Experimental study on polysaccharide of Cipangopaludina chinensis against HBV in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To evaluate the biological activity of polysaccharide of Cipangopaludina chinensis (PCC) against HBV in vitro.</p><p><b>METHOD</b>HepG2 2. 2. 15 cells were taken as the in vitro experimental model. The cell toxicity was observed by MTT. PCC of different safe concentrations and positive control medicine 3TC were added into the cells. Cell control without medicine was set at the same time. Cultural supernatants were collected at 9 d. HBsAg and HBeAg in cultural supernatants were tested by ELISA. The content of HBV-DNA was detected by TaqMan probe fluorescence quantitative PCR.</p><p><b>RESULT</b>TC0 and TC50 of PCC in HepG2 2. 2. 15 cell culture were 1 g . L-1 and >10 g . L-1, respectively, suggesting low toxicity in cells. IC50 of PCC in HepG2 2. 2. 15 cells HBsAg and HBeAg were 0. 501, 0. 401 g. L-1, with SI being >19.96 and >24. 94, respectively. PCC could effectively inhibit the secretion of HBsAg and HBeAg, and have a better effect on HBeAg than on HBsAg. PCC had a significant inhibitory effect on HBV-DNA in HepG2 2. 2. 15 cells at concentrations of 0. 1, 1 g . L-1 P <0.05).</p><p><b>CONCLUSION</b>PCC has the effect against HBV activity in vitro to some extent, with low toxicity, thereby having a good prospect for application.</p>

Development of a GeXP based multiplex RT-PCR assay for simultaneous detection of eight arboviruses related to encephalitis / 病毒学报

Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) is currently available in virus detection and defined as the simultaneous amplification of two or more DNA/RNA targets in a single reaction vessel. In this study, we attempted to modify the conventional mRT-PCR technique on a basis of GenomeLab Genetic Analysis System (GeXP). Initially, we optimized the analytical validation of the GeXP analyzer and its design of workflow and simultaneously detected eight arboviruses that related to epidemic encephalitis by verifying the specificity of mRT-PCR with Japanese encephalitis virus(JEV) cell cultures and positive strains identified previously and determining the sensitivity with in vitro-transcribed RNA of serial dilutions. The GeXP system after optimization could amplify the specific fragments related to the viruses and exposed specifically a total of 13 target genes out of eight types of arboviruses at the level of 10(2) copies/microL, and the findings suggest that the novel protocol we developed can be high-throughput and highly specific and sensitive as well as quickness in screening of the encephalitis viruses, and is promising in detection of encephalitis-associated viruses for molecular epidemiological studies.

Human enterovirus 71 (EV71) resisted type I interferon induced antiviral effect / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate human enterovirus 71 (EV71) resistance to type I interferon induced antiviral effect.</p><p><b>METHODS</b>After type I interferons (alpha, beta) were incubated with HeLa cells, recombinant type I herpes simple virus (HSV-1) with green fluorescent protein (GFP) was inoculated onto the HeLa cells. HSV-1 proliferation was observed by GFP expression and PCR. After EV71 was inoculated onto HeLa cells incubated with the same quantity of interferon, proliferation of EV71 were detected by RT-PCR of 2A gene.</p><p><b>RESULTS</b>Recombinant HSV-1 GFP expression and viral DNA replication obviously decreased in HeLa cells incubated with type I interferon (alpha, beta). However, EV71 effectively proliferated in the interferon irritated HeLa cell by RT-PCR.</p><p><b>CONCLUSION</b>HeLa cell irritated by type I interferon (alpha, beta) produced antiviral substance that inhibits HSV-1 proliferation. EV71 resisted the antiviral substance induced by type I interferon and could significantly replicate in the HeLa cells.</p>

