Prohibitin plays a critical role in Enterovirus 71 neuropathogenesis.

A close relative of poliovirus, enterovirus 71 (EV71) is regarded as an important neurotropic virus of serious public health concern. EV71 causes Hand, Foot and Mouth Disease and has been associated with neurological complications in young children. Our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. Here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during EV71 infection of motor-neuron NSC-34 cells, which are found at the neuromuscular junctions where EV71 is believed to enter the central nervous system. Meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in NSC-34 cells. Among the candidate proteins, we focused our attention on prohibitin (PHB), a protein that is involved in multiple cellular functions and the target of anti-cancer drug Rocaglamide (Roc-A). We demonstrated that cell surface-expressed PHB is involved in EV71 entry into neuronal cells specifically, while membrane-bound mitochondrial PHB associates with the virus replication complex and facilitates viral replication. Furthermore, Roc-A treatment of EV71-infected neuronal cells reduced significantly virus yields. However, the inhibitory effect of Roc-A on PHB in NSC-34 cells was not through blocking the CRAF/MEK/ERK pathway as previously reported. Instead, Roc-A treated NSC-34 cells had lower mitochondria-associated PHB and lower ATP levels that correlated with impaired mitochondria integrity. In vivo, EV71-infected mice treated with Roc-A survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. Together, this study uncovers PHB as the first host factor that is specifically involved in EV71 neuropathogenesis and a potential drug target to limit neurological complications.

Effects of increased daily physical activity on mental health and depression biomarkers in postmenopausal women.

[Purpose] Little is known about the effectiveness of daily physical activity on depression biomarkers in older adults. This study aimed to investigate the effects of increased daily physical activity for 8 weeks on depression biomarkers in postmenopausal women. [Participants and Methods] Thirty-eight postmenopausal females were randomly assigned into a control or an active group and were asked to wear a uniaxial accelerometer for 8 weeks. Blood samples were obtained at baseline and at the end of the intervention. During the intervention, the active group was asked to increase their physical activity level above their usual lifestyle whereas those in the control group maintained their daily lifestyle. [Results] After the 8-week intervention, the step counts of the participants in the active group increased. The serum concentration of the brain-derived neurotrophic factor and serotonin increased significantly in the active group, but not in the control group, as compared with baseline values. The serum concentration of derivatives of reactive oxygen metabolites and biological antioxidant potential did not change after the intervention in either group. [Conclusion] These findings may suggest that promotion of daily physical activity in postmenopausal women has a positive impact on depression without any change in oxidative stress.

Clinical and Molecular Epidemiology of Carbapenem-Resistant Enterobacteriaceae Among Adult Inpatients in Singapore.

BACKGROUND: Since 2010, the incidence of carbapenem-resistant Enterobacteriaceae (CRE) has been increasing in Singapore. We analyzed the clinical and molecular epidemiology of CRE among adult inpatients in Singapore. METHODS: Quarterly incidence of unique subjects (per 100000 patient-days) with positive clinical and surveillance cultures for CRE were estimated based on mandatory data submitted to the National Public Health Laboratory by public hospitals between 2010 and 2015. CRE-positive adult inpatients were prospectively recruited from 6 public sector hospitals between December 2013 and April 2015. Subjects answered a standardized epidemiologic questionnaire and provided samples for this study. Further clinical information was extracted from subjects' electronic medical records. Whole-genome sequencing was performed on study isolates to determine transmission clusters. RESULTS: Incidence of CRE clinical cultures among adult inpatients plateaued from 2013 (range: 7.73 to 10.32 per 100000 patient-days) following an initial increase between 2010 and end-2012. We prospectively recruited 249 subjects. Their median age was 65 years, 108 (43%) were female, and 161 (64.7%) had carbapenemase-producing Enterobacteriaceae (CPE). On multivariate analysis, prior carbapenem exposure (OR: 3.23; 95% CI: 1.67-6.25) and hematological malignancies (OR: 2.85; 95% CI: 1.10-7.41) were associated with non-carbapenemase-producing CRE (NCPE) (n = 88) compared with CPE (n = 161) subjects. Among 430 CRE isolates from the 249 subjects, 307(71.3%) were CPE, of which 154(50.2%) were blaKPC-positive, 97(31.6%) blaNDM-positive, and 42 (13.7%) blaOXA-positive. Klebsiella pneumoniae (n = 180, 41.9%), Escherichia coli (n = 129, 30.0%) and Enterobacter cloacae (n = 62, 14.4%) were the main Enterobacteriaceae species. WGS (n = 206) revealed diverse bacterial strain type (STs). The predominant blaKPC-positive plasmid was pHS102707 (n = 62, 55.4%) and the predominant blaNDM-positive plasmid was pNDM-ECS01 (n = 46, 48.9%). Five transmission clusters involving 13 subjects were detected. CONCLUSIONS: Clinical CRE trend among adult inpatients showed stabilization following a rapid rise since introduction in 2010 potentially due to infection prevention measures and antimicrobial stewardship. More work is needed on understanding CPE transmission dynamics.

