severe EV-71 infection is young age and poor cellular immunity. Rapid laboratory diagnosis and molecular surveillance is important to closely monitor the emergence of new EV-71 subgenotypes. Since vaccine and anti-virals for EV-71 are not available, control and prevention strategies remain the only ways to combat the infection.

Nipah virus: Phylogeny and replication

Phylogenetic analysis of Nipah virus isolates from Malaysia, Bangladesh and Cambodia suggested the presence of at least two different clusters of NiV strains. Based on the major glycoprotein (G) gene, the Nipah virus-Tambun isolate clustered with Nipah virus isolates from Cambodia and Bangladesh, whereas the remaining isolates from Malaysia clustered in a separate cluster. Sequence heterogeneity among the Nipah virus isolates from Malaysia was noted but the overall genomic sequence divergence value was small, suggesting a possible recent introduction of the virus. Nipah virus replicated well in porcine stable kidney cells and human lung fibroblast cells. Human monocytes, on the other hand were infected with Nipah virus but the cells did not support productive infection. Similarly, infection of human neuronal cells did not result in release of high infectious virus yield. The monocytes can serve to disseminate Nipah virus from site of infection including across the blood-brain barrier. And in the brain, Nipah virus is probably spread through cell-to-cell spread mechanism.

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.

Outlook of dengue in Malaysia: a century later

Dengue continues to be a major health threat to Malaysia a century after its first reported outbreak in 1902. Examination of the available outbreak data suggested that a major DF/DHF outbreak occurred in Malaysia in a cyclical pattern of approximately every 8 years. All four dengue virus serotypes are found co-circulating in Malaysia, but after the first and only major outbreak involving DEN-4 in 1960's, only DEN-1, DEN-2 and DEN-3 were associated with DF/DHF outbreaks. It is argued that perhaps the spread of the later dengue virus serotypes followed the pattern of spread of the mosquito vector Aedes aegypti, whereas the former was associated with Aedes albopictus, the outdoor and rural area dwelling mosquito. Estimating from the trend and pattern of dengue and the associated dengue virus serotypes, unless there is a major breakthrough in dengue vaccine development, it is likely that dengue outbreaks will continue to occur in Malaysia throughout the 21st century.

Antigenic cell associated dengue 2 virus proteins detected in vitro using dengue fever patients sera

At least three major antigenic dengue 2 virus proteins were recognized by pooled dengue fever patients' sera in infected Aedes albopictus (C6/36) mosquito cells. Dengue virus envelope (E), premembrane (PrM) and non-structural protein 1 (NS 1) dimer were detected beginning on day 3 postinfection in both the cell membrane and cytosolic fractions. Using the patients' sera, the presence of antigenic intermediate core protein (C)-PrM and NS1-non-structural protein 2a (NS2a) in the cytoplasmic fraction of dengue 2 virus infected cells was revealed. The presence of a approximately 92 and approximately 84 kDa NS 1 dimer in the membrane (NS 1m) and cytosolic (NS 1c) fractions of C6/36 cells, respectively, was also recognized. Using individual patient's serum, it was further confirmed that all patients' sera contained antibodies that specifically recognized E, NS 1 and PrM present in the dengue 2 virus-infected cell membrane fractions, suggesting that these glycosylated virus proteins were the main antigenic proteins recognized in vivo. Detection of dengue 2 virus C antibody in some patients further suggested that C could be antigenic if presented in vivo.