用鼠疫菌攻击动物制备感染蚤的试验分析

宿主、媒介、病原体是构成鼠疫流行的三个重要环节。有关媒介蚤的实验研究中常常需要制备一定数量的感染蚤供试。1989～1996年,我们在进行云南家野两型疫源地七种主要蚤类传播鼠疫的研究过程中,对七种蚤类进行了人工实验感染,因感染蚤的制备是整个传播试验的关键步骤,本文旨在介绍感染蚤的制备方法及应掌握的环节。

The ubiquitin-specific protease 17 is involved in virus-triggered type I IFN signaling

Viral infection initiates a series of signaling cascades that activate the transcription factors nuclear factor kappa B and interferon regulatory factor 3, which collaborate to induce transcription of genes for type I interferons （IFNs） and other cytokines. Here we report that the deubiquitinating enzyme ubiquitin-specific protease 17 （USP17） is required for virus-induced RIG-I- and melanoma differentiation-associated protein-5 （MDA5）-mediated type I IFN signaling. Knockdown of endogenous USP17 inhibited virus-, cytoplasmic poly（I：C）- and poly（dA：dT）-induced activation of the IFN-β promoter and cellular antiviral responses. We further found that knockdown of USP17 inhibited RIG-I- and MDA5-induced but not downstream activator-induced activation of the IFN-β promoter, which was correlated with an increase in ubiquitination levels of RIG-I and MDA5. Taken together, our findings suggest that USP17 functions through deubiquitination of RIG-I and MDA5 to regulate virus-induced type I IFN signaling.

The Flavivirus Protease As a Target for Drug Discovery

Many flaviviruses are significant human pathogens causing considerable disease burdens,including encephalitis and hemorrhagic fever,in the regions in which they are endemic.A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication,such as the viral protease.During viral replication,the flavivirus genome is translated as a single polyprotein precursor,which must be cleaved into individual proteins by a complex of the viral protease,NS3,and its cofactor,NS2B.Because this cleavage is an obligate step of the viral life-cycle,the flavivirus protease is an attractive target for antiviral drug development.In this review,we will survey recent drug development studies targeting the NS3 active site,as well as studies targeting an NS2B/NS3interaction site determined from flavivirus protease crystal structures.

Inhibition of Japanese Encephalitis Virus Infection by Flavivirus Recombinant E Protein Domain Ⅲ

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.

Isolation of Prawn(Exopalaemon carinicauda) Lipopolysaccharide and β-1,3-Glucan Binding Protein Gene and Its Expression in Responding to Bacterial and Viral Infections

The pattern recognition proteins （PRPs） play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and 13-1, 3-glucan binding protein （LGBP） gene in the ridge tail white prawn （Exopalaemon carinicauda） （EcLGBP） was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crus- taceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at dif- ferent levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus （WSSV）. The expression of EcLGBP in response to E parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.

Globulin-platelet model predicts minimal fibrosis and cirrhosis in chronic hepatitis B virus infected patients

