Staphylococcus epidermidis (S. epidermidis) is an opportunistic pathogen that colonizes on the surface of human skin and in mucous membranes. It can form biofilms by adhering to indwelling medical devices, often causing biofilm-associated infection. Two-component signal transduction systems (TCSs) play an important role in modulating biofilm formation in bacteria. However, regulation mechanisms of TCSs in S. epidermidis are still poorly understood. In this study, the expression of response regulator ArlR of arlRS TCS in S. epidermidis growth phases was investigated. The prokaryotic expression plasmid pET28a-arlR was constructed, and ArlR protein was expressed and purified. The purified ArlR was used to immunize mice for inducing anti-ArlR antibodies. The titers of anti-ArlR in the immune sera were over 1:100000, as determined by immune dot blot technique. ArlR expression in the different growth phases in bacteria was detected by Western blot analysis. Results showed that the expression level of ArlR was lowest at 2-hour culture and highest at 4-hour culture, and declined at 6-hour and 10-hour cultures. Western blot analysis results were confirmed by the transcriptional levels of arlR determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. It would help to get additional insight in to arlRS TCS regulation functions in the formation of biofilms in S. epidermidis.

Serine-threonine protein kinases (STPKs) are eukaryotic-like protein kinases, which are important regulator of bacterial growth and metabolism, involved in various cellular processes of mycobacteria, including cell shape and morphology, glucose and glutamine transport, phagosome-lysosome fusion, and/or activity of transcription factors. Mycobacterium tuberculosis encodes 11 eukaryotic-like STPKs (PknA, PknB, PknD to PknL), which are divided into five clusters, ABL cluster, HED cluster, FIJ cluster, PknG and PknK. PknA and PknB of ABL cluster may play a role in morphology and cell division, and PknL can phosphorylate a DNA-binding protein Rv2175c. HED cluster may be associated with several physiological process and pathogenesis. PknF of FIJ cluster may participate in glucose transport and cell division, PknI may regulate bacterial growth against hostile environment, and PknJ may regulate bacterial metabolism by phosphorylating many functional protein. PknG may play a role in glutamate/glutamine metabolism and pathogenesis. PknK may be associated with regulating bacterial growth in hostile environment in vivo. In general，STPKs may participate in many steps in the pathogenesis of Mycobacterium tuberculosis, but more details of the mechanisms involved remain unclear to further investigation. Thus further studies are needed to uncover the mystery.

The objective of the current study is to evaluate sera lipoarabinomannan-38kD-IgG (LAM-38kD-IgG) test in the serological diagnosis of tuberculosis(TB). 128 active tuberculosis patients (107 pulmonary TB, 21 extrapulmonary TB )，51 with latent TB infection and 68 healthy people were enrolled and diagnosed by clinic, bacteriology and X-ray examination. All specimens were studied for LAM-38kD-IgG by using Enzyme-Linked Immunosorbent Assay(ELISA). The results demonstrated that the total sensitivity and specificity of LAM-38kD-IgG test of active tuberculosis patients were 46.88% and 98.53%. The positive predict value and the negative predict value of LAM-38kD-IgG test were 98.36% and 49.63% respectively. The sensitivity of LAM-38kD-IgG test in the diagnosis of pulmonary tuberculosis and extra-pulmonary tuberculosis were 46.73% and 47.62% (P>0.05) respectively; the sensitivities in the patients with smear-positive and smear-negative pulmonary tuberculosis were 52.54% and 39.58% (P>0.05) respectively. The specificity of LAM-38kD-IgG test was higher than tuberculin test (TST) (41.46%). It was concluded that LAM-38kD-IgG test has a very good specificity and an acceptable sensitivity and positive predictive value. It is thought that it could be used in combination with other methods to increase diagnostic accuracy, especially for culture-negative and extrapulmonary tuberculosis cases, which are difficult to diagnose.

According to World Health Qrganization (WHO)’s global tuberculosis control report, up to one-third of the global population is estimated to carry latent Mycobacterium tuberculosis （M.tb）infection. Although following M.tb infection, only 5 to 10% of immunocompetent individuals develop active tuberculosis （TB）, this disease remains a major health problem particularly in low-income developing countries, which currently causes over 2 million deaths annually and is predicted to be among the 10 leading causes of disease burden even in the year 2020. India, China, Pakistan and Indonesia together account for 50% of the global TB epidemic. In sub-Sahara Africa, because of high HIV prevalence the TB incidence reaches 290 per 100 000 which is a hundred times of developed countries. Molecular epidemiology studies have shown that diverse strains of different genotypes were prevalence in different regions such as the predominance of W-Beijing family, one of the most successful M.tb families, in countries of East Asia. The notion that some strains of a single genotype, such as the W-Beijing genotype, are more virulent than others is supported by results obtained with animal and cellular models. The inhibition of the host immune response by mycobacterial strains of the W-Beijing family has recently been confirmed in vivo. This review discusses the phylogenetic studies that have been made in discovering how certain M.tb has achieved its virulence as a successful pathogen.

Hand foot and mouth disease is a global infectious disease caused by human enterovirus. Since late 1990s, outbreaks of EV71-asscoiated hand foot and mouth disease have been reported frequently throughout Asia and severe neurological central nerves system involvement and neurogenic pulmonary edema lead to long-term sequela and fatality. Annual national outbreak of hand foot and mouth disease has occurred since 2008 and EV71 is the major causative agent for the outbreak and associated death. Currently, hand foot and mouth disease is a national priority disease and becomes a growing public health concern in China. Hand foot and mouth is usually a benign self-limited disease, however, 0.05% of sick children succumb to severe complications such as brain stem encephalitis and cardiopulmonary collapse based on national data. To date, no effective antiviral drugs and vaccine are available for treatment and prevention of disease. Early diagnosis and recognition of severe complications, timely clinical intervention for severe cases at an earlier stage and implementation of public health measures are the current mainstay of reducing the impact of outbreaks and its associated mortalities. EV71 vaccine under development in China is a promising strategy to prevent EV71 epidemics and severe diseases and reduce its socioeconomic impact.

