Military tropical medicine in the 21st century / 第二军医大学学报

Tropical medicine is defined by an association with geographic location, and it is a branch of medicine integrating preclinical medicine, clinical medicine and preventive medicine and investigating the diagnosis, treatment and prevention of diseases of tropical and subtropical zones. Military tropical medicine is a new interdiscipline based on tropical medicine and military medicine. With the improvement of health condition and the development of global economy, some tropical infectious diseases have been gradually controlled. However, factors such as increasingly frequent international communication and extreme changes in global climate induced by overproduction activity of human are leading to a redistribution of infectious diseases, which inevitably has impact on military strategies and tactics. This article reviews the past and prospect of military tropical medicine.

Infectious disease-related pathogens and their prevention and control strategies after earthquakes / 第二军医大学学报

Epidemic diseases often occur following natural disasters, such as earthquakes. The most commonly seen epidemics after an earthquake include: enteric diseases (dysentery, typoid and paratypoid fever, cholera, hand foot-mouth disease, hepatitis A, hepatitis E, etc), arthropod-borne infectious diseases (malaria, Kala-Azar, Japanese encephalitis, etc), zoonosis (plague, hemorrhagic fever with renal syndrome, anthrax, etc), soil and epidemic water transmitted diseases (tetanus, gas gangrene, leptospirosis, etc), respiratory diseases (measles, rubella, influenza, etc), food-borne diseases (food poisoning caused by bacteria or bacterial toxin). This article reviews the controlling principles and measures for major infectious pathogens and epidemic diseases after earthquake.

The prevalence of hepatitis C virus (HCV) subtypes in Chinese HIV-1/HCV co-infected individuals / 中华流行病学杂志

Objective To better understand the prevalence and geographic distribution of genotypes/subtypes on HCV and the relationship between HCV genotypes/subtypes and HIV infection disease progression in the HIV-1/HCV co-infected individuals living in high HIV-1 prevalent areas in China. Methods 186 plasma samples were collected from HIV-1 seropositive individuals infected through paid blood donors (PBD), injecting drug users (IDUs) or sexual contact, living in most severely affected provinces, Henan, Yunnan, Xinjiang, Jilin and Liaoning provinces. Samples with HCV viral load >1000 cop/ml were amplified by RT-nested PCR, sequenced and phylogenetically analyzed for genotyping/subtyping of HCV. HIV-1, HCV viral loads and CD4 T lymphocytes were measured for all subjects. Results (1) HCV were identified as 1 a (1.7%), 1 b (39.9%), 2a (17.9%), 3a (10.4%), 3b (15.6%), 6a (1.2%), 6n (6.4%), and a newly unclassified subtype (7.5%). HCV 2a and lb subtypes predominated in PBD in Henan, 3a and 3b in IDUs in Xinjiang and Yunnan, and 6 genotype/subtypes in IDU in Yunnan. (2) There were no significant differences in CD4 T cell counts among the different HCV subtypes. (3) The viral load of HCV RNA in lb subtype was higher than that of non-1b subtype, however, no significant differences in HIV-1 viral loads and CD4 T cell counts were found between Ib and non-1b subtype. Both HIV and HCV viral loads were lower in 2a than non-2a subtype. Conclusion The prevalence of HCV genotype/subtype in HIV-1/FICV co-infected individuals was associated with geographic areas and transmission routes. HCV subtypes had no direct correlation with HIV infection disease progression.

Proteomic analysis of differently expressed proteins in human hepatocellular carcinoma cell lines HepG2 with transfecting hepatitis B virus X gene / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hepatitis B virus encoded X protein (HBx) is a trans-activating protein that may be involved in hepatocarcinogenesis, although few natural effectors of HBx that participate in this process have been identified. We screened, by comparative proteomics method, effectors of HBx associated with hepatocarcinogenesis.</p><p><b>METHODS</b>HBx positive and negative HepG2 cells were constructed and expression patterns of cellular proteins were obtained by high resolution, two dimensional electrophoresis. Comprehensive analyses of proteins associated with hepatocellular carcinoma (HCC) were focused on the differently expressed proteins (more than two-fold increase or decrease, P < 0.05) from HBx positive and negative HepG2 cells. For peptide mass fingerprinting, protein spots with different intensity between HBx positive and negative HepG2 cells were directly cut out of gels and processed for matrix assisted, laser desorption/ionization, time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) analysis.</p><p><b>RESULTS</b>The mean number of protein spots for HBx negative and HBx positive HepG2 cells were 2095 +/- 137 and 2188 +/- 105, respectively. The analysis of paired cells showed 75 spots with significant differences in expression between HBx negative and HBx positive cells: 37 spots corresponding to 32 different proteins; 25 proteins were upregulated, 7 downregulated. We found 7 proteins not previously reported differentially expressed in HBx positive HepG2 cells. Variations in protein accumulation were confirmed for four (HSP90AB1, BCL2 associated athanogene 2, nucleophosmin and chloride intracellular channel 1) by Western blotting in HBx positive HepG2 cells.</p><p><b>CONCLUSIONS</b>Numerous effectors of HBx that may promote the development of HCC are identified, of which 7 are newly noted in HepG2 cells. Several of these effectors of HBx may help in elucidating the roles of HBx in hepatocarcinogenesis and diagnostics or targets for therapeutic intervention.</p>

