Effects of traditional korean medicine Lurong Dabu Decoction on TLR4/WNT-5A signaling pathway in guinea pigs with cough variant asthma / 中国药理学通报

Aim To discuss the mechanism of Lurong Dabu Decoction on cough variant asthma. Methods Guinea pigs were divided into normal group(CON), model group(OVA), Lurong Dabu Decoction high-dose group(HIGH),low-dose group(LOW), and dexamethasone group(DEX)at random. The CVA model was established by smoking plus injection of OVA, aluminum hydroxide solution and nebulized inhalation to stimulate cough. Gguinea pigs were dissected 24 hours after the last challenge to obtain alveolar lavage fluid(BALF)and lung tissues. Immunoadsorption(ELISA)method was applied to detect the types of inflammatory cells and the content of inflammatory cytokines in BALF; HE and Masson staining of the middle lobe of the left lung were used to observe the pathological changes in lung tissues; immunohistochemical staining was used to observe TLR4 and WNT-5A protein expression and distribution of lung tissues; the protein extracted from the upper lobe of the left lung was used to measure the level of TLR4 and WNT-5A protein in lung tissues by Western blot; immunofluorescence was employed to measure the fluorescence intensity of TLR4 and WNT-5A in lung tissues; flow cytometry was used to detect IL-4 and IFN-γ in guinea pig lung tissues. Results Lurong Dabu Decoction could improve guinea pig airway inflammation, inhibit collagen fiber deposition, reduce the content of IL-4, IL-5, and IL-13 in BALF, and inhibit the protein expression of TLR4 and WNT-5A in lung tissues and increase IFN-γ levels in lung tissues while decreasing IL-4 levels. Conclusion Lurong Dabu Decoction may inhibit the occurrence of CVA through TLR4/WNT-5A signaling pathway.

Hepatitis B Immunoprophylactic Failure and Characteristics of the Hepatitis B Virus Gene in Mother-Infant Pairs in Parts of China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To determine the hepatitis B immunoprophylactic failure rate in infants born to hepatitis B virus (HBV) infected mothers and to characterize HBV genes.</p><p><b>METHODS</b>HBV-serological testing was conducted for pregnant women and infants. The complete genomes of 30 HBV isolates were sequenced, and genetic characteristics were analyzed using MEGA 5 software.</p><p><b>RESULTS</b>The immunoprophylactic failure rate for infants who had completed the scheduled hepatitis B vaccination program was 5.76% (32/556). High sequence homology (99.8%-100%) was observed in 8 of the 10 mother-infant pairs. We identified 19 subgenotype C2 strains, 9 subgenotype B2 strains, and 2 subgenotype C1 strains. Three serotypes were detected: adr (19/30), adw (9/30), and ayw (2/30). The frequency of amino acid mutation of the 'a' determinant region was 16.67% (5/30), including that of Q129H, F134Y, S136Y, and G145E. We detected 67 amino acid mutations in the basal core promoter, precore, and core regions of the genome.</p><p><b>CONCLUSION</b>The immunoprophylactic failure rate in infants born to HBV-infected mothers is low in the regions of China examined during this study. Moreover, HBV mutation in the 'a' determinant region could not account for immunoprophylactic failure for all infants.</p>

Comparison of ELISA diagnostic kits used in China for hepatitis C virus IgG antibody detection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To compare the commercial diagnostic reagent available in China for hepatitis C virus ( HCV) IgG antibody detection in order to provide some useful information for HCV prevention.</p><p><b>METHODS</b>The HCV-IgG detection reagents produced by six different Enterprises named A, B, C, D, E and F were chosen and applied to detect 160 HCV specious sera samples. HCV-IgG reagent from ABBOTT was adopted as gold-standard and the samples in gray zone were determined by RIBA method finally.</p><p><b>RESULTS</b>160 sera samples comprised 88 positive samples and 72 negative samples. The total conformity ranged from 88.13% to 98.13% and the Youden indexes ranged from 0.74 to 0.96 when the reagents from six different Enterprises were compared with gold-standard. The highest conformity was 98.13%, observed in D reagent with the highest Youden index of 0.96.</p><p><b>CONCLUSION</b>The total conformity rates were more than 88% when the HCV-IgG antibody detection reagents from six different Enterprises were compared with ABBOTT reagent. It was highly conformable. However, some reagent proved to be less conformable in negative samples detection.</p>