Clarity™ digital PCR system: a novel platform for absolute quantification of nucleic acids.

In recent years, digital polymerase chain reaction (dPCR) has gained recognition in biomedical research as it provides a platform for precise and accurate quantification of nucleic acids without the need for a standard curve. However, this technology has not yet been widely adopted as compared to real-time quantitative PCR due to its more cumbersome workflow arising from the need to sub-divide a PCR sample into a large number of smaller partitions prior to thermal cycling to achieve zero or at least one copy of the target RNA/DNA per partition. A recently launched platform, the Clarity™ system from JN Medsys, simplifies dPCR workflow through the use of a novel chip-in-a-tube technology for sample partitioning. In this study, the performance of Clarity™ was evaluated through quantification of the single-copy human RNase P gene. The system demonstrated high precision and accuracy and also excellent linearity across a range of over 4 orders of magnitude for the absolute quantification of the target gene. Moreover, consistent DNA copy measurements were also attained using a panel of different probe- and dye-based master mixes, demonstrating the system's compatibility with commercial master mixes. The Clarity™ was then compared to the QX100™ droplet dPCR system from Bio-Rad using a set of DNA reference materials, and the copy number concentrations derived from both systems were found to be closely associated. Collectively, the results showed that Clarity™ is a reliable, robust and flexible platform for next-generation genetic analysis.

Local transmission and global dissemination of New Delhi Metallo-Beta-Lactamase (NDM): a whole genome analysis.

BACKGROUND: New Delhi metallo-ß-lactamase (bla NDM), a plasmid-borne carbapenemase gene associated with significant mortality and severely limited treatment options, is of global public health concern as it is found in extremely diverse Gram-negative bacterial strains. This study thus aims to genetically characterize local and global spread of bla NDM. METHODS: To investigate local transmission patterns in the context of a single hospital, whole genome sequencing data of the first 11 bla NDM-positive bacteria isolated in a local hospital were analyzed to: (1) identify and compare bla NDM-positive plasmids; and (2) study the phylogenetic relationship of the bacteria chromosomes. The global analysis was conducted by analyzing 2749 complete plasmid sequences (including 39 bla NDM-positive plasmids) in the NCBI database, where: (1) the plasmids were clustered based on their gene composition similarity; (2) phylogenetic study was conducted for each bla NDM-positive plasmid cluster to infer the phylogenetic relationship within each cluster; (3) gene transposition events introducing bla NDM into different plasmid backbones were identified; and (4) clustering pattern was correlated with the plasmids' incompatibility group and geographical distribution. RESULTS: Analysis of the first 11 bla NDM-positive isolates from a single hospital revealed very low bla NDM-positive plasmid diversity. Local transmission was characterized by clonal spread of a predominant plasmid with 2 sporadic instances of plasmid introduction. In contrast to the low diversity locally, global bla NDM spread involved marked plasmid diversity with no predominant bacterial clone. Thirty-nine (1.4 %) out of the 2749 complete plasmid sequences were bla NDM-positive, and could be resolved into 7 clusters, which were associated with plasmid incompatibility group and geographical distribution. The bla NDM gene module was witnessed to mobilize between different plasmid backbones on at least 6 independent occasions. CONCLUSIONS: Our analysis revealed the complex genetic pathways of bla NDM spread, with global dissemination characterized mainly by transposition of the bla NDM gene cassette into varied plasmids. Early local transmission following plasmid introduction is characterized by plasmid conjugation and bacterial spread. Our findings emphasize the importance of plasmid molecular epidemiology in understanding bla NDM spread.