Cross reactivity of mimotopes of hepatitis C virus hypervariable region 1 / 病毒学报

The cross reactivity of mimotopes of hepatitis C virus (HCV) hypervariable region 1 (HVR1) was investigated to obtain epitopes that have high cross reactivity. Five expression vectors encoding B cell mimotopes fused with Trx were constructed, and the mimotope proteins were purified. The cross reactivity of mimotope proteins with HCV positive sera was determined by ELISA. HCV pseudotype particles (HCVpp) were generated and applied to evaluate neutralization effects of the sera of BALB/c mice immuned with the mimotope proteins on infection of Huh7. 5 cells. Our data showed that the mimotope proteins (P1, P2, P5, P6, P8) could react to the HCV positive sera. The HCVpp infection inhibition of the sera of BALB/c mice immuned with P6 or P8 was detectable. These results suggest that the mimotopes may be valuable in the studies of anti-HCV infection and development of HCV vaccines.

Comparison of immune responses induced by recombinant attenuated Salmonella typhi carrying eukaryotic expression plasmid or prokaryotic expression plasmid of HCV core protein / 生物工程学报

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for development of protective HCV vaccines. However, this protein may attenuate the induction of systemic immune responses due to its immunomodulatory properties. In this study, we constructed a HCV core gene-containing eukaryotic expression plamid pCI-C, and an in vivo-inducible prokaryotic expression plasmid pZW-C, and transformed the recombinant plasmids into an attenuated Salmonella typhimurium aroA strain SL7207. The resulting bacterial strains SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and the immune responses specific to HCV core protein were assessed. Immunization with the recombinant bacteria SL7207/pCI-C led to a persistent drop in percentage of CD3 CD4 T cells, and induced a weak anti-core IgG production. Splenocytes from SL7207/pCI-C immunized mice developed a relatively weak proliferation response and inferior cytotoxic activity compared to those from the mice immunized with bacteria SL7207/pZW-C. Boost immunization with SL7207/ pCI-C yielded limited improvement in immune strength, while the boost with bacteria SL7207/pZW-C significantly enhanced the immune response. These results suggest that de novo synthesis of native HCV core protein may blunt the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.

Rapid detection of Pseudomonas aeruginosa by the fluorescence quantitative PCR assay targeting 16S rDNA / 生物工程学报

The 16S rDNA specific primers were designed for rapid detection of Pseudomonas aeruginosa (PA) by the fluorescence quantitative PCR (FQ-PCR) assay, based upon multiple sequence alignment and phylogenetic tree analysis of the 16S rDNAs of over 20 bacteria. After extraction of PA genomic DNA, the target 16S rDNA fragment was amplified by PCR with specific primers, and used to construct recombinant pMDT-Pfr plasmid, the dilution gradients of which were subjected to the standard quantitation curve in FQ-PCR assay. Different concentrations of PA genomic DNA were detected by FQ-PCR in a 20microL of reaction system with SYBR Green I. At the same time, various genomic DNAs of Staphylococcus aureus, Salmonella typhi, Shigella flexneri, Proteus vulgaris, Staphylococcus epidermidis, Escherichia coli, and Mycobacterium tuberculosis were used as negative controls to confirm specificity of the FQ-PCR detection assay. Results demonstrated that the predicted amplified product of designed primers was of high homology only with PA 16S rDNA, and that sensitivity of the FQ-PCR assay was of 3.6pg/microL of bacterial DNA or (2.1 x 10(3) +/- 3.1 x 10(2)) copies/microL of 16S rDNA, accompanied with high specificity, and that the whole detection process including DNA extraction could be completed in about two hours. In contrast to traditional culture method, the FQ-PCR assay targeting 16S rDNA gene can be used to detect PA rapidly, which exhibits perfect application prospect in future.