AIM： To establish a simple model consisting of the rou- tine laboratory variables to predict both minimal fibrosis and cirrhosis in chronic hepatitis B virus （HBV）-infected patients. METHODS： We retrospectively investigated 114 chron- ic HBV-infected patients who underwent liver biopsy in two different hospitals. Thirteen parameters were analyzed by step-wise regression analysis and correla- tion analysis. A new fibrosis index [globulin/platelet （GP） model] was developed, including globulin （GLOB） and platelet count （PLT）. GP model = GLOB （g/mL） x 100/PLT （x 109/L）. We evaluated the receiver operating characteristics analysis used to predict minimal fibrosis and compared six other available models. RESULTS： Thirteen clinical biochemical and hemato- logical variables [sex, age, PLT, alanine aminotransfer- ase, aspartate aminotransferase （AST）, albumin, GLOB, total bilirubin （T.bil）, direct bilirubin （D.bil）, glutamyl-transferase, alkaline phosphatase, HBV DNA and pro- thrombin time （PT）] were analyzed according to three stages of liver fibrosis （F0-F1, F2-F3 and F4）. Bivariate Spearman＇s rank correlation analysis showed that six variables, including age, PLT, T.bil, D.bil, GLOB and PT, were correlated with the three fibrosis stages （FS）. Cor- relation coefficients were 0.23, -0.412, 0.208, 0.220, 0.314 and 0.212; and P value was 0.014, 〈 0.001, 0.026, 0.018, 0.001 and 0.024, respectively. Univariate analysis revealed that only PLT and GLOB were signifi- cantly different in the three FS （PLT： F = 11.772, P 〈 0.001; GLOB： F = 6.612, P = 0.002）. Step-wise multiple regression analysis showed that PLT and GLOB were also independently correlated with FS （R2 = 0.237）. By Spearman＇s rank correlation analysis, GP model was significantly correlated with the three FS （r = 0.466, P 〈 0.001）. The median values in F0-F1, F2-F3 and F4 were 1.461, 1.720 and 2.634. Compared with the six available models （fibrosis index, AST-platelet ratio, FIB-4, fibrosis-cirrhosis index and age-AST model and age-PLT ratio）, GP model showed a highest correlation coefficient. The sensitivity and positive predictive value at a cutoff value 〈 1.68 for predicting minimal fibrosis F0-F1 were 72.4% and 71.2%, respectively. The speci- ficity and negative predictive value at a cutoff value 〈 2.53 for the prediction of cirrhosis were 84.5% and 96.7%. The area under the curve （AUC） of GP model for predicting minimal fibrosis and cirrhosis was 0.762 [95% confidence interval （CI）： 0.676-0.848] and 0.781 （95% CI： 0.638-0.924）. Although the differences were not statistically significant between GP model and the other models （P all 〉 0.05）, the AUC of GP model was the largest among the seven models. CONCLUSION： By establishing a simple model using available laboratory variables, chronic HBV-infected patients with minimal fibrosis and cirrhosis can be di- agnosed accurately, and the clinical application of this model may reduce the need for liver biopsy in HBV- infected patients.

The Herpes Simplex Virus Type 1 Infected Cell Protein 22

As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction with the UL13 kinase,mediates the phosphorylation of RNA polymerase Ⅱ.Both ICP22 and UL13 are required for the activation of cdc2,the degradation of cyclins A and B and the acquisition of a new cdc2 partner,the UL42 DNA polymerase processivity factor.The cdc2-UL42 complex mediates postranscriptional modification of topoisomerase Ⅱα in an ICP22-dependent manner to promote L gene expression.In addition,ICP22 interacts with cdk9 in a Us3 kinase dependent fashion to phosphorylate RNA polymerase Ⅱ.

Involvement of aquaporins in a mouse model of rotavirus diarrhea

Rotavirus diarrhea is a major worldwide cause of infantile gastroenteritis; however, the mechanism responsible for intestinal fluid loss remains unclear. Water transfer across the intestinal epithelial membrane seems to occur because of aquaporins(AQPs). Accumulating evidence indicates that alterations in AQPs may play an important role in pathogenesis. Here, we focus on changes in AQPs in a mouse model of rotavirus diarrhea. In the present study, 32 of 35 mice developed diarrhea and mild dehydration within 24 hours after infection with rotavirus strain SA11. Intestinal epithelial cells demonstrated cytoplasmic vacuolation, malaligned villi, and atrophy. AQP1 expression was significantly attenuated in the ileum and colon in comparison with controls; likewise, AQP4 and-8 protein expression were significantly decreased in the colon of rotavirus diarrhea-infected mice. In contrast, AQP3 protein expression was significantly increased in the colon of rotavirus-infected mice in comparison with controls. These results indicate that rotavirus diarrhea is associated with the downregulation of AQP1,-4, and-8 expression. Therefore, AQPs play an important role in rotavirus diarrhea.

Characterization of a Putative Filovirus Vaccine:Virus-Like Particles

Filoviruses are hemorrhagic fever viruses endemic to parts of Africa and the Philippines. Infection carries with it a mortality rate of up to 90% and currently there are no effective vaccines or therapeutics available to combat infection. However, the filovirus virus-like particles (VLP), which are currently under development, have been shown to be a promising vaccine candidate. They provide protection from infection in the mouse, guinea pig, and nonhuman primate models of infection, eliciting high anti-glycoprotein antibody titers and T cell responses to viral proteins. In this review, we will highlight the development of the filovirus VLP and describe the current understanding of VLP immunogenicity and correlates of protection.

Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity

AIM： To construct a live attenuated Salmonella typhimurium （S. typhimurium） strain harboring the H pylori neutrophil activating protein （HP-NAP） gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity. METHODS： By genetic engineering methods, the genomic DNA of Hpylori was extracted as a template. The total length of the HP-NAP gene was amplified by polymerase chain reaction （PCR） and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immunogenicity was evaluated with animal experiments. RESULTS： A 435 bp product was cloned using high homology with HP-NAP gene in GenBank （more than 98%）. With identification by PCR and restriction enzyme digestion, a recomoinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with Hpyloril whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 （pIRES-NAP） also induced a specific immune response. CONCLUSION： The successful construction of HP-NAP oral DNA vaccine with good immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against Hpylori infection.

Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions

Active host-pathogen interactions take place during infection of human immunodeficiency virus type 1 (HIV-1). Outcomes of these interactions determine the efficiency of viral infection and subsequent disease progression. HIV- infected cells respond to viral invasion with various defensive strategies such as innate, cellular and humoral immune antiviral mechanisms. On the other hand, the virus has also developed various offensive tactics to suppress these host cellular responses. Among many of the viral offensive strategies, HIV-1 viral auxiliary proteins (Tat, Rev, Nef, Vif, Vpr and Vpu) play important roles in the host-pathogen interaction and thus have significant impacts on the outcome of HIV infection. One of the best examples is the interaction of Vif with a host cytidine deaminase APOBEC3G. Although specific roles of other auxiliary proteins are not as well described as Vif-APOBEC3G interaction, it is the goal of this brief review to summarize some of the preliminary findings with the hope to stimulate further discussion and investiga- tion in this exhilarating area of research.

Indinavir Resistance Evolution in One Human Immunodeficiency Virus Type 1 Infected Patient Revealed by Single-Genome Amplification

Human Immunodeficiency Virus Type 1 exists in vivo as quasispecies, and one of the genome's characteristics is its diversity. During the antiretroviral therapy, drug resistance is the main obstacle to effective viral prevention. Understanding the molecular evolution process is fundamental to analyze the mechanism of drug resistance and develop a strategy to minimize resistance. Objective: The molecular evolution of drug resistance of one patient who had received reverse transcriptase inhibitors for a long time and had treatment which replaced Nevirapine with Indinavir was analyzed, with the aim of observing the drug resistance evolution pathway. Methods: The patient, XLF, was followed-up for six successive times. The viral populations were amplified and sequenced by single-genome amplification. All the sequences were submitted to the Stanford HIV Drug Resistance Database for the analysis of genotypic drug resistance. Results: 149 entire protease and 171 entire reverse transcriptase sequences were obtained from these samples, and all sequences were identified as subtype B. Before the patient received Indinavir, the viral population only had some polymorphisms in the protease sequences. After the patient began Indinavir treatment, the variants carrying polymorphisms declined while variants carrying the secondary mutation G73S gained the advantage. As therapy was prolonged, G73S was combined with M46I/L90M to form a resistance pattern M46I/G73S/L90M, which then became the dominant population. 97.9% of variants had the M46I/G73S/L90M pattern at XLF6. During the emergence of protease inhibitors resistance, reverse transcriptase inhibitors resistance maintained high levels. Conclusion: Indinavirresistance evolution was observed by single-genome amplification. During the course of changing the regimen to incorporate Indinavir, the G73S mutation occurred and was combined with M46I/L90M.

Increased presence of effector lymphocytes during Helicobacter hepaticus-induced colitis

AIM： To identify and characterize drosophila mothers against decapentaplegic （SMAD）3-dependent changes in immune cell populations following infection with He- Iicobacter hepaticus （H. hepaticus）. METHODS： SMAD3/ （n = L9） and colitis-resistant SMAD3＋/ （n = 24） mice （8-10 wk of age） were in- fected with/-/, hepaticus and changes in immune cell populations [T lymphocytes, natural killer （NK） cells, T regulatory cells] were measured in the spleen and mesenteric lymph nodes （MsLNs） at 0 d, 3 d, 7 d and 28 d post-infection using flow cytometry. Genotype-dependent changes in T lymphocytes and granzyme B＋ cells were also assessed after 28 d in proximal colon tissue using immunohistochemistry. RESULTS： As previously observed, SMAD3＋, but not SMAD3＋/- mice, developed colitis, peaking at 4 wk post-infection. No significant changes in T cell subsets were observed in the spleen or in the MsLNs between genotypes at any time point. However, CD4＋ and CD8＋/ CD62L＋＋ cells, an effector T lymphocyte population, as well as NK cells （NKp46/DX5＋） were significantly higher in the MsLNs of SMAD3/ mice at 7 d and 28 d post-in- fection. In the colon, a higher number of CD3＋ cells were present in SMAD3＋ compared to SMAD3＋/- mice at base- line, which did not significantly change during infection. However, the number of granzyme B＋ cells, a marker of cytolytic lymphoo/tes, significantly increased in SMAD3＋ mice 28 d post-infection compared to both SMAD3＋/- mice and to baseline values. This was consistent with more severe colitis development in these animals. CONCLUSION： Data suggest that defects in SMAD3 signaling increase susceptibility to H. hepaticus-induced colitis through aberrant activation and/or dysregulation of effector lymphoo/tes.

Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique

AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection. METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BamH I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used. The mRNA of HepG2 cells transfected with pcDNA3.Incomplete S and pcDNA3.1(-) empty vector was isolated, and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR) method, and cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification. RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete s was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86 positive clones. Colony PCR showed that 86 clones contained DNA inserts of 200-1 000 bp, respectively. Sequence analysis was performed in 35 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 33 coding sequences were obtained. These cDNA sequences might be target genes transactivated by complete S protein of HBV. Moreover, two unknown genes were discovered, full length coding sequences were obtained by bioinformatics techniques, one of them was named complete S transactivated protein 1 (CSTP1) and registered in GenBank (AY553877). CONCLUSION: The complete S gene of HBV has a transactivating effect on SV40 early promoter. A subtractive cDNA library of genes transactivated by HBV complete S protein using SSH technique has been constructed successfully. The obtained sequences may be target genes transactivated by HBV complete S protein among which some genes coding proteins are involved in cell cycle regulation, metabolism, immunity, signal transduction, cell apoptosis and formation mechanism of hepatic carcinoma.

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c(Hsp70_(359–610)) fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.

Research progress on Helicobacter pyloriouter membrane protein

Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It includes 32 members. These OMP are important for the diagnosis, protective immunity, pathogenicity of H pylori and so on. They are significantly associated with high H pylori density,the damage of gastric mucosa, high mucosal IL-8 levels and severe neutrophil infiltration. We introduce their research progress on pathogenicity.

C-terminal domain of hepatitis C virus core protein is essential for secretion

AIM: We have previously demonstrated that hepatitis C virus (HCV) core protein is efficiently released into the culture medium in insect cells. The objective of this study is to characterize the HCV core secretion in insect cells. METHODS: We constructed recombinant baculoviruses expressing various-length of mutant core proteins, expressed these proteins in insect cells, and examined core protein secretion in insect cells. RESULTS: Only wild type core was efficiently released into the culture medium, although the protein expression level of wild type core was lower than those of other mutant core proteins. We found that the shorter form of the core construct expressed the higher level of protein. However, if more than 18 amino acids of the core were truncated at the C-terminus, core proteins were no longer secreted into the culture medium. Membrane flotation data show that the secreted core proteins are associated with the cellular membrane protein, indicating that HCV core is secreted as a membrane complex. CONCLUSION: The C-terminal 18 amino acids of HCV core were crucial for core secretion into the culture media. Since HCV replication occurs on lipid raft membrane structure, these results suggest that HCV may utilize a unique core release mechanism to escape immune surveillance, thereby potentially representing the feature of HCV morphogenesis.

Helicobacter pylori specific immune response induced by conservative flagellin linear B-cell epitope

AIM:To testify the immunogenicity of a conservative B-cell linear epitope of Helicobacter pylori ( H pylori) flagellin A. METHODS: Different programs were used to analyze the secondary structure, molecular hydropathy, and surface accessibility of Hpyloriflagellin A. Linear B-cell epitopes were estimated based on the structural and physiochemical information. Analysis of residue divergence was proposed to screen a conservative linear epitope. The 29-peptide (Pep29mer) synthesized by chemical method, including the predicted conservative B-cell epitope and a known K^2d compatible T-cell epitope, was used to immunize mice, and then H pylori-specific antibodies were detected by ELISA. RESULTS: Based on the analyses of divergent amino acid residues, structural and physiochemical characteristics, it was strongly suggested that the short fragment NDSDGR was the core of a conservative linear epitope in flagellin A. Animals immunized by Pep29mer acquired efficient immune response. In detail, serum Hpylori-specific IgA and IgGl increased significantly in immunized group, while IgG2a only had an insignificant change. Hpylori-specific IgA in gastrointestinal flushing fluid also increased significantly. CONCLUSION: The conservative short fragment NDSDGR is the core of a linear B-cell epitope of flagellin A.