Study on the phenomenon of splashes and sprays from virology / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the phenomenon of accidental splashes and sprays from manipulation of recombinant virus material and to measure the approximate spilled distance when recombinant virus material inadvertently dropped in the biosafety laboratory.</p><p><b>METHODS</b>first, two groups owning different experience simulated the course of accidental spills and splashes by recombinant adenovirus (rADV) which expressed green fluorescence protein (GFP), the GFP signal were observed in 96 well cell plate after spills appeared; Second, the routine two heights (75 cm and 110 cm) and capacity (1 ml, 1.5 ml, 4 ml and 8 ml) of virus were chose to simulate the experiment of unexpected dropping.</p><p><b>RESULTS</b>First, the positive quantity of the first group owning 5 years' experience is much less than the second group owning 2 years' work experience, the former was 7 positive wells, the latter was 81 positive when they used the pipette to operation. Second, when the unclosed test tubes (1 ml, 1.5 ml, 4 ml and 8 ml recombinant virus) inadvertently dropped, the largest spill distance was 0.92 m, 1.57 m, 2.63 m and2.68 m respectively.</p><p><b>CONCLUSION</b>The better experience is important to make sure safety when we make infectious material; the contaminated distance increased with the amount of recombinant virus material.</p>

Molecular interaction between PrP protein and the signal protein 14-3-3 beta / 病毒学报

The molecular interaction between PrP and 14-3-3 beta and the possible interactional domain between two proteins were studied by co-immunoprecipitation, pull down and FRET assays. The results showed that PrP protein could interact with 14-3-3 beta in vitro and in vivo. The domain which responded for the interaction was located at C-terminal of PrP (amino acid residues 106 to 126). This study of the interaction between PrP and 14-3-3 protein further provided the insight into the potential role of 14-3-3 in the biological function of PrP and the pathogenesis of prion disease.

The construction and identification of the PRNP vectors with ubiquitin or the lysosome-targeting signal / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate PrP expression characteristic of PRNP nucleic acid vaccine vector with ubiquitin or the lysosome-targeting signal.</p><p><b>METHODS</b>The gene of ubiquitin and lysosome-targeting signal were ligated to PRNP and pcDNA3.1 vector that is, pcDNA3.1-UPrP and pcDNA3.1-PrPL were constructed. The expression characteristics of PrP with two signals were evaluated by Western Blot and the localization was observed by indirect immune fluorescence.</p><p><b>RESULTS</b>The protein expressed by pcDNA3.1-UPrP and pcDNA3.1-PrPL with ubiquitin and lysosome-targeting signal can be recognized by prion-specific antibody. The protein has three glycosylation molecules form as native PrP.PrP with ubiquitin was degraded gradually with time extension,whereas quantity of PrP with lysosome signal reduced in 48 h after transfection. The protein with two location signals can direct fusion proteins to cytoplasm.</p><p><b>CONCLUSION</b>The PRNP vectors with ubiquitin or the lysosome-targeting signal were constructed and expressed in eukaryocyte successfully. There will be one of good foundation on PRNP nucleic acid vaccine.</p>

Investigating the risk of two model virus in the laboratory / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the survival time of recombination rival in environment and inactivation ability of different disinfectant and ultraviolet radiation against virus.</p><p><b>METHODS</b>NC membranes absorbed the recombinant adenovirus (rADV) or herpes simplex virus (rHSV) with green fluorescence protein (GFP) were laid, or immersed in various concentration of different disinfectants such as ethanol, sodium hypochlorite, lysol and geramine and then taked out them every 15 min, or exposed under ultraviolet radiation, then the NC membranes were adsorbed 1 h in cell, 37 degrees C 5% CO2 48 h. The results were observed under the fluorescence microscope.</p><p><b>RESULTS</b>(1) the average survival time of rHSV under environment is less than 60 min, rADV is almost up to 2 h. (2) The infection ability of rHSV and rADV was inactived 15 min by both ethanol (100%, 70% and 50%) and sodium hypochlorite (5%, 2.5% and 1.25%). (3) Two virus can be killed by 0.1% bromogeramine. (4) Both 5% and 2.5% lysol, but rADV can not lost the infection on Vero Cell until 75 min by 1.25% Lysol. (5) The rHSV was inactivated under ultraviolet radiation, but rADV was not.</p><p><b>CONCLUSION</b>The survival time of is different from both envelope rival and the no-envelope viral under nature environment and the inactivate ability of disinfectant also is different between two model virus; Disinfectant should be choose according to virus type.</p>