Tracking inter-institutional spread of NDM and identification of a novel NDM-positive plasmid, pSg1-NDM, using next-generation sequencing approaches.

OBJECTIVES: Owing to gene transposition and plasmid conjugation, New Delhi metallo-ß-lactamase (NDM) is typically identified among varied Enterobacteriaceae species and STs. We used WGS to characterize the chromosomal and plasmid molecular epidemiology of NDM transmission involving four institutions in Singapore. METHODS: Thirty-three Enterobacteriaceae isolates (collection years 2010-14) were sequenced using short-read sequencing-by-synthesis and analysed. Long-read single molecule, real-time sequencing (SMRTS) was used to characterize genetically a novel plasmid pSg1-NDM carried on Klebsiella pneumoniae ST147. RESULTS: In 20 (61%) isolates, blaNDM was located on the pNDM-ECS01 plasmid in the background of multiple bacterial STs, including eight K. pneumoniae STs and five Escherichia coli STs. In six (18%) isolates, a novel blaNDM-positive plasmid, pSg1-NDM, was found only in K. pneumoniae ST147. The pSg1-NDM-K. pneumoniae ST147 clone (Sg1-NDM) was fully sequenced using SMRTS. pSg1-NDM, a 90 103 bp IncR plasmid, carried genes responsible for resistance to six classes of antimicrobials. A large portion of pSg1-NDM had no significant homology to any known plasmids in GenBank. pSg1-NDM had no conjugative transfer region. Combined chromosomal-plasmid phylogenetic analysis revealed five clusters of clonal bacterial NDM-positive plasmid transmission, of which two were inter-institution clusters. The largest inter-institution cluster involved six K. pneumoniae ST147-pSg1-NDM isolates. Fifteen patients were involved in transmission clusters, of which four had ward contact, six had hospital contact and five had an unknown transmission link. CONCLUSIONS: A combined sequencing-by-synthesis and SMRTS approach can determine effectively the transmission clusters of blaNDM and genetically characterize novel plasmids. Plasmid molecular epidemiology is important to understanding NDM spread as blaNDM-positive plasmids can conjugate extensively across species and STs.

Antiviral activity of Lactobacillus reuteri Protectis against Coxsackievirus A and Enterovirus 71 infection in human skeletal muscle and colon cell lines.

BACKGROUND: Recurrence of hand, foot and mouth disease (HFMD) pandemics continues to threaten public health. Despite increasing awareness and efforts, effective vaccine and drug treatment have yet to be available. Probiotics have gained recognition in the field of healthcare worldwide, and have been extensively prescribed to babies and young children to relieve gastrointestinal (GI) disturbances and diseases, associated or not with microbial infections. Since the faecal-oral axis represents the major route of HFMD transmission, transient persistence of probiotic bacteria in the GI tract may confer some protection against HFMD and limit transmission among children. METHODS: In this work, the antiviral activity of two commercially available probiotics, namely Lactobacillus reuteri Protectis (L. reuteri Protectis) and Lactobacillus casei Shirota (L. casei Shirota), was assayed against Coxsackieviruses and Enterovirus 71 (EV71), the main agents responsible for HFMD. In vitro infection set-ups using human skeletal muscle and colon cell lines were designed to assess the antiviral effect of the probiotic bacteria during entry and post-entry steps of the infection cycle. RESULTS: Our findings indicate that L. reuteri Protectis displays a significant dose-dependent antiviral activity against Coxsackievirus type A (CA) strain 6 (CA6), CA16 and EV71, but not against Coxsackievirus type B strain 2. Our data support that the antiviral effect is likely achieved through direct physical interaction between bacteria and virus particles, which impairs virus entry into its mammalian host cell. In contrast, no significant antiviral effect was observed with L. casei Shirota. CONCLUSIONS: Should the antiviral activity of L. reuteri Protectis observed in vitro be translated in vivo, such probiotics-based therapeutic approach may have the potential to address the urgent need for a safe and effective means to protect against HFMD and limit its transmission among children.