Establishment and application of TaqMan real-time RT-PCR for the detection of hepatitis E virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis E virus (HEV).</p><p><b>METHODS</b>According to the references, primers-probe sets which were located in ORF2, the conservative part of HEV genome were designed and therefore we established a HEV TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HEV RNA in clinical samples.</p><p><b>RESULTS</b>The HEV Real-time RT-PCR assay established in this study were able to detect HEV RNA with a detection limit of 10 copies/reaction. When the detection of a same sample was repeated for several times, coefficients of variation (CV) was all less than 1.53%. Our data also suggested that there were 1.87 x 10(6)-8.12 x 10(9) RNA copies in 1 ml of the clinical samples.</p><p><b>CONCLUSION</b>The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HEV RNA. It was applied successfully in the pathogen detection of clinical samples.</p>

Investigation of the relationship between apolipoprotein gene polymorphism and hepatitis B virus infection in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the gene polymorphisms of ApoAI-75 Msp1, ApoB Msp1, ApoCIII Sst1, LRP5, and ApoE genotypes in two pairs of semi different modes of hepatitis B for HBV markers.</p><p><b>METHODS</b>The patients are divided into 9 groups. There were a total of 720 cases, 80 patients in each group, The patients was carried out by SnaPshot method (single-base multilocus micro-sequencing), and different genotypes of each locus were conducted by the method of sequencing in order to support the final evidence of the accuracy of test results.</p><p><b>RESULTS</b>There was association between gene polymorphisms of ApoAI-75Msp1 and ApoE and different modes of two pairs of semi-hepatitis B (P < 0.05), while there wasn't any association between gene polymorphisms of ApoB-Msp1, ApoCIII-Sst1, LRP5 and different modes of two pairs of semi-hepatitis B (P > 0.05).</p><p><b>CONCLUSION</b>The gene polymorphism of ApoAI-75Msp1 and ApoE was associated with the different modes of HBV markers.</p>

Value of combined detection of GP73 and alpha-fetoprotein in diagonosis of hepatocellular carcinoma / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the diagnostic value of the measurement of serum Golgi protein 73 (GP73) in the diagnosis of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum GP73 in the 81 cases of HCC, 71 cases of chronic hepatitis or cirrhosis (CH/LC) and 65 cases of healthy blood donors, and to evaluate the sensitivity and specificity in the diagnosis of HCC through the ROC curves.</p><p><b>RESULTS</b>The average levels of serum GP73 in HCC, CH/LC and Normal groups were (152.67 +/- 33.59) ng/ml, (93.15 +/- 20.02) ng/ml and (58.95 +/- 17.29) ng/ml(o) After calculating through the ROC curves, 120 ng/ml was set as the optimal cut-off point, GP73 has a sensitivity of 77.80% and a specificity of 78.00%.</p><p><b>CONCLUSIONS</b>GP73 as a serum marker in the diagnosis of HCC had a higher sensitivity than AFP, and the combined detection of GP73 and AFP could improve HCC diagnosis.</p>

Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering.</p><p><b>METHODS</b>Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software.</p><p><b>RESULTS</b>SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene.</p><p><b>CONCLUSION</b>Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.</p>

The assemblage, purification and characterization of EV71 VLPs expressed in baculovirus / 病毒学报

To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.

The characterization analysis of HAV recombinant antigen was expressed by vaccinia virus vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To find a suitable cell line for hepatitis A antigen expressed by vaccinia virus vector and to find a way of inactivation and preservation of the HAV recombinant antigen. Methods Series of cell lines such as K4,143, HEL, Hep-2 and Vero were inoculated with vaccinia virus that can express HAV recombinant antigen. ELISA was used to determine the contents of expression antigen. The characterization of the HAV antigen expressed by vaccinia virus was then analyzed after it was treated with different methods.</p><p><b>RESULTS</b>The expression of HAV recombinant antigen in K4,143 and HEL cell lines was a little more than expression in Hep-2 and Vero cell lines. The antigenicity is obviously higher when HAV recombinant antigen was inactivated by beta-propiolactone other than it was inactivated by formalin. It was best to preserve the prepared HAV recombinant antigen under -40 degrees C condition.</p><p><b>CONCLUSIONS</b>The application of vaccinia virus vector in hepatitis A antigen preparation was very useful and promising.</p>