To find the variable number of tandemrepeat ( VNTR) locus combinations that adapt to study the microevolution of Mycobacterium tuberculosis ( M. tuberculosis ) strain of Beijing genotype and the molecular markers to distinguish diff erent sublineages , VNTR and single nucleotide polymorphism( SNP) genotyping were usedto genotype 135 strains fromculture-positive sputumof tuberculosis patients ( collected and preserved by Shanghai Municipal Center f or Disease Control and Prevention) in Chongming Island , Shanghai . Based on the data, the mini mumspanning tree ( MST) was constructed . The results showed that the genotyping inf ormation of 16-locus VNTR best matched the SNP genotyping data, indicating that it was applicable in the microevolution study of M. tuberculosis strain of Beijing genotype in Chongming Island ,
Shanghai . SNP hapl otypes were mainly concentrated in ST10 ( 42. 2%) , ST19 ( 30. 4%) , and ST22 (14.1 %) . Four VNTRloci ( Mtub21 , QUB26 , MI RU16 and QUB4156) may be the molecular markers to distinguish different sublineages of Beijing genotype strains .

Objective To establish a molecular biology method for detecting sequences of unknown viruses. Methods The hepatitis B and hepatitis C viruses were used as hypothetically unrecognized DNA, RNA viruses respectively to test the efficacy of this random PCR-based method. Virus-containing serum was first filtered through a 0.22-μm disk filter followed by DNase I treatment to eliminate the host genomic DNA. After the 26mer 5’ end anchored random primer was annealed to the extracted nucleic acids either by Klenow polymerase extension (DNA as template) or by reverse transcription (RNA as template), the virus genomes were randomly amplified with the 20mer anchor primer. Purified PCR products were cloned, sequenced and subjected to BLAST search for further analysis. Results HBV and HCV genomes were amplified by random PCR and their fragments could be detected in the picked clones. The detective rate was about 15% at 1106 copies/ml viral load. Positive clones could still be obtained with as few as 1104 copies/ml viral load. Conclusion This culture-independent random PCR-based method can be used to investigate unknown viruses and will be useful for the early diagnosis of emerging infectious disease.

Objective To explore the characteristic and reason for indeterminate HIV Western Blot(WB),the limitation of WB and the possible amendatory measures.Methods Summing up and analysis the distribution of the indeterminate HIV WB among the tested people,the laboratory test results,the follow-up and the final HIV antibody outcome.Results The relative healthy people including the people of voluntary counseling and testing,the blood donors and pregnant women make up 50% of the indeterminate HIV WB,the follow-up for the indeterminate HIV WB is difficult and the there are few people of the indeterminate HIV WB have HIV antibody test again.There is false-positive in WB test,especially the false-positive of P24 is serious.Conclusion The indeterminate HIV WB is related to the false positive of WB test,measures should be taken to reduce the indeterminate HIV WB and to interpret the result accurately.

In oeder to investigate the effects of C terminal SPRY domain of TRIM22 on its transcription, translation and subcellular localization, SPRY domain was deleted (ΔSPRY) and amplified through polymerase chain reaction (PCR) and was then inserted into the eukaryotic expression plasmid, pcDNA3.1. Plasmids expressing wild type TRIM22 or TRIM22-ΔSPRY were transfected into HepG2 cells. The mRNA expression level was determined by semiquantitative reverse transcriptase PCR (RT-PCR), the protein expression level was determined by Western blot analysis, and the subcellular localization was determined by immunofluorescence staining. The results showed that plasmid expressing TRIM22-ΔSPRY was successfully constructed. After transfection into HepG2 cells, the TRIM22-ΔSPRY showed no difference from the wild type TRIM22 at both mRNA and protein levels. However TRIM22-ΔSPRY was localized exclusively to the cytoplasm of HepG2 cells in contrast to the nuclear localization of wild type TRIM22. These results may be useful for studying the role of SPRY domain in TRIM22-mediated antiviral activities.

Objective To construct Staphylococcus epidermidis gene knock-out mutants with different plasmids. Methods Recombinant plasmids were constructed for homologous recombination of the two component signal transduction system genes (arlS、saeRS and lytS) of S. epidermidis with two different shuttle plasmids, pMAD and pBT2, transformed to S. aureus RN4220 by electroporation and then transformed into S. epidermidis 1457. S. epidermidis mutants were selected using various antibiotics and biomarkers and identified by PCR and biochemistry experiments. Results With pMAD or pBT2 plasmid, we separately constructed S. epidermids genes knock-out homologous recombinant plasmids, pMAD-ΔsaeRS, pBT2-ΔarlS and pBT2-ΔlytS. S. epidermidis 1457 was transformed with the recombinant plasmids and the gene knocking out mutant was screened out, respectively. Only one cycle was needed for screening out SE1457-ΔsaeRS mutant and 5-10 cycles were required for screening out SE1457-ΔarlS and SE1457-ΔlytS mutant. The biofilm forming of SE1457-ΔarlS mutant decreased by 90.96% and no significant difference was observed between SE1457-ΔlytS、SE1457-ΔsaeRS mutants and SE1457 parent strain. Conclusion pMAD method can be used to construct S. epidermidis mutant strain simply and effectively.

Objective To verify the changes of a circadian gene dbp ( coding for the D site albumin promoter binding protein) in liver tissues of HBsAg positive transgenic mice. Methods Real-time fluorescent quantitative PCR was used to compare the transcriptional levels of dbp in male and female HBsAg positive transgenic mice versus their controls. Individual transgenic mouse and mice at different time points were used to study the regulation of dbp. Results In the liver tissues from both #59 and #10 lineages of HBsAg positive transgenic mice, dbp was up-regulated, compared to their control counterparts, but there was marked individual variability. Preliminary results showed that in HBsAg negative female mice, the transcriptional level of dbp was higher than that in male mice. This difference was more marked in HBsAg positive female transgenic mice. At 8:00 and 2:00, dbp was up-regulated in HBsAg positive transgenic mice, compared to the controls. However, at 8:00 and 2:00, the dbp transcriptional levels were similar between HBsAg positive transgenic mice and the controls. Conclusions For the first time it is reported that dbp was up-regulated in HBsAg positive transgenic mice, which provided evidence that HBV protein expression could be associated with changes in a circadian gene. Further studies are needed to confirm whether this up-regulation can also be found in patients, and clarify the implications of this observation.