Beta-actin variant is necessary for Enterovirus 71 replication.

Enterovirus 71 (EV71) is one of the main etiological agents of the Hand, Foot and Mouth Disease (HFMD) and has been known to cause fatal neurological complications such as herpangina, aseptic meningitis, poliomyelitis-like paralysis and encephalitis. EV71 is endemic in the Asia-Pacific region and causes occasional epidemics. In order to better understand EV71 infection, we compared the proteome between EV71-susceptible and EV71-resistant human Rhabdomyosarcoma (RD) cell line. We found significant differences in the ß-actin variants between the EV71-susceptible RD cells and EV71-resistant RD cells, suggesting that ß-actin, in association with other proteins such as annexin 2 is required in vesicular transport of EV71. This finding further support our previous study that actin potentially plays a role in pathogenesis and the establishment of the disease in HFMD.

A non-mouse-adapted enterovirus 71 (EV71) strain exhibits neurotropism, causing neurological manifestations in a novel mouse model of EV71 infection.

Enterovirus 71 (EV71) is a neurotropic pathogen that has been consistently associated with the severe neurological forms of hand, foot, and mouth disease. The lack of a relevant animal model has hampered our understanding of EV71 pathogenesis, in particular the route and mode of viral dissemination. It has also hindered the development of effective prophylactic and therapeutic approaches, making EV71 one of the most pressing public health concerns in Southeast Asia. Here we report a novel mouse model of EV71 infection. We demonstrate that 2-week-old and younger immunodeficient AG129 mice, which lack type I and II interferon receptors, are susceptible to infection with a non-mouse-adapted EV71 strain via both the intraperitoneal (i.p.) and oral routes of inoculation. The infected mice displayed progressive limb paralysis prior to death. The dissemination of the virus was dependent on the route of inoculation but eventually resulted in virus accumulation in the central nervous systems of both animal groups, indicating a clear neurotropism of the virus. Histopathological examination revealed massive damage in the limb muscles, brainstem, and anterior horn areas. However, the minute amount of infectious viral particles in the limbs from orally infected animals argues against a direct viral cytopathic effect in this tissue and suggests that limb paralysis is a consequence of EV71 neuroinvasion. Together, our observations support that young AG129 mice display polio-like neuropathogenesis upon infection with a non-mouse-adapted EV71 strain, making this mouse model relevant for EV71 pathogenesis studies and an attractive platform for EV71 vaccine and drug testing.

Sustained high levels of interleukin-6 contribute to the pathogenesis of enterovirus 71 in a neonate mouse model.

Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD) in young children and has been consistently associated with the most severe complications of the disease, including central nervous system inflammation and pulmonary edema. Increasing frequency and amplitude of EV71 outbreaks have raised awareness and concerns worldwide. Previous reports proposed that overwhelming virus replication combined with the induction of massive proinflammatory cytokines is responsible for the pathogenicity of EV71. Specifically, elevated interleukin-6 (IL-6) levels were observed consistently in patients and strongly correlated with disease severity. In this study, we show in the neonate mouse model that sustained high levels of IL-6 produced upon EV71 infection lead to severe tissue damage and eventually death of the animals. Administration of anti-IL-6 neutralizing antibodies after the onset of the clinical symptoms successfully improved the survival rates and clinical scores of the infected hosts. Compared to untreated infected controls, anti-IL-6-treated mice displayed reduced tissue damage, absence of splenic atrophy, and increased immune cell activation. In addition, markedly elevated systemic levels of IL-10 were measured in the protected animals. Furthermore, there was no significant difference in virus titers between anti-IL-6-treated mice and untreated mice, indicating that the anti-IL-6 antibody-mediated protection is independent of the virus load. Our findings thus demonstrate that IL-6 plays a major role in EV71-induced immunopathogenesis. As there is still neither vaccine nor treatment available against EV71, anti-IL-6 antibody treatment represents a potential therapeutic approach to providing protection from the most severe complications of the disease.

Comparative in vitro evaluation of the antimicrobial activities of povidone-iodine and other commercially available antiseptics against clinically relevant pathogens.

