Isolation of a novel disintegrin containing Arg-Gly-Asp tripeptide sequence from Gloydius brevicaudus venom and investigation of its biological activities / 药学学报

Snake venom has special pharmacological activities and contains a array of small polypeptides that can antagonize integrins, therefore called disintegrins. Disintegrins can block integrin-dependent platelet aggregation, tumor growth, and tumor metastasis. A disintegrin fraction was isolated and purified from the venom of snake Gloydius brevicaudus (GBV). Its physical and chemical properties were characterized, and its biological activities were investigated. The crude venom of GBV were isolated by Superdex 75 gel filtration chromatography. The anti-platelet aggregation activity of the fractions was screened by the Born method. The fraction that shown anti-platelet activity was further purified with Sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion exchange chromatography, and Lichrospher C18 reversed-phase chromatography respectively. The purity of the active component was analyzed with SDS-PAGE (Tris-Tricine system) and high-performance liquid-phase chromatography (HPLC), with protein concentration determined by the Bradford method. The molecular weight was evaluated by the gel imaging method and mass spectrometry, and the isoelectric point was measured by disc isoelectric focusing electrophoresis. The protease activity was measured with the Rick method. The phospholipase A activity was determined by the automatic potentiometric titration method. Amino acid sequencing results were subjected to homology comparison using the BLAST program. Seven fractions (Ⅰ-Ⅶ) were isolated from GBV by gel filtration chromatography on Superdex 75 column. The fraction Ⅳ inhibited the platelet aggregation induced by ADP with molecular weight lower than 10 000 Da, suggesting a disintegrin component. A disintegrin named GBV-Ⅳ4 was purified from the fraction by Sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18 reverse chromatography. It was homogeneous shown as a single band on SDS-polyacrylmide gel electrophoresis (SDS-PAGE, Tris-Tricine system) with molecular weight 8 746 Da as calculated by Image Master VDS system. The isoelectric point of GBV-Ⅳ4 was 6.3 by disc polyacrylamide gel electrophoresis. GBV-Ⅳ4 exhibited no detectable phospholipase A2 (PLA2) activity with the pH-stat technique or proteinase activity according to the method of Rick. GBV-Ⅳ4 is composed of 70 amino acids with RGD (Arg-Gly-Asp) active region and a molecular weight of 7 442 Dalton as assayed by Mass Spectrography. Characterization of GBV-Ⅳ4 is consistent with meta-chain disintegrin (70 amino acid sequence, six pairs of disulfide bond). Retrieved by Genbank, GBV-Ⅳ4 has high homology with other disintegrins. We concluded that GBV-Ⅳ4 is a novel disintegrin contained RGD. GBV-Ⅳ4 showed dose-dependent inhibition of ADP- or thrombin-induced platelet aggregation with IC50 0.339 or 0.577 μg·mL-1 respectively. In conclusion, a new disintegrin derived from the GBV snake venom and named GBV-Ⅳ4 containing RGD tripeptide sequence could inhibit platelet aggregation.

Risk evaluation on H7N9 avian influenza in Guangzhou, China / 中华流行病学杂志

<p><b>OBJECTIVE</b>We conducted both quick surveillance and evaluation programs within one week after the novel H7N9 influenza cases had been released by the Ministry of Health (MOH), to get the basic information on H7N9 virus in Guangzhou.</p><p><b>METHODS</b>We sampled live birds from food markets and the natural habitat of birds to detect H7N9, H5 and H9 viruses. We interviewed workers from both markets and natural habitats. We also reviewed records on pneumonia patients with unknown causes from the surveillance system, to find clues related to the identification of severe pneumonia.</p><p><b>RESULTS</b>We sampled 300 specimens from 49 stalls in 13 food markets and a natural habitat but none showed H7N9 positive result. A chopping block was detected positive of carrying H5 avian influenza virus, while another 4 specimens including a chicken cage, a duck cage, a chopping block and a pigeon cage were detected positive of carrying H9 avian influenza virus. In the past month, no sick, dead birds or ILI cases among the workers were discovered. 21.2% (7/33) of the stalls did not follow the set regulations for prevention. 10.3% (4/39) of the stalls had the cages cleaned, 4 days after the inspection. 3.7% (2/54) of the workers wore masks and 40.7% (22/54) of them wore gloves during the slaughtering process. 102 bird feces specimens were tested negative on H7N9 virus. No pneumonia cases with unknown reason were identified. From April 3(rd) to 17(th), we found 26 severe pneumonia cases but with negative results on influenza A (H7N9).</p><p><b>CONCLUSION</b>According to the data and information from 1) lab tests, 2) pneumonia cases with unknown reasons under the surveillance system, 3) the identification of severe pneumonia cases, and 4) preventive measures and actions taken by the workers, we inferred that no H7N9 virus or related cases were found prior to April in Guangzhou. However, the risk of H7N9 epidemic does exist because of the following reasons:1) improper market management process, 2) negligent behavior of the workers and 3) potential trend of the national situation, suggesting strategies related to poultry markets management, health education and preventive measures against the avian influenza need to be strengthened.</p>