Objective To investigate the antimicrobial resistance of enterococcus faecalis and enterococcus faecium in our hospital for the rational use of antibiotic. Methods GPI plate was applied for bacterial identification and susceptibility testing.Whonet5 software was used for data analysis. Results Less than 50% of enterococcus faecalis and enterococcus faecium isolates were resistant to chloramphenicol and nitrofurantoin. Less than 1% of enterococcus faecalis and enterococcus faecium isolates were resistant to vancomycin. The resistance rate of enterococcus faecalis to penicillin G, high concentration gentamicin, ciprofloxacin, rifampicin and erythromycin descend year by year, and that of enterococcus faecium to ciprofloxacin, rifampicin and nitrofurantoin was opposite. In addition, the resistance rate of enterococcus faecium to most antibiotic was higher than enterococcus faecalis. Conclusions The enterococcus faecalis and enterococcus faecium is becoming multi-resistant bacteria. Antimicrobial therapy should be decided according to the results of susceptibility testing.

Objective To explore the mechanismand possible drug target of the new anti-tuberculosis compound, S28, which is effective on Mycobacterium tuberculosis in vitro.Methods Two dimensional gel electrophoresis ( 2 - DE) was employed to determine the effects of this compound on the expression of Mycobacterium tuberculosis proteome. Global proteome of Mycobacterium tuberculosis strain H37Ra was treated with S28 and compared with control ( untreated) . Results The expression of 13 proteins was decreased; six
proteins, which were remarkably decreased in Mycobacterium tuberculosis treated with S28, were subjected to matrix assisted laser adsorption/ionization time-of-flight mass spectrometry ( MALDI - TOF- MS) analysis and the nature of two proteins was successfully determined. They were identified as elongation factor Tu and short chain dehydrogenase, which play important roles in protein translation and energy metabolism. Conclusion These data provide insight into potential novel anti-tuberculosis mechanisms and drug targets.

Objective The goal of the current study is to develop infectious porcine reproductive and respiratory syndrome virus(PRRSV) cDNA clones as expression vectors for porcine circovirus type Ⅱ(PCV2) ORF2 antigens and to use this new vector to dissect the replication and transcription abilities of PRRSV.Methods ORF2 from PCV2 was inserted into our previously established infectious clone of PRRSV,designated pAPRRS,at three locations:1) between ORF1b and ORF2a;2) between ORF5 and ORF6;and 3) between ORF6 and ORF7.The three full length mutant clones were then transfected into MARC-145 cells, resulting in three recombinant viruses. The genetic instability of recombinant virus was further examined via PRRSV’s mRNA2-specific RT-PCR sequence analysis of sgmRNA2, which enabled us to determine the origin of the sequence at the junction site. Results Our results demonstrated that the sgmRNA2 of this recombinant PRRSV-PCV2 has one TRS derived from the PRRSV ORF2 TRS, and two TRS segments were derived from the sequence of PCV2. One TRS was derived from the PRRSV ORF2 noncanonical TRS located at the downstream of PRRSV ORF2 initiation cordon. Conclusion Two recombinant PRRSV – PCV viruses are stable for more than ten passages, which makes them useful as marker vaccines. The most common reason for genetic instability of the PRRSV-PCV recombinant virus is an insertion of a sequence that resembles TRS and a subsequent change of the RNA structure for PRRSV ORF2 TRS and its flanking sequence. PRRSV can use a PCV2 sequence as its own TRS, which is useful for dissecting the mechanism behind PRRSV replication and transcription.

Interferon-β plays an important role in innate immunity against viral infection. Hepatocytes, as hepatitis viruses-harboring cells, are reported to possess the potential to inductively express interferon-β. However, a practical in vitro cell model for investigating the interplay between hepatitis B virus and host cells is rarely reported. Here, we determined the inductive expression of interferon-β by interferon-β agonists〔(Newcastle disease virus, NDV) and poly(I∶C)〕in immortalized primary hepatic cell line, PH5CH8,and hepatocarcinoma cell lines, Huh-7 and HepG2. The data demonstrated that inductive expression of interferon-β in PH5CH8 cells was significantly higher than that in Huh7 or HepG2 cells. In addition, the expression level of key molecules critical for interferon-β induction was investigated to clarify the underlying mechanism. The results showed that the background expression level was fairly low in Huh7 and HepG2 cell lines, compared to that in PH5CH8 cells. It is suggested that PH5CH8 cells possess intact potential to produce interferon-β and reconstitution of a selective interferon-deficient hepatic cell line might be achieved via introduction of related molecules critical for interferon induction.

Objective In order to investigate the mechanism of tuberculosis pathology, in particular the mechanism of liquefaction, studies are conducted to stablish a tuberculosis pathology model in rabbit skin using Mycobacterium bovis attenuated strain BCG, Mycobacterium tuberculosis avirulent strain H37Ra, and Mycobacterium smegmatis( M. smegmatis) . Methods BCG, H37Ra, and M. smegmatis were injected separately into the flank of New Zealand white rabbits by intradermal route twice every one and half months. Fourteen days after the last injection, lesions were removed for further histological analysis. Results Apparent liquefaction and ulceration were produced in the skin of rabbits injected with high dose( 5 ×106CFU/ml) of BCG, H37Ra, and M. smegmatis, with ulceration happening around 10 days after the first injection and three days after second immunization. The inflammation induced by BCG was stronger than the lesion induced by H37Ra, and the latter was stronger than that induced by M. smegmatis. Moderate( 5 ×104CFU/ml) and low doses( 5 ×102CFU/ml) of H37Ra and M. smegmatis did not induce obvious lesions, while the moderate and low doses of BCG induced granulomas. Conclusion High dose of BCG, H37Ra and M. smegmatis could induce caseous necrosis and liquefaction in rabbit skin. These results demonstrate the usefulness of this model for further pathogenesis research.