Aims: Antiseptics, such as povidone-iodine (PVP-I), play an important role in infection control across a wide range of clinical settings. This study aimed to evaluate the comparative in vitro efficacy and rate of onset of action of a range of formulations of PVP-I and other commonly used antiseptics. Methods: The antimicrobial efficacy of a range of antiseptics and antimicrobial agents used for skin, wound, vagina and oral antisepsis was evaluated according to the EU Standards DIN EN1276 and EN14476. The panel of organisms tested included bacterial and fungal pathogens and two enteroviruses (Coxsackievirus A16 [CA16] and Enterovirus 71 [EV71]). Results: All PVP-I products tested were highly efficacious in vitro (>99.99% kill rate) against a range of clinically relevant bacterial and fungal pathogens with rapid onset of action (30-60 seconds), at both high and low concentrations. By comparison, the efficacy of other antiseptics tested was generally reduced upon dilution. PVP-I products used in wound and oral care were found to be more effective in vitro against CA16 and EV71, and had a faster onset of action than most other agents tested. Conclusion: This study provides valuable insights into the in vitro efficacy of a range of commonly used antiseptics and may help inform the selection of appropriate antiseptics by healthcare professionals.

Rapid detection of enterovirus 71 by real-time TaqMan RT-PCR.

BACKGROUND: Enterovirus 71 (EV71) is the main etiological agent of Hand, Foot and Mouth Disease (HFMD) and has been associated with neurological complications which resulted in fatalities during recent outbreaks in Asia Pacific region. OBJECTIVE: Develop a real-time TaqMan RT-PCR for rapid detection of EV71. STUDY DESIGN: Specific primers and probe were designed based on highly conserved VP1 region of EV71. The sensitivity of the real-time RT-PCR was evaluated with 67 clinical specimens collected from pediatric patients with suspected HFMD. RESULTS: Our real-time TaqMan RT-PCR showed 100% specificity in detecting EV71 and showed an analytical sensitivity of 5 viral copies. High sensitivity was also achieved in detecting EV71 directly from clinical specimens. CONCLUSIONS: Real-time TaqMan RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks.

Development of multiplex real-time hybridization probe reverse transcriptase polymerase chain reaction for specific detection and differentiation of Enterovirus 71 and Coxsackievirus A16.

Large outbreaks of hand, foot, and mouth disease have been reported in the Asia Pacific region over the last few years and resulted in significant fatalities. The 2 main etiologic agents are Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16). Both viruses are closely related genetically and show similar clinical symptoms. However, EV71 are associated with neurologic complications and can lead to fatalities. In this study, we developed a multiplex real-time hybridization probe reverse transcriptase polymerase chain reaction to detect and differentiate EV71 from CA16 using the LightCycler (Roche Molecular Biochemicals). Specific primers and hybridization probes were designed based on highly conserved VP1 region of EV71 or CA16. Our results showed high specificity and sensitivities in detecting EV71 or CA16 from 67 clinical specimens, and no other enterovirus serotype was detected. Rapid diagnosis to differentiate EV71 from CA16 in outbreak situations will enable pediatricians to identify and manage the patients more effectively.

Prevalence, Antimicrobial Resistance, and Genetic Diversity of Listeria spp. Isolated from Raw Chicken Meat and Chicken-Related Products in Malaysia.

Listeria spp. are ubiquitous in nature and can be found in various environmental niches such as soil, sewage, river water, plants, and foods, but the most frequently isolated species are Listeria monocytogenes and Listeria innocua. In this study, the presence of Listeria spp. in raw chicken meat and chicken-related products sold in local markets in Klang Valley, Malaysia was investigated. A total of 44 Listeria strains (42 L. innocua and 2 L. welshimeri) were isolated from 106 samples. Antibiotic susceptibility tests of the L. innocua strains revealed a high prevalence of resistance to clindamycin (92.9%), ceftriaxone (76.2%), ampicillin (73.8%), tetracycline (69%), and penicillin G (66.7%). Overall, 31 L. innocua and 1 L. welshimeri strain were multidrug resistant, i.e., nonsusceptible to at least one antimicrobial agent in three or more antibiotic classes. The majority of the L. innocua strains were placed into five AscI pulsogroups, and overall 26 distinct AscI pulsotypes were identified. The detection of multidrug-resistant Listeria strains from different food sources and locations warrants attention because these strains could serve as reservoirs for antimicrobial resistance genes and may facilitate the spread and emergence of other drug-resistant strains.