Genotypes distribution of hepatitis C virus through multi-center, large sample studies among chronic hepatitis C patients in Chinese Han population / 中华流行病学杂志

Objective To understand the distribution of hepatitis C virus (HCV) genotypes in China.Methods A national multi-center,large sample epidemiological survey was carried out and certified by an international third-party testing organization.Internationally accepted method for sequencing analysis of viral genotype was used.Results Patients were those suffered from the 4 major genotypes of chronic hepatitis C,including nine subtypes,which were dominated by genotype 1 (69.6％),with type lb in particular (accounting for 68.38％ ).Among the rare gene-6,we saw more subtypes of 6a.In the south,central,and north areas,there was no significant difference seen between the south and the north areas in the distribution of the genotype 1.However,the rate of gene type 2a increased gradually from south to north.In terms of less common type of gene 3b and gene-6,they were seen mainly in the southern provinces.Conclusion In China,Patients with chronic hepatitis C had 4 HCV genotypes,with 9 subtypes.The rarely seen genotypes 3b and 6 were mainly distributed in the southern provinces.

Relation between clinical detection rates of the novel influenza virus A/H1N1 RNA in confirmed patients in Guangzhou and the disease course / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the relation of the detection rates of the novel influenza virus A/H1N1 RNA in clinically confirmed patients in the 2009 pandemic with the age distribution of the patients and the disease course.</p><p><b>METHODS</b>A total of 151 clinical patients with H1N1 infection were enrolled in this study, from whom 833 dynamic throat swab samples were obtained for detecting the H1N1 RNA using real-time PCR. A statistical analysis of the age distribution was performed among the patients with different disease courses. Chi-square for trend test was used to study the correlation between the detection rates of H1N1 RNA and the time of disease onset.</p><p><b>RESULTS</b>The majority of patients were young with their ages ranging from 10 to 20 years (57.26%) and 20 to 30 years (22.18%). Chi-square for trend test revealed that the positivity rates of the throat swabs in the patients decreased with the prolongation of the disease course (chi(2)=9.784, P=0.002).</p><p><b>CONCLUSION</b>Most of the H1N1 patients are young within the age range of 10-30 years, and the longest disease course can exceed 10 days. The positivity rates of throat swabs from the H1N1 patients decreases with the prolongation of the disease course.</p>

Epidemiological characteristic of first case of locally identified A/H1N1 secondary cases caused by imported source of infection in China / 中华流行病学杂志

Objective To study the first locally identifcd A/HINI secondary cases outbreak in China. Methods Interview and field investigation were integrated to describe the whole process of transmission on each case and to illustrate the relationships between the onset of the disease and the retated factors. Results Two contact persons appearanced fever and whose throat swabs were tested positive to H1N1 viral nucleic acid. The two had a history of contact in a short distance with the initial imported case without any protective measure in the poor air ventilation. The patients clinical situation was slight. The incubation was between 37 hours and 57 hours. No other new case was found after intervention as isolation and antisepsis were taken. Conclusion This event was proved to be an outbreak of local A/H1N1 secondary cases caused by the imported case. The main mode of transmission was personal contact in a short distance without protection, through air and droplet. The locus with poor air ventilation was high risk place. Contact persons should be observed seven days and tested continuously.Infectivity and pathogenicity of the A/H1N1 virus were limited and appeared weakened by generations. Patient's condition was related with persistence and frequency of contact with the infection sources. Enhancing management of contact persons, health education, early diagnose, early treatment and early insulation were effective measures of controling and prenventing the spread A/H1N1.

An analysis on genetic characteristics of hemagglutinin and neuraminidase of type B influenza viruses in Guangzhou in 2006 / 病毒学报

To understand the genetic characteristics of hemagglutinin (HA) and neuraminidase (NA) of type B influenza viruses in Guangzhou in 2006, three virus strains from etiology surveillance and seven strains from outbreaks were investigated. Genome RNAs of type B influenza viruses were extracted and reverse-transcripted into cDNAs using random primers. The whole-length DNA of HA and NA were amplified by polymerase chain reaction (PCR), cloned into T-A plasmid and sequenced, and analyzed phylogenetically by DNAstar software. The results showed that the HA of type B influenza viruses were similar and the homology were more than 99%. The type B influenza viruses belong to Victoria lineage. The NA of the type B influenza viruses were similar and the homology were more than 98%. Phylogenetic trees of HA and NA showed that the isolates from etiology surveillance formed a cluster, and the isolates from outbreaks were separated from the cluster. The homology of the type B influenza viruses with B/Shanghai/361/2002, which is the WHO recommended influenza vaccine strain in 2005-2006, were 88.9%-89.7%. It suggested the protective effect of influenza vaccine against type B influenza viruses used in 2005-2006 in Guangzhou may not be afforded.