Antigenicity of hepatitis C virus F protein and serum prevalence of anti-F in HCV-infected patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To examine the antigenicity of hepatitis C virus (HCV) F protein and investigate serum prevalence of anti-F in HCV-infected patients.</p><p><b>METHODS</b>Eleven pairs of overlapping primers were used to synthesize the full-length HCV f gene, from which the truncated HCV f65 gene fragment was amplified by PCR. HCV f65 gene was then cloned into pET32a(+), and transformed into E. coli strain Plyss (DE3). This recombinant E.coli was induced by IPTG for the production of HCV F65 protein. The expressed HCV F65 protein, purified by Ni-NTA agarose, was further used in ELISA to detect serum anti-F, and to immunize rabbits for making polyclonal anti-F. The rabbit polyclonal anti-F was purified by Staphylococcus aureus protein A agarose.</p><p><b>RESULTS</b>After recombinant pET32a(+)-f65 was constructed successfully, HCV F65 protein was expressed and purified. The purified HCV F65 protein was used as a capture antigen in ELISA to detect serum anti-F in HCV infected patients (n = 30). The result showed that the mean A450 value and the positive rate of serum anti-F were 0.125+/-0.061 and 63.3%, respectively. The rabbit-derived polyclonal anti-F reacted specifically with HCV F65 protein, of which the titer was 1:30,000.</p><p><b>CONCLUSION</b>Our expressed HCV F65 protein is of antigenicity, and can be used to determine serum anti-F. Anti-F IgG does exist in the sera of the HCV-infected patients. Moreover, the rabbit-derived polyclonal anti-F can be used to detect HCV F protein.</p>

Enhancement of cellular immune response to DNA vaccine encoding hepatitis C virus core and envelope 2 fusion antigen by murine Fms-like tyrosine kinase 3 ligand / 生物工程学报

Hepatitis C virus (HCV) is an important human pathogen that causes chronic liver disease worldwide. It is desirable to develop vaccines to prevent HCV infection, or at least to prevent progression to chronicity. We once constructed an optimized hepatitis C virus core and envelope 2 fusion antigen DNA vaccine, which could induce humoral and cellular immune responses against HCV core and E2 protein in BALB/c mice efficiently. Flt3 (Fms-like tyrosine kinase 3) -ligand has been identified as an important cytokine for the generation of professional antigen-presenting cells, particularly dendritic cells. We reasoned that a DNA vaccine coexpressing the antigen and FL may activate immune responses more effectually. In this study, The influence of FL on this HCV DNA vaccine was evaluated. The cDNA encoding signal peptide and extracellular domain of murine FL was inserted into the plasmid pST-CE2t, and the resulting plasmid pST-CE2t/FL was transfected into COS7 cells. The HCV core and E2 protein were detected by Western blotting, and the soluble murine FL was detected by ELISA. Eight-week-old female BALB/c mice were inoculated intramuscularly with 100 microg pST-CE2t, pST-CE2t/FL or mock vector, respectively, and boosted at the same dosage 3 weeks later. Anti-HCV core and E2 total IgG and isotypes were measured at weeks 1,3,5,7. Splenocyte proliferative response to recombinant HCV core and E2 protrein were detected at week 7. SP2/0 cells expressing HCV core protein were used as target cells for the detection of cytotoxic T lymphocyte (CTL) response. Western blot analysis showed that a protein band with molecular weight about 70 kD from lysate of COS7 cells transfected with plasmid pST-CE2t/FL could be detected by anti-HCV core or E2 monoclonal antibodies, which indicated that pST-CE2t could express glucosylated HCV core and E2 fusion protein. Murine FL could be detected in the culture supernatant of COS7 cells transfected with pST-CE2t/FL. Plasmid pST-CE2t immunized mice developed higher anti-HCV core and E2 IgG seroconversion rates and titers than pST-CE2t/FL group did at different various times, but the IgG2a/IgG1 ratio of anti-HCV E2 protein in pST-CE2t/FL group is much higher than pST-CE2t group. Splenocytes from pST-CE2t or pST-CE2t/FL immunized mice could proliferate with stimulation of HCV core or E2 protein in vitro, although pST-CE2t/FL group showed much stronger response. Splenocytes from mice immunized with pST-CE2t/FL induced 79.03% +/- 9.95% of target cell lysis at the effector/target ratio of 100:1, which was significantly greater than the lysis (62.2% +/- 8.62%) observed in mice immunized with pST-CE2t. Our data demonstrated that the incorporation of FL can preferentially enhance the cellular response to this HCV fusion antigen DNA vaccine. In contrast, HCV specific antibodies were inhibited by FL in vaccinated mice. More and more data supports that recovery from acute HCV infection may depend upon the generation of broad-based cellular immune responses to viral proteins. So, FL may be of potential value as an adjuvant in the development of DNA-based immunization for prophylactic and therapeutic vaccine against HCV infection.