Enhanced potency and efficacy of 29-mer shRNAs in inhibition of Enterovirus 71.

Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) in young children. It has been associated with severe neurological complications and has caused significant mortalities in large-scale outbreaks in Asia. In this study, we demonstrated an enhanced silencing of EV71 through the use of chemically synthesized 29-mer shRNAs. The 29-mer shRNAs were designed to target three highly conserved regions of EV71 genome. Transfection of rhabdomyosarcoma (RD) cells with the 29-mer shRNAs significantly inhibited EV71 replication in a dose-dependent manner as demonstrated by reduction of viral RNA, VP1 protein and plaque forming units. The inhibitory effects were more potent and were achieved at 10-fold lower concentrations when compared to 19-mer siRNAs reported previously [Sim, A.C.N., Luhur, A., Tan, T.M.C., Chow, V.T.K., Poh, C.L., 2005. RNA interference against Enterovirus 71 infection. Virology 341, 72-79]. The viral inhibitory effects lasted 72 h post-infection and there was no adverse off-target silencing effect. Gene silencing by 29-mer shRNAs targeted at the 3D(pol) region (sh-3D) was the most effective, achieving 91% viral inhibition. Further evaluation found that no enhanced inhibitory effects were observed when sh-3D was cotransfected with each of the other two candidates. This study showed an improvement in triggering RNAi using the more potent 29-mer shRNAs, indicating its therapeutic potential against EV71.

Identification of HIV Mutation as Diagnostic Biomarker through Next Generation Sequencing.

INTRODUCTION: Current clinical detection of Human immunodeficiency virus 1 (HIV-1) is used to target viral genes and proteins. However, the immunoassay, such as viral culture or Polymerase Chain Reaction (PCR), lacks accuracy in the diagnosis, as these conventional assays rely on the stable genome and HIV-1 is a highly-mutated virus. Next generation sequencing (NGS) promises to be transformative for the practice of infectious disease, and the rapidly reducing cost and processing time mean that this will become a feasible technology in diagnostic and research laboratories in the near future. The technology offers the superior sensitivity to detect the pathogenic viruses, including unknown and unexpected strains. AIM: To leverage the NGS technology in order to improve current HIV-1 diagnosis and genotyping methods. MATERIALS AND METHODS: Ten blood samples were collected from HIV-1 infected patients which were diagnosed by RT PCR at Singapore Communicable Disease Centre, Tan Tock Seng Hospital from October 2014 to March 2015. Viral RNAs were extracted from blood plasma and reversed into cDNA. The HIV-1 cDNA samples were cleaned up using a PCR purification kit and the sequencing library was prepared and identified through MiSeq. RESULTS: Two common mutations were observed in all ten samples. The common mutations were identified at genome locations 1908 and 2104 as missense and silent mutations respectively, conferring S37N and S3S found on aspartic protease and reverse transcriptase subunits. CONCLUSION: The common mutations identified in this study were not previously reported, therefore suggesting the potential for them to be used for identification of viral infection, disease transmission and drug resistance. This was especially the case for, missense mutation S37N which could cause an amino acid change in viral proteases thus reducing the binding affinity of some protease inhibitors. Thus, the unique common mutations identified in this study could be used as diagnostic biomarkers to indicate the origin of infection as being from Singapore.

Droplet digital PCR as a useful tool for the quantitative detection of Enterovirus 71.

Hand, foot and mouth disease (HFMD) is a contagious viral disease that frequently affects infants and children and present with blisters and flu-like symptoms. This disease is caused by a group of enteroviruses such as enterovirus 71 (EV71) and coxsackievirus A16 (CA16). However, unlike other HFMD causing enteroviruses, EV71 have also been shown to be associated with more severe clinical manifestation such as aseptic meningitis, brainstem and cerebellar encephalitis which may lead to cardiopulmonary failure and death. Clinically, HFMD caused by EV71 is indistinguishable from other HFMD causing enteroviruses such as CA16. Molecular diagnosis methods such as the use of real-time PCR has been used commonly for the identification of EV71. In this study, two platforms namely the real-time PCR and the droplet digital PCR were compared for the detection quantitation of known EV71 viral copy number. The results reveal accurate and consistent results between the two platforms. In summary, the droplet digital PCR was demonstrated to be a promising technology for the identification and quantitation of EV71 viral copy number.

