The role of lipid rafts in the early stage of Enterovirus 71 infection.

BACKGROUND/AIMS: Although it has been widely accepted that Enterovirus 71 (EV71) enters permissive cells via receptor-mediated endocytosis, the details of entry mechanism for EV71 still need more exploration. This study aimed to investigate the role of lipid rafts in the early stage of EV71 Infection. METHODS: The effect of cholesterol depletion or addition of exogenous cholesterol was detected by immunofluorescence assays and quantitative real-time PCR. Effects of cholesterol depletion on the association of EV71 with lipid rafts were determined by flow cytometry and co-immunoprecipitation assays. Localization and internalization of EV71 and its receptor were assayed by confocal microscpoy and sucrose gradient analysis. The impact of cholesterol on the activation of phosphoinositide 3'-kinase/Akt signaling pathway during initial virus infection was analyzed by Western-blotting. RESULTS: Disruption of membrane cholesterol by a pharmacological agent resulted in a significant reduction in the infectivity of EV71. The inhibitory effect could be reversed by the addition of exogenous cholesterol. Cholesterol depletion post-infection did not affect EV71 infection. While virus bound equally to cholesterol-depleted cells, EV71 particles failed to be internalized by cholesterol-depleted cells. EV71 capsid protein co-localized with cholera toxin B, a lipid-raft-dependent internalization marker. CONCLUSION: Lipid rafts play a critical role in virus endocytosis and in the activation of PI3K/Akt signaling pathway in the early stage of EV71 infection.

An immunochromatographic test for serological diagnosis of scrub typhus.

A fluorescent immunochromatographic test (FM-ICT) was developed for rapid detection of anti-Orientia tsutsugamushi antibodies in serum samples. The FM-ICT was constructed based on the dual-antigen sandwich method. Truncated 56 kDa outer membrane protein of O. tsutsugamushi strain SJ, was expressed in E. coli and mixed with those of Ptan and Gillam strains. A thin line of the protein mixture was precisely sprayed across a nitrocellulose membrane making this the "Test" line. Polyclonal antibodies (pAbs) to O.tsutsugamushi were sprayed in another line across the membrane making this the "Control" line. Fluorescent microspheres conjugated 56 kDa proteins reacting with sample serum will be captured on the "Test" line if the sample contains antibodies to O.tsutsugamushi. Several experimental parameters were optimized. After optimizing the reaction procedure, the results are visible, within 6 min, with the naked eye under ultraviolet light. The limit of detection (LOD) was determined to be 7.63 ng/mL with prepared polyclonal antibodies. No cross-reaction was observed with sera samples from other febrile diseases. In clinical evaluations, the strips showed 94.92% sensitivity (106/112) and 93.75% specificity (56/60). The FM-ICT we developed will provide a new tool for on-site diagnosis of scrub typhus.

Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent, dynamin and caveola-mediated endocytosis pathway.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of the most common agents of viral encephalitis. The infectious entry process of JEV into host cells remains largely unknown. Here, we present a systemic study concerning the cellular entry mechanism of JEV to B104 rat neuroblastoma cells. It was observed that JEV internalization was inhibited by chloroquine and ammonium chloride, both of which can elevate the pH of acidic organelles. However, JEV entry was not affected by chlorpromazine, overexpression of a dominant-negative form of EPS 15 protein, or silencing of the clathrin heavy chain by small interfering RNA (siRNA). These results suggested that JEV entry depended on the acidic intracellular pH but was independent of clathrin. We found that endocytosis of JEV was dependent on membrane cholesterol and was inhibited by inactivation of caveolin-1 with siRNA or dominant-negative mutants. It was also shown, by using the inhibitor dynasore, the K44A mutant, and specific siRNA, that dynamin was required for JEV entry. Phagocytosis or macropinocytosis did not play a role in JEV internalization. In addition, we showed that JEV entry into the neuroblastoma cells is not virus strain specific by assessing the effect of the pharmacological inhibitors on the internalization of JEV belonging to different genotypes. Taken together, our results demonstrate that JEV enters B104 cells through a dynamin-dependent caveola-mediated uptake with a pH-dependent step, which is distinct from the clathrin-mediated endocytosis used by most flaviviruses.

NCAPH is negatively associated with Mcl­1 in non­small cell lung cancer.