The purpose of the current study is to investigate the anti-IFN-αeffects and to determine the functional region of the novel protein TS′X′encoded by the 3022-1787nt deletion mutant of hepatitis B virus ( HBV) . Regions
coding for TS′X′( in frame with the opening reading frame of DNA polymerase, in which T stands for terminal protein, S′for partially deleted spacer region, and X′for truncated X protein) and TS′( N-terminal of TS′X′containing terminal protein and partially deleted spacer region) were amplified by PCR and cloned into the pcDNA3. 1/HisC vector separately. The recombinant vector was transfected into Huh7 hepatocytes individually by FuGENE6 transfection reagent, and the expression of the fusion protein was verified by Western blot analysis. The recombinant or
empty vector was co-transfected to Huh7 hepatocytes with IFN-α response reporter plasmid p6-16CAT at the molar ratio of 5∶1, 10∶1,15∶1 and 30∶1, and cells were treated with IFN-α2a ( 100 IU/ml) 48 h post-transfection. After 24 h of stimulation, the cells were lysed and the intracellular CAT value was calculated by ELISA assay. The results
demonstrated that, as compared to the empty vector, the intracellular CAT values were gradually reduced in parallel with an increasing amount of TS′X′or TS′recombinant ( n = 6, P < 0. 05) , while no significant differences were observed between TS′X′and TS′recombinants ( n = 6, P > 0. 05) . It was concluded that the novel protein TS′X′
encoded by the 3022-1787nt deletion mutant of HBV suppressed the response of Huh7 hepatocytes to IFN-α, and the N- terminal region was a functional domain for its anti-IFN-α effects.

Objective To establish the HHV-6-specific CD4⁺ T cell clones and further study the character of HHV-6-specific CD4⁺ T cells. Methods We acquired CD4⁺ T cell clones by the liquid microwell limiting dilution technique, and selected HHV-6-specific CD4⁺ T cell clones by means of 3H-thymidine uptake. The phenotype of HHV-6-specific T cell clones were analyzed with FACS. We tested the IL-10, IL-4 or IFN-γ secretion from HHV-6-specific CD4⁺ T cell clones by ELISA. Results Of the five HHV-6-specific CD4⁺ T cell clones, four showed proliferative responses to the stimulation with HHV-6 infected JJHAN lysates. Moreover, proliferation responses of HHV-6-specific CD4⁺T cell clones depended on the concentration of the HHV-6-infected JJHAN lysates. The phenotypic analysis with FACS demonstrated that HHV-6-specific T cell clones consisted of CD3⁺ CD4⁺ T cells. CD4⁺ T cell clones-W-2 and W-4 possessed the feature of high levels of IL-10 production, while CD4⁺ T cell clones-W-1 possessed the feature of high levels of IL-10 and IFN-γ production and W-3 possessed the feature of high levels of IFN-γ production. Conclusions The HHV-6-specific CD4⁺ T cell clones, were established in our study so as to further study their pneotype, the character of cytokine secrection, and to provide experimental basis for selecting HHV-6-specific CD4⁺ Treg cell clones.

Objective Chlamydia trchomatis and Neisserria gonorrhoeae are the primary bacterial pathogens causing urethral infection in men.Methlend blue stain is used to detect urethral inflammation, suggestive of infection, in men and guide threapeutic dicisions. In the absence of signs, symptoms or polymorphonuclear leukocyte on methlend blue stain(PMNLs).The tctreatment is deferred on management. Studing relation about Chlamydia-infected,Gonorrhea-infeced and PMNLs who compare their clical characteristics of urethral inflammation in men. Metholds 3,000 were presenting for routine sexually transmitted disease care with uethral PMNLS count, the direct qualitive detection of Chlamydia trachomatis antigen and culture. Results: Among 3,000 eligible men,387(12.0%) had chlamydia and 415(13.8%)had gonorrhea. Among chlamydia-infected men,PMNLS per oil-immersion field were ≥5 in 242(62.5%), 1 to 4 in 59(15.2%),and none in 86(22.2%)，36(9.3%) and 141(36.4%) in men were asympomati or signs at that stage oa their infection. Among gonorrhea-infected, PMNs per oil-immersion field were ≥5 in 397(95.7%), 1 to 4 in 10(2.4%), none in 8(1.9%), 5(1.2%) and 46(11.1%) in men were asympomati or signs at that stage oa their infection. And an additional 86 were coinfeced with both organisms. PMNLs were≥5 in 76,1 to 4 in 5,none in 5. Conclusions:The paper used to analyze the ralationship of urethral inflammation PMNs,Chlamydia trachomatis and Neisseria gonorrhoeae infetion.The pations of chlamydia、gonorrhea and PMNLs in men infected have obvious difference(P<0.001). 86(22.2%) of chlamydial and 8(1.2%)of gonococcal infection had no evidence of PMNLs. It is strenuous that both of clical and testing teehnicality is important to increase dignosis for urthral inflammation in men and control sexually transmitted disease.

Objective Duck hepatitis B virus (DHBV) infected animal model was used to evaluate the inactivation of DNA virus by methylene blue photodynamic method. Methods DHBV was purified by Ultra-centrifugation. Purified DHBV was sequentially diluted and added to human plasma or RBC components respectively, followed by inactivation with methylene blue photodynamic method. These samples were inactivated into one-day-old ducklings by intravenous injection. Sera from ducks were collected at different intervals and detected by dot hybridization with DHBV probe labeled with 32P for DHBV DNA, and confirmed by southern blot hybridization for DHBV DNA in duck livers. Results The infectivity of DHBV (ID50) in the blood components was calculated before and after the treatment with methylene blue photodynamic method. Before viral inactivation, the ID50 of DHBV mixed with human plasma for one-day-old duckling was 1033.3 copies, and the ID50 of DHBV mixed with human RBC in one-day-old duckling was 103.50 copies. After inactivation with methylene blue photodynamic, the ID50 of DHBV mixed with plasma in one-day-old duckling was 1010 copies, while the ID50 of DHBV-RBC in one-day-old duckling was 108.35 copies. Treatement of methylene blue photodynamic the viral titer of DHBV-plasma decreased 6 logs, and DHBV-RBC decreased 5 logs. Conclusion Methylene blue photodynamic can inactivate DNA virus(using DHBV as a marker) in human blood or RBC, and the inactivation effect is better for virus in the plasma than that in RBC. The DHBV infected ducks could be used as a model for evaluating the effect of DNA virus inactivation in vivo.

