Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle – nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses. Importance JEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.
One hundred and six patients suffering from severe dehydrating diarrhoea were studied of whom 36 patients were positive for Vibrio cholerae. Out of 36, 15 were positive for V. cholerae O1, 10 for V. cholerae O139 and 11 for V. cholerae non-O1 non-O139. O1 and O139 were positive for the 301-bp ctxA amplicon and 471-bp tcpA amplicon indicating that the strains possessed toxigenic capability whereas no non-O1 non-O139 strain possessed ctxA or tcpA genes. Post-admission severity of purging and amount of ORS required were less in the V. cholerae non-O1 non-O139 group (P < 0.05) compared to the V. cholerae O1 and O139 groups. It appears from this study that a cholera-like clinical condition can be caused in the absence of CT as exemplified by strains of non-O1 non-O139.
Background Cholera, an acute diarrheal disease is a major public health problem in many developing countries. Several rapid diagnostic tests (RDT) are available for the detection of cholera, but their efficacies are not compared in an endemic setting. In this study, we have compared the specificity and sensitivity of three RDT kits for the detection of Vibrio cholerae O1 and compared their efficiency with culture and polymerase chain reaction (PCR) methods. Methods Five hundred six diarrheal stool samples collected from patients from two different hospitals in Kolkata, India were tested using SD Bioline Cholera, SMART-II Cholera O1 and Crystal-VC RDT kits. All the stool samples were screened for the presence of V. cholerae by direct and enrichment culture methods. Stool DNA-based PCR assay was made to target the cholera toxin (ctxAB) and O1 somatic antigen (rfb) encoding genes. Statistical evaluation of the RDTs has been made using STATA software with stool culture and PCR results as the gold standards. The Bayesian latent class model (LCM) was used to evaluate the diagnostic tests in the absence of the gold standard. Results Involving culture technique as gold standard, the sensitivity and specificity of the cholera RDT kits in the direct testing of stools was highest with SAMRT-II (86.1%) and SD-Cholera (94.4%), respectively. The DNA based PCR assays gave very high sensitivity (98.4%) but the specificity was comparatively low (75.3%). After enrichment, the high sensitivity and specificity was detected with SAMRT-II (78.8%) and SD-Cholera (99.1%), respectively. Considering PCR as the gold standard, the sensitivity and specificity of the RDTs remained between 52.3–58.2% and 92.3–96.8%, respectively. In the LCM, the sensitivity of direct and enrichment testing was high in SAMRT-II (88% and 92%, respectively), but the specificity was high in SD cholera for both the methods (97% and 100%, respectively). The sensitivity/specificity of RDTs and direct culture have also been analyzed considering the age, gender and diarrheal disease severity of the patients. Conclusion Overall, the performance of the RDT kits remained almost similar in terms of specificity and sensitivity. Performance of PCR was superior to the antibody-based RDTs. The RTDs are very useful in identifying cholera cases during outbreak/epidemic situations and for making them as a point-of-care (POC) testing tool needs more improvement.
With changes in livestock management practices and food processing industry, along with changes in people's food habits, many diseases have emerged. Infection with verotoxin-producing Escherichia coli (VTEC) is one such illness. In the present study an attempt was made to isolate, identify and characterize VTEC strains with reference to the O157:H7 serotype from animal, human sources and some food products with the aid of the available modern methods. A total of 876 samples (330 animal, 184 human, 362 food samples) were screened for the presence of VTEC by conventional as well as PCR technique. Seventeen VTEC strains (12 animal, one human and four food samples) were isolated. The isolation rate was higher in diarrhoeic animals (6.02%), followed by diarrhoeic handler (3.12%) and raw beef (1.78%) samples. All strains showed the presence of the VT gene by PCR tests and were uniformly sensitive to common antibiotics except tetracycline, cephalexin, dicloxacillin, erythromycin and lincomycin. Since all strains were isolated from various sources of animal and human origin and all strains showed the presence of the VT gene and uniform antibiogram, a zoonotic association is suggested. This study marks the first report of isolation of VTEC strains from animal sources in India.
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