Proteomic profiling of chikungunya virus-infected human muscle cells: reveal the role of cytoskeleton network in CHIKV replication.

Chikungunya virus (CHIKV) is an arthropod-borne, positive-sense, single-stranded RNA virus belonging to genus Alphavirus and family Togaviridae. The clinical manifestations developed upon CHIKV-infection include fever, myositis, arthralgia and maculopapular rash. Thus, the re-emergence of CHIKV has posed serious health threats worldwide. Due to the fact that myositis is induced upon CHIKV-infection, we sought to understand the dynamic proteomic regulation in SJCRH30, a human rhabdomyosarcoma cell line, to gain insights on CHIKV pathogenesis. Two-dimensional gel electrophoresis (2DE) in combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to profile differential cellular proteins expression in CHIKV-infected SJCRH30 cells. 2DE analysis on CHIKV-infected cells has revealed 44 protein spots. These spots are found to be involved in various biological pathways such as biomolecules synthesis and metabolism, cell signaling and cellular reorganization. siRNA-mediated gene silencing on selected genes has elucidated the biological significance of these gene-translated host proteins involved in CHIKV-infection. More importantly, the interaction of vimentin with non-structural protein (nsP3) of CHIKV was shown, suggesting the role played by vimentin during CHIKV replication by forming an anchorage network with the CHIKV replication complexes (RCs). BIOLOGICAL SIGNIFICANCE: Chikungunya virus (CHIKV) is a re-emerging virus that has caused various disease outbreaks in Africa and Asia. The clinical symptoms of CHIKV-infection include fever, skin rash, recurrent joint paint, and myositis. Neuronal implications and death may be resulted from the severe viral infection. Up to date, there are no effective treatments and vaccines against CHIKV-infection. More importantly, little is known about the differential regulation of host proteins upon CHIKV infection, hence deciphering the viral-host cell interactions during viral infection provide critical information on our understanding on the mechanisms of virus infection and its dependency of host proteins for replication. In light of the muscle-related clinical manifestations of myositis resulting from CHIKV-infection, human rhabdomyosarcoma cells, SJCRH30 were utilized in this protein profiling study, in order to decipher the pathogenesis of CHIKV. This study has identified an arrays of host proteins that are differentially regulated upon CHIKV infection including that of the cytoskeletal protein, vimentin that plays significant role in aiding the replication of CHIKV within the host cells through 2DE assay. Immunofluorescence assay further shows that the novel interaction between cytoskeleton structure and CHIKV replication complex by forming an intercalating network around the replication complexes and facilitating various stages of the virus life cycle. This novel finding has inevitably led to a deeper understanding of CHIKV pathogenesis in revealing the importance of host proteins during CHIKV replication, as well as contributing to the development of specific antiviral strategies against this medically important viral pathogen.

Enterovirus71 (EV71) utilise host microRNAs to mediate host immune system enhancing survival during infection.

Hand, Foot and Mouth Disease (HFMD) is a self-limiting viral disease that mainly affects infants and children. In contrast with other HFMD causing enteroviruses, Enterovirus71 (EV71) has commonly been associated with severe clinical manifestation leading to death. Currently, due to a lack in understanding of EV71 pathogenesis, there is no antiviral therapeutics for the treatment of HFMD patients. Therefore the need to better understand the mechanism of EV71 pathogenesis is warranted. We have previously reported a human colorectal adenocarcinoma cell line (HT29) based model to study the pathogenesis of EV71. Using this system, we showed that knockdown of DGCR8, an essential cofactor for microRNAs biogenesis resulted in a reduction of EV71 replication. We also demonstrated that there are miRNAs changes during EV71 pathogenesis and EV71 utilise host miRNAs to attenuate antiviral pathways during infection. Together, data from this study provide critical information on the role of miRNAs during EV71 infection.