To investigate the immunogenicity of eukaryotic expression plasmid of duck hepatitis B virus core antigen ( DHBcAg) in mice, we first analyzed the hydrophilic and hydrophobic characteristics of the amino acid sequence of
DHBcAg by bioinformatics with the Vector NTI Suite software, and constructed the eukaryotic expression plasmids of the complete ( E-DHBc 263) and partial sequences ( only the hydrophilic part, E-DHBc 180) of DHBcAg gene. The
results showed that both plasmids expressed DHBcAg in transfected COS7 cells detected by indirect immunofluorescence. The prokaryotic expression plasmids, p-DHBc 263 and p-DHBc 180, were constructed to express DHBcAg for DHBcAb detection, but only p-DHBc180-expressed DHBcAg could be detected by Western blot. Then we immunized BALB/ c mice with E-DHBc 180 and E-DHBc 263. DHBcAb titers in sera from immunized mice were detected by enzyme-linked immunosorbent assay ( ELISA) on the plates coated with prokaryotically expressed DHBcAg. The mice primed and boosted with E-DHBc180 produced DHBcAb, and titers were 1∶100-1∶400, while in
E-DHBc 263-immunized mice DHBcAb level was belowthe detection level. It suggests that E-DHBc 180 could be used as DHBcAg DNA vaccine for further research in its effects on DHBV infection.

Campylobacter jejuni ( C. jejuni) is a common causative pathogen of bacterial gastroenteritis in humans. Chemotaxis, adirectional movement of bacteria towards suitable parasitic positions, is the initial key step for C. jejuni to achieve colonization on jejunal mucosa of hosts. The chemotactic migration was controlled and regulated by bacterial chemotaxis-associated two-component signaling system( che-TCS) in a MCPs→Ches→Flis/Mots manner. FliG, amember of Fli protein family, has been confirmed in some other bacterial pathogens to be a flagellar motor protein as well as an essential component of switch complex in bacterial flagellar motor. However, the function of FliG protein in chemotaxis of C. jejuni remains unknown. In the present study we generated a fliG gene knockout ( fliG^- ) mutant from C. jejuni strain NCTC11168 based on homologous recombination, and the motility, chemotaxis and colonization of fliG^- mutant were subsequently determined. The motility test and chemotaxis test in vitro demonstrated that the diameters of colonies on semisolid agar plate and chemotactic rings towards 0. 2 mol/L sodiumdeoxycholate ( SDC) in hard agar plus (HAP) of fliG^- mutant were significantly smaller than those of the wild type strain ( P < 0. 05) . Compared to the wild type strain, the numbers of fliG^- mutant adhering to the surface of jejunal mucosa and existing in jejunal content of BALB/c-ByJ mice were also significantly decreased ( P <0. 05) . All the results of this study lead a conclusion that fliG is an essential gene for flagellar motility and chemotactic movement towards jejunal
mucosa of C. jejuni during infection.

Objective To investigate the epidemiology, clinical characteristics, presence of extra-intestinal injury and the direct costs resulting from diarrhea caused by rotavirus and astrovirus infection in children below the age of 5 years in Shanghai. Methods Stool specimens saved from inpatients and outpatients of the Fudan University Children’s Hospital during the period June 2006 to March 2007 were analysed. Initially, the presence of rotavirus was detected using the colloidal gold immunochromatographic assay. Specimens negative for rotavirus were then tested for astrovirus using emzyme-linked immunosorbent assay (ELISA). The target population in this study was children below the age of five, with ≤ 2 weeks of duration of diarrhea, which were negative for bacterial culture. Results A total of 724 stool specimens (5 years old or younger) were collected. Out of 724 samples, 42.5% were positive for rotavirus, of which 85% were from patients of two years old or younger. Cases were distributed throughout the year peaking in December 2006—January 2007. As an account of the economic burden this type of illness places on families, 50% of community-acquired inpatients have hospital bills ranging from RMB 1 473.7—4 029.6 (25th percentile to 75th percentile) and 50% of hospital-acquired inpatients have hospital bills ranging from RMB 3 096.8—1 0552.3 (25th percentile to 75th percentile). Of 276 stool specimens negative for rotavirus (age 5 years or younger), astrovirus was detected in 11.6%, of which 53.6% were from patients of two years old or youger. Cases were distributed all through the year, with a peak from October 2006 to January 2007. Conclusion Both rotavirus and astrovirus are important pathogens which cause viral gastroenteritis in young children in Shanghai.

Objective To detect the subcellular location of Rv2629 in Mycobactrium tuberculosis, and the drug susceptibility testing of transformed M. smegmatis. Methods Different component of Mycobacterium tuberculosis were separated by differential velocity centrifugation, and subcellular location of Rv2629 was examined by Western-Blot. pMV261 plasmid was used to transformed the Rv2629 gene to M. smegmatis. RIF resistant was assayed by BACTEC MGIT 960 instrument. Results The subcellular location of Rv2629 was major at cell wall and cell membrane. The MIC of RIF for M. smegmatis transformed with mutated Rv2629 was 160μg/mL. While the MIC for wild type Rv2629 gene was 20μg/mL similar to the parental strain. Conclution These results indicate that the 191A/C mutation of Rv2629 gene is associated with RIF resistance.

Infection with hepatitis C virus (HCV) is currently treated with interferon-α( IFN-α) , but the molecular mechanismby which IFN inhibits HCV replication still remains elusive. Therefore, in this study, we attempted to elucidate the possible mechanismfor the IFN-inducible protein, Viperin, against HCV. Over-expression of Viper in could significantly down-regulate intracellular levels of HCV protein and RNA in both the replicon and HCVcc systems. Membrane flotation analysis indicated that expression of Viperin could interfere with the association of HCV NS3 and NS5A with the lipid raft, resulting in increased sensitivity of these two proteins to the treatment of non-ionic detergent. Furthermore, Viperinwas able to interact with FPPS because co-transfection of Viperin with FPPS could partially reverse the anti-HCV effects of Viperin in replicon cells. The above results indicate a possible novel mechanismof IFN-induced antiviral activity, that is, over-expression of Viperin can interfere with the microdomain of intracellular lipid raft and, therefore, further disrupt the stability of viral replicase complex to inhibit its functions.