Lung cancer has a high mortality rate worldwide. Non­SMC condensin I complex subunit H (NCAPH) has been identified to be one of the regulatory subunits of the condensin I complex, which is essential for the correct packaging and segregation of chromosomes in eukaryotes. NCAPH is abnormally overexpressed in various types of cancer. A pro­survival member of the Bcl­2 family, myeloid cell leukemia sequence 1 (Mcl­1) is also frequently overexpressed in multiple cancers and is associated with poorer clinical outcomes for patients. The association of NCAPH and Mcl­1 proteins with the clinical and pathological features of non­small cell lung cancer (NSCLC) remains to be elucidated. In the current study, the positive percentage of NCAPH in the non­cancerous lung tissues was revealed to be higher compared with that in NSCLC. However, the positive percentage of Mcl­1 in the non­cancerous lung tissues was lower compared with NSCLC. In addition, NCAPH high­expression patients had a higher overall survival rate compared with patients exhibiting low expression, whereas the Mcl­1 high­expression group had a lower survival rate. Pairwise association in 260 cases of NSCLC revealed that overexpression of the NCAPH protein was negatively associated with Mcl­1 expression and vice versa. The results of multivariate Cox proportional hazard regression analysis also indicated that NCAPH and Mcl­1 demonstrated potential as distinct prognostic factors that may be used in NSCLC. The expression of NCAPH and Mcl­1 may be associated with, and act as distinct molecular marks for the prediction of a poor prognosis in patients with NSCLC.

Serologic assay for avian-origin influenza A (H7N9) virus in adults of Shanghai, Guangzhou and Yunnan, China.

BACKGROUND AND OBJECTIVE: To investigate serologic status for novel avian influenza A (H7N9) virus in different areas in China, we examined serum samples collected in winter of 2011 from adult population of Shanghai (eastern China), Guangzhou (southern China) and Yunnan (southwest China) for the antibody responses to this virus. STUDY DESIGN: A total of 900 stored serum samples of adult outpatients (300 samples for each area) were subjected to anti-hemagglutinin (HA) antibodies assay using enzyme-linked immunosorbent assay (ELISA) and neutralizing antibodies assay using H7N9 pseudotyped particles (H7N9pp) and authentic H7N9 virus based neutralization test. RESULTS: Anti-H7 antibodies were detected in 164, 186 and 123 samples from three areas above, respectively. Among anti-H7 positive sera, 20, 42 and 13 samples had neutralizing titers of ≥10 when 8×10(2) focus-forming units (FFU) of H7N9 pseudotyped particles (pp) were adopted in neutralizing assay, respectively. When neutralizing antibodies were assayed using classic microneutralization (MN) test, MN titers of ≥10 were found in 7 samples from Guangzhou, but none from Shanghai and Yunnan. CONCLUSION: Low levels of protective immunity pre-existed in some general adult population of the three areas, and pre-existing immunity against H7N9 in Guangzhou appears stronger than that in Shanghai and Yunnan.

Application of HBx-induced anti-URGs as early warning biomarker of cirrhosis and HCC.

BACKGROUND: Hepatitis B virus (HBV) carriers are at high risk for the development of hepatocellular carcinoma (HCC), but there are no reliable markers that will identify such high-risk patients. HBV up-regulates the expression of selected genes (URGs) in the liver during chronic infection. These aberrantly expressed proteins trigger corresponding antibodies (anti-URGs) that appear prior to the detection of HCC. This study was undertaken to see if the anti-URGs could be used as early warning biomarker of HBV-induced liver cirrhosis and HCC. METHODS: A cross sectional study using a total of 625 serum samples from HBV infected and uninfected controls were tested for the anti-URGs using specific ELISAs. RESULTS: The number and specificity of anti-URGs correlated with the severity of liver disease Anti-URGs were predominantly present among patients with HBV-associated HCC (55.2%) and cirrhosis (60.7%), and at a lower frequency among patients with chronic hepatitis (35.8%), and at still lower frequencies in most asymptomatic carriers (12.3%) with normal ALT, among patients with chronic hepatitis C (38.5%) and blood donors (0.9%). These anti-URGs were rarely detected in sera from those with tumors other than HCC, except among HBV infected patients with cholangioicarcinoma and in some patients with drug induced hepatitis. 3 or more anti-URGs could precede the diagnosis of cirrhosis or HCC 11.8 months on average, and HBV hepatitis patients with 3 or more anti-URGs have much higher risk (5/20 vs 0/30) to develop cirrhosis and HCC than those patients with less anti-URGs. As the early warning biomarker, 3 or more anti-URGs were served as the threshold to separate the cirrhosis and HCC from others with a moderate sensitivity (58.3%) and specificity (80.0%), which was better than other biomarkers (AFP, AFP-L3, GPC3 and GP73) and would improve up to 70.3% when combined with another biomarker. CONCLUSIONS: The results of this clinical validation study suggest that the anti-URGs might have diagnostic/prognostic utility among patients at high risk for the development of cirrhosis and HCC.