Induction of immune responses in mice by hepatitis B virus large envelope DNA vaccine delivered by attenuated Salmonella typhimurium / 生物工程学报

The enhanced green fluorescent protein (EGFP) expression plasmid was transformed into an attenuated AroA- autotrophic mutant of Salmonella typhimurium SL7207, the resultant bacteria was administered orally to BALB/c mice. EGFP expressed in spleen cells was detected by flow cytometry. A DNA vaccine encoding HBV large envelope protein was immunized BALB/c mice by oral delivery through SL7207 or by direct intramuscular injection. The serum antibodies, T lymphocyte proliferative response and cytotoxic T lymphocyte response of mice were detected. The results showed that both DNA immunization methods could induce cellular and humoral immune responses, whereas oral vaccination elicited stronger immune responses than intramuscular vaccination did. Therefor, oral administration with HBV DNA vaccine using attenuated Salmonella may be a simple and effective method for the therapy of hepatitis B.

Detection and genetic analysis of TT virus in hemodialysis patients / 第二军医大学学报

Objective: To study the prevalence and pathogenesis of TT virus (TTV) in hemodialysis patients. Methods: Serum TTV DNA was tested in 69 hemodialysis patients from our hospital by nested-PCR using primers from a conservative region of TTV genenome, genetic analysis and detection of hepatitis C virus antibody (anti-HCV) and the levels of alanine aminotransferase (ALT) were also carried out simultaneously. Results: The overall prevalence of TTV viremia was 27.5%. The PCR-amplified gene fragment from one patient was sequenced, and its gene sequence homologies with GH1,TA278, TTVCHN1 and TTVCHN2 ranged from 89% to 100%, its deduced amino acid sequence ranged from 87% to 100%. There was no significant difference of TTV prevalence between anti-HCV positive and negative patients. No significant elevation of ALT was found in all patients. Conclusion: High prevalence of TTV infection is found among hemodialysis patients, and TTV infection has no significant association with HCV infection or elevation of ALT.

Detection and genetic analysis of TT virus in hemodialysis patients / 第二军医大学学报

Objective: To study the prevalence and pathogenesis of TT virus (TTV) in hemodialysis patients. Methods: Serum TTV DNA was tested in 69 hemodialysis patients from our hospital by nested-PCR using primers from a conservative region of TTV genenome, genetic analysis and detection of hepatitis C virus antibody (anti-HCV) and the levels of alanine aminotransferase (ALT) were also carried out simultaneously. Results: The overall prevalence of TTV viremia was 27.5%. The PCR-amplified gene fragment from one patient was sequenced, and its gene sequence homologies with GH1,TA278, TTVCHN1 and TTVCHN2 ranged from 89% to 100%, its deduced amino acid sequence ranged from 87% to 100%. There was no significant difference of TTV prevalence between anti-HCV positive and negative patients. No significant elevation of ALT was found in all patients. Conclusion: High prevalence of TTV infection is found among hemodialysis patients, and TTV infection has no significant association with HCV infection or elevation of ALT.

STUDIES OF HEPG2 CELLS INFECTED WITH HGV RNA GENOME / 微生物学通报

In order to observe the replication and expression of HGV RNA genome in HepG2 cells and establish a cell model of HGV infection, HGV RNA genome was prepared in vitro and transfected HepG2 cells with lipofec-tamin. HGV RNA-positive supernatants were used to infect fresh HepG2 cells. RT-PCR, immunohistochemistry and Western blot assays were carried out to detect the replication and expression of HGV in HepG2 cells. Both positive and negative strands of HGV RNA could be detectable in cell culture supernatants and cells at 24h post-transfection. During the culture periods of 90 days, the cells were maintained by changing the medium every 3 or 5 days, and cultured for more than 20 passages. Both strands of HGV could be detectable in culture supernatants and cells. Immunohistochemistry and Western blot results also confirmed that HGV E2 protein could be expressed in the infected HepG2 cells. HGV RNA could also be detectable in the frozen-thawed HepG2 cells infected with HGV RNA genome. Therefore, HGV RNA genome can replicate and express in HepG2 cells, this HGV RNA genome transfected cells model could be used as a cell model in the studies of replication and infection of HGV.

Expression Diversity of Quorum-sensing-Related Genes in Pseudomonas aeruginosa / 微生物学通报

One hundred Pseudomonas aeruginosa quorum-sensing-related genes were selected and their primers were synthesized. The fragments of specific sequences which are related QS genes were amplified by PCR. These verified sequences were inserted into the vector pMD-18T for sequencing. These DNA fragments were dotted onto glass slides to make cDNA microarray. Hybridization was performed with cy3/cy5-dCTP labeled probes. The scanning data of early stationary phase and mid-logarithmic phase indicated that 9 genes were up-regulated and 6 genes were down-regulated. Undergoing the different medicines,we took tobramycin as an example to compare the expression diversity. The results confirm that the QS cDNA chip is useful,and may contribute to better understand the mechanism of quorum-sensing,and can help us find the new targets for restraining the growth of Pseudomonas aeruginosa.