目的探讨T细胞酶联免疫斑点法(TSPOT)在我国人类免疫缺陷病毒(HIV)感染人群中用于诊断结核潜伏感染的应用价值。方法应用TSPOT-TB试剂盒对68例明确诊断的HIV感染者血液标本进行结核分枝杆菌(Mtb)特异性T细胞的检测,同时对所有病例做结核菌素纯蛋白生物(PPD)试验。结果在HIV感染者总体、CD4<200/μl和CD4>200/μl各组中,TSPOT检测阳性率分别为67.65%、44.44%和70.69%,PPD试验阳性率分别为41.18%、11.11%和46.55%,其中在HIV感染者总体及CD4>200/μl组中TSPOT检测阳性率均高于PPD试验,差异有统计学意义(P均<0.005)。TSPOT检测在CD4<200/μl组中的阳性率低于CD4>200/μl组,但差异无统计学意义(P>0.05);PPD试验在CD4<200/μl组中的阳性率远低于CD4>200/μl组,差异有统计学意义(P<0.05)。结论TSPOT检测在我国HIV感染合并结核潜伏感染的早期快速诊断中有较大应用价值,尤其是在CD4细胞计数>200/μl的HIV感染人群中,阳性率高于目前常用的PPD试验。PPD试验阳性率受CD4细胞计数水平的显著影响，而T SPOT检测不首次此因素影响。

The present paper aims to investigate the clinical effects of combination of metronidazole, clotrimazole and chlorhexidine acetate effervescent tablets and living preparationof lactobacillus on bacterial vaginosis. A total of 100 patients with bacterial vaginosis from the Department of Obstetrics and Gynecology, Hospital of Chinese and Western Integrated Medicine of Jiangsu Province were selected and equally divided into the control group and experimental group. The patients in the control group were only treated with metronidazole, clotrimazole and chlorhexidine acetate effervescent tablets, and the patients in the experimental group were treated with combination of metronidazole, clotrimazole and chlorhexidine acetate effervescent tablets and living preparationof lactobacillus. The curative effects in the two groups were compared after treatment. In the experimental group, 2 cases were lost to follow-up, 35 markedly effective, 8 effective. The markedly effective rate was 72.92% and the total effective rate was 89.58%. In the control group, 1 case was lost to follow-up, 31 markedly effective, 7 effective. The markedly effective rate was 63.27% and the total effective rate was 77.56%. The total effective rate was significantly different in two groups (P<0.05). The recurrence rate in the experimental group was 6.25%, significantly lower than 20.41% in the control group (P<0.05). The results suggest that combination of metronidazole, clotrimazole and chlorhexidine acetate effervescent tablets and living preparation of lactobacillus is safe and effective in treatment of bacterial vaginosis.

Objective To elucidate the need of reassessing the diagnosis when residual pulmonary tuberculosis( TB) cavities still exist. Methods FromJan2003 to Jun2007, sixty-seven patients who were initially diagnosed with pulmonary TB and had already completed a short course treatment were enrolled in the current study. All the patients were negative for the sputumTB tests, but still had residual pulmonary cavities. Within three months after the short course treatment, these patients were re-evaluated by bronchoalveolar lavage ( BAL) . Fifty-nine of themwere also re - evaluated by CT-guided percutaneous lung needle biopsy ( PLB) after receiving the informed consent fromthe patients. Results None of the sixty-seven patients was found TB positive by BAL. Among 59 of these patients who had CT-guided PLB, 62 PLB specimens were collected: two specimens were positive for cancer, five for fungi and five for TB, and three for various bacteria. Conclusion Even when the diagnosis of pulmonary TB infection is confirmed and the anti - TB therapy is effective, the diagnosis of other diseases complicated with TB infection should still be considered, especially when there are residual pulmonary TB cavities. To avoid delayed diagnosis of cancer or other infectious disease in pulmonary TB patients, repeated evaluation by using PLB or BAL is encouraged.

There has been a sharp increase in the incidence of hand, foot and mouth disease in China, an unfavorable trend that deserved public attention. In order to gain insight into the epidemiological and clinical characteristics of
hand, foot and mouth disease, data were descriptively analyzed for 98 patients who were in a hospital in China between May and June in 2008. A high incidence of hand, foot and mouth disease occurred mostly in infants aged 1 to 5 years ( 80. 61% ) living in village or suburban areas. The children living at home ( 73. 47% ) were more likely to acquire
the disease than the children attending boarding kindergartens ( 26. 53% ) . Typical clinical presentations included fever and rash. All 98 patients presented with a rash, and 88 patients ( 89. 80% ) had a fever. The rash was present mostly on the hands, feet, mouth, buttocks, and knees. Routine laboratory studies showed either no change or increase
in white blood cell counts. Reverse transcriptase-polymerase chain reaction( RT-PCR) examination showed positive EV71 ( 74 cases) or CA16 ( 4 cases) infections. Previous studies demonstrated that antivirus treatment and symptomatic treatment for hand, foot and mouth disease are effective, with the infection typically being controlled in
approximately one week. Without treatment, the course of the disease leads to grave complications, including death. Integrated control measures including active treatment of infections, controlling the source of infection and health education should be carried out.

The present study was aimed to investigate major histocompatibility complex Ⅱ ( MHC Ⅱ ) antigen presentation induced by interferon-γ ( IFN-γ) in macrophages after exposure to Mycobacterium tuberculosis ( M. uberculosis) . The expressions of MHCⅡ, CD86, and CD80 on macrophages, after exposure to M. tuberculosis for 24 h prior to the addition of IFN-γ, were detected by flow cytometry. The mRNA levels of CⅡTA, PⅠ, PⅢ and PⅣ were assayed by reverse transcriptase-polymerase chain reaction. MHCⅡ antigen presentation was detected by enzyme-linked immunosorbent assay ( ELISA) . The results showed that M. tuberculosis inhibited the expressions of MHCⅡ and CD86 but not CD80 induced by IFN-γon macrophages. The ability of antigen presentation was also inhibited. The mRNA levels of CⅡTA, PⅠ, PⅢ and PⅣ induced by IFN-γwere also decreased. It suggests that M. tuberculosis inhibits MHCⅡ antigen presentation induced by IFN-γon macrophages.