Genetic analysis of HAV strains isolated from patients with acute hepatitis in Hebei Shijiazhuang of China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To characterize the hepatitis A virus (HAV) wild type strains circulating in Hebei Shijiazhuang of China during 2005-2007, to provide the bases for further investigation of the sources of HAV infection.</p><p><b>METHODS</b>The VP1/P2A junction regions were detected by RT-PCR from HAV IgM positives serum samples during 2005 and 2007, the 34 RT-PCR positive samples were sequenced and subjected to phylogenetic analysis by Neighbor Joining (NJ) method.</p><p><b>RESULTS</b>All the detected HAV strains were identified as sub-genotype I A, the homology of nucleotide sequence in the VP1-2A imation region ranged from 95%-100%, the amino acid sequences of HAV strains almost had no difference.</p><p><b>CONCLUSION</b>There are different HAV strains existing in Hebei Shijiazhuang of China, same HAV strain may exist in different areas; or in one area, identical or different HAV strains may be detected. This work provides the bases for further investigation of the sources of HAV infection and also for effectively control measures to prevent the spread of the disease.</p>

Genotyping of hepatitis A virus prevalent strains in Xinjiang Hetian of China in 2006 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analysis the genotypes of wild type hepatitis A virus circulated in Xinjiang Hetian of China in 2006.</p><p><b>METHODS</b>The Vp1-2A region of HAV genome was amplified and sequenced from serum samples collected in Xinjiang Hetian of China in 2006, and subjected to phylogenetic analysis by Neighbor Joining (NJ) method.</p><p><b>RESULTS</b>The nucleotide sequence differences in the VP1-2A region among Xinjiang Hetian HAV strains ranged from 0%-3.9%, all belonged to sub-genotype 1A. Genetically similar strains were identified among Xinjiang Hetian 2006 and Xinjiang Yili 2005 of China isolates. Only 0-2 amino acid differences were found among the Xinjiang Hetian HAV isolates in the VP1-2A region.</p><p><b>CONCLUSION</b>There were different HAV strains existing in the investigated areas, these strains may have different transmission pathways for the spread of the disease. The results indicate the usefulness of molecular epidemiological methods in studying changes in the circulating HAV strains and in tracing transmission routes, and also for effectively control measures to prevent the spread of the disease.</p>

Development of an extraction and concentration method for the detection of hepatitis A virus in different samples / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop an extraction and concentration method for the detection of hepatitis A virus (HAV) in shellfish, water, serum and saliva samples by nested RT-PCR.</p><p><b>METHODS</b>HAV were artificially inoculated into the above samples, calm sample was extracted using glycine buffer pH9.5, PEG precipitation; water sample was PEG precipitated directly; then all the samples including serum and saliva samples were extracted using Trizol regent, followed by nested RT-PCR detection using primers from HAV VP1-2A region.</p><p><b>RESULTS</b>The detection limit for HAV in cultured cell lysis was 0.1TCID50; in water, serum or salva sample was 1TCID50 respectively, in calm sample was 1-10 TCID50. HAV RNA was detected in water and sera samples collected from the HAV outbreak region, sequenced and analysis.</p><p><b>CONCLUSION</b>The method developed here is convenient, specific and capable of detecting low levels of HAV in different samples, would be useful for diagnostic laboratories in order to perform HAV analysis in cases of foodborne infections or for molecular epidemiology investigation of HAV outbreaks.</p>

Baculovirus expression of two human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.</p><p><b>METHODS</b>The heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.</p><p><b>RESULTS</b>The baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.</p><p><b>CONCLUSION</b>The recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.</p>

ISOLATION AND IDENTIFICATION OF XJ STRAIN OF CANINE DISTEMPER VIRUS / 微生物学通报

A canine distemper virus strain was isolated from the lung of dog coming from Aksu in Xing Jiang using lung primary M cell during the CDV molecular epidemiological study. It was demonstrated to be a virulent strain of CDV by a series of systematic identification such as morphology , serology neutralization test, canine infection test, and molecular virology test.