Objective　To investigate the role of Hepatitis B surface antigen (HBsAg) in the immune escape of innate immunity by Hepatitis B virus (HBV).　Methods After treated with PMA, THP-1 was differentiated into macrophage- like cells. Macrophage- like cells were treated further with LPS and the pam3csk4 in the presence or absence of HBsAg. Cytokine IL-10 , IL-12 protein, IL-10, IL-12 mRNA levels , NF-κB p65 protein nuclear translocation as well as IκB-α degradation and ERK protein phosphorylation were detected to monitor the activation of the TLR signaling pathway. Results LPS and the pam3csk4 induced production of cytokine IL-10 , IL-12 protein, NF-κB p65 protein nuclear translocation as well as IκB-α degradation and ERK protein phosphorylation were inhibited by HBsAg in a dose-dependent manner. Conclusion HBsAg inhibit the activation of TLR2 and TLR4 signaling pathway.

The genes encoding 19kD lipoprotein and early secreting antigenic target-6( ESAT-6) were amplified from genome of the standard Mycobacterium tuberculosis H37Rv using polymerase chain reaction, and then cloned into the vector pMD18-T followed by subcloning into the expression vector pET-21a, respectively. Recombinant 19kD-ESAT6 was expressed in E. coli, and then purified by Ni-NTA. The antigenicity of the fusion protein was analyzed by Western blot analysis. The recombinant 19kD-ESAT6 plasmid was constructed successfully, and could be expressed efficiently in E. coli BL21 ( DE3) . The relative molecular mass of the fusion protein was approximately 29 ×10³ by SDS-PAGE. The recombinant 19kD-ESAT6 protein showed specific antigenicity as determined by Western blot, and can be used for the development of serodiagnostic reagents.

Objective To investigate the mechanism of MyD88 inhibition of hepatitis B virus (HBV) replication. Methods Two truncated versions of MyD88, M (1-151) and M (152-296) were constructed. Plasmid IκBα-SR encoding NF-κB super-repressor or plasmid IKKα/IKKβ which can strongly activate NF-κB signaling were used. Huh7 cells were transiently transfected with HBV dimer, MyD88 or the above plasmids. Assays of HBsAg, HBeAg, core protein, HBV DNA and NF-κB activation were performed. Results The expression of the full length MyD88, and the two truncated forms, M (1-151) and M (152-296), showed different induction patterns of NF-κB activity; patterns that are consistent with their inhibitory effect on viral protein synthesis and core particle-associated HBV DNA replication. Similarly, the expression of MyD88 resulted in a significant reduction of core protein and the co-expression of IKKα/IKKβ dramatically inhibited the core protein level. Furthermore, the co-expression of IκBα-SR dramatically restored the core protein level. Similar results were also observed for HBeAg, HBsAg and HBV core particle DNA. Conclusion Results from these studies support a role of NF-κB signaling activation in HBV viral replication and suggest a novel mechanism for the inhibition of HBV replication by MyD88. if(document.getElementById('ChDivSummary').innerHTML!=""){CutSpan('ChDivSummary',500);DisplaySpanDiv('ChDivSummary');ClearSummaryOnLoad('SummaryLinkChID','SummaryLinkEnID');}else{CutSpan('EnDivSummary',1000);DisplaySpanDiv('EnDivSummary');ClearSummaryOnLoad('SummaryLinkEnID','SummaryLinkChID');}

Objective To establish a rapid adenosine triphosphate ( ATP) bioluminescence assay for assessing drug resistance of Mycobacterium tuberculosis( M. tuberculosis) . Methods In this study mycobacterial growth was monitored using a bioluminescence assay of mycobacterial ATP. Cultures of M. tuberculosis H37Rv and of 10 clinical isolates of the same species were exposed to serial dilutions of rifampicin. Results Suppression of ATP, which indicates growth inhibition, occurred for susceptible but not for resistant strains, within five to seven days of incubation. Breakpoint concentrations between susceptible and resistant strains were determined by comparing these results with those obtained by the reference method ( BACTEC 3D) . Full agreement was found in 100% of the assays with the resistance ratio method on Lowenstein-Jensen ( L-J) medium, and 100% of the assays were in full agreement with the radiometric system( BACTEC 3D) . Conclusion The bioluminescence assay of mycobacterial ATP has comparable ability to detect drug resistant strains of M. tuberculosis; A main advantage of the bioluminescence method is its rapidity; Results are available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium.

Objective To study whether HBsAg-HBIG immunogenic complex therapeutic vaccine (YIC) could induce S gene immune escape mutants.Methods Five chronic hepatitis B patients who had been treated with 6 injections of either 30 or 60 micrograms of YIC were recruited for this study. These patients were those who responded to YIC treatment, shown as a drop in serum HBV DNA virus load >2 log10, with serum HBeAg converted to negative at 24 weeks. However, their virus load rebound at the end of 24 weeks follow-up. As controls, one was a patient who did not respond to YIC treatment and remained serum HBV DNA positive at similar level during and after treatment, while the other one was a patient immunized with the alum placebo. Serum samples before treatment and 6 months after termination of treatment were collected, and DNA extracted. PCR was used to amplify HBV S gene, the precore/core gene and the core promoter regions followed by sequencing.Results In all 7 patients treated with YIC or alum no S gene mutation was found in the “a” determinant, nor was precore stop codon mutation detected. However, three patients had mutations in the 1762/1764 core-promoter region, other two patients had mutations in the core promoter region other than the 1762/1764 double mutation.Conclusion The rebound of HBV replication in five YIC-treated patients was not due to the emergence of HBV S immune-escape mutants. if(document.getElementById('ChDivSummary').innerHTML!=""){CutSpan('ChDivSummary',500);DisplaySpanDiv('ChDivSummary');ClearSummaryOnLoad('SummaryLinkChID','SummaryLinkEnID');}else{CutSpan('EnDivSummary',1000);DisplaySpanDiv('EnDivSummary');ClearSummaryOnLoad('SummaryLinkEnID','SummaryLinkChID');}

Objective To survey the distribution of nontuberculous mycobacteria in drinking water distribution systems in Shanghai. Methods Samples of tap water, source water, and treated water from water corporations were collected. The bacteria were collected through filtrating and culturing on Lowenstein-Jensen medium. The nontuberculous mycobacteria were identified by sequencing 16S rRNA. Results The positive rates of nontuberculous mycobacteria in source water, treated water, and tap water were 60% , 25% , and 10. 3% , respectively. Mycobacterium gordonae and Mycobacterium fortuitum were identified, of which Mycobacterium gordonae was the most frequently identified species. Conclusion Nontuberculous mycobacteria are widely distributed in drinking water distribution systems in Shanghai. Thus, effective control measures should be taken to protect the health of the people.

Sphingomonas paucimobilis (S. paucimobilis) is Gram-negative bacillus that is widely distributed in the natural environment. It has been reported to cause opportunistic infection in immunocompromized hosts. The infection has been reported in the postoperative wound, post-traumatic leg ulcer, septicemia, meningitis, chronic cellulitis and postoperative endophthalmitis. No previous cases of infective endocarditis caused by S. paucimobilis have been reported. Here a case of infective endocarditis caused by S. paucimobilis was reported to help understand this disease. The middle-aged male patient was admitted to the hospital because of recurrent fever for more than two months. The first episode was high fever companied with pain in the left-upper abdomen. Computer topography (CT) suggested an infarction in spleen. Physical examination revealed that skin petechiae and heart murmur. Laboratory examination revealed transient anemia, microscopic hematuria, positive rheumatoid  factor, and echocardiography suggested emerging valvular regurgitation. Anti-infection treatment was effective. The same S. paucimobilis was isolated from peripheral blood culture four times and thus the diagnosis of endocarditis by S. paucimobilis was confirmed. The results of drug sensitivity testing showed pan-susceptible. The patient fully recovered by anti-infection treatment with mitral valve replacement operation. He was a healthy man without systematic diseases and the infection was community-aquired, which was different from most reports.

Objective Utilize the transgenic mouse lineage # 59 which mimics human HBsAg positive carriers, to investigate mechanisms of cellular response involved in HBsAg persistence. Methods Affymertix DNA microarray was used to observe the differential gene expression of the transgenic mouse liver compared to the sibling controls which were HBsAg negative. Proteomic experiments were conducted to identify the alterations of enzymes involved in metabolism in e transgenic mouse liver. Results Microarray showed that 43 genes were significantly (≥2 folds) up regulated and 104 genes were significantly down regulated in the transgenic mouse liver. Eighteen enzymes associated with metabolisms were identified by proteomic studies with increased levels (≥1.5 folds) and 9 enzymes with decreased levels in the transgenic mouse liver. Conclusion The persistence of HBsAg markedly affected host liver cell metabolism. Specifically, cholesterol biosynthesis was increased while bile acid synthesis was diminished. While glycolysis activity was reduced, the gluconeogenesis activity was increased. Amino acid catabolism was increased and urea synthesis was decreased. The potential biological impact of these metabolic alterations in HBV persistent infection was discussed.

The present paper aims to develop a rapid, accurate and efficient technique to detect enterovirus 71 (EV71) and coxsackievirus A16 (CA16) simultaneously for the etiological investigation of hand, foot and mouth disease (HFMD) in children during epidemic seasons in Beijing. Primers including one pair of universal enterovirus primers based on highly conserved region of 5’UTR of enteroviruses and two pairs of type-specific primers based on highly conserved VP1 region of EV71 and CA16 were designed and used at different concentrations in one single reaction tube to develop a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) which was carried out with two annealing temperatures. After the sensitivity and specificity of the multiplex RT-PCR were evaluated, the technique was applied to use for detecting EV71 and CA16 from clinical specimens collected from pediatric patients with HFMD who visited the Children’s Hospital Affiliated to Capital Institute of Pediatrics during the period of March to October in 2010. All the specimens were also inoculated into the Vero cells for virus isolation, which was used as a golden standard for this study. The minimum detectable concentration for CA16 and EV71 were 5.32 pg/ml and 0.64 pg/ml, respectively. The specificity of multiplex RT-PCR was 100%. The application of multiplex RT-PCR in 381 clinical samples collected from 371 HFMD cases showed that the total positive rate was 78.4%, of which the detection positive rates of CA16 and EV71 were 32.6% and 35.8% (the tested positive ratio of CV16:EV71 was 1:1.1). Comparative analysis with virus isolation demonstrated that the detection accuracy of this method was up to 95.2% for CA16 and 98.6% for EV71. The results indicate that this novel multiplex RT-PCR offers a rapid, sensitive and time-saving method to detect EV71 and CA16 from clinical specimens and can be used for the etiological surveillance of HFMD. Both CA16 and EV71 were still the major pathogens of HFMD in Beijing in 2010.

Objective To clone and express the Leptospira interrogans(L.interrogans)vaccine candidate gene,LB061 and to analysis the immunogenicity and conservation of the recombinant protein in different serovars of L.interrogans.Methods The characters of LB061 gene were predicted by bioinformatics software.The prokaryotic expression system of the LB061 was constructed by routine molecular biological techniques.Expression of the recombinant LB061 protein was induced by IPTG,and confirmed by Western blot.Balb/C mice were immunized with ercombinant LB061 and immune serum was used to study the immunogenicity of LB061 and the conservation of expressed LB061 in sixteen different serovars.In addition,antibodies against LB061 in rabbit serum infected with L.interrogans strain Lai(56601)were examined by Western blot.Results LB061 is a putative outer membrane protein in L.interrogans as predicted by bioinformatical analysiss.The prokaryotic expression system of LB061 was constructed successfully.Serum titers of anti-recombinant LB061 antibodies in immunized BALB/c mice were 1:32000.LB061 was detected in sixteen different serovars of L.interrogans.LB061-specific antibodies were detected in rabbit serum infected wit L.interrogans strain Lai by Western blot.Conclusion The vaccine candidate gene,LB061,of L.interrogans serogroup,icterohemorrhagic serovar,Lai strain can elicit antibody responses in rabbit during infection with Leptospira interrogans.Our studies provide a basis for further investigation on the role of LB061 as a vaccine candidate gene.