Association of IL-28B, TBX21 gene polymorphisms and predictors of virological response for chronic hepatitis C.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. The outcomes of both spontaneous HCV clearance and response to therapy depend on both viral and host factors. To investigate the influence of polymorphisms of IL-28B rs12979860 and TBX21 rs17250932, rs4794067 as well as viral factors (HCV genotype, F protein) on the outcome of HCV infection, we genotyped 565 patients with chronic HCV infection, 191 patients spontaneously resolved from HCV infection, 359 healthy controls and 383 treatment-naïve CHC patients with pegylated interferon-α and ribavirin (PEG IFN-α/RBV). Results showed that TBX21 rs4794067 variant genotypes significantly correlated with increased risk of HCV chronic infection (dominant model: OR = 5.690, 95% CI = 2.024-16.000) and susceptibility (dominant model: OR = 5.658, 95% CI = 2.514-12.735). We also found that the rs12979860, rs2227982 and rs36084323 polymorphisms showed no significant associations with susceptibility or spontaneous clearance of HCV in the anti-F antibody subgroup; however, the anti-F antibody positive subgroup might show an increased risk of N-SVR (all P < 0.001). Our results demonstrate that variant factors in both the host and pathogen are commonly important for HCV clearance. In addition rs4794067 and F protein status may be strong predictive markers in the Chinese population.

PD-1/PD-L1 signal pathway participates in HCV F protein-induced T cell dysfunction in chronic HCV infection.

Programmed cell death-1/programmed cell death-1 ligand 1 (PD-1/PD-L1) inhibitory signal pathway has been verified to be involved in the establishment of persistent viral infections. Blockade of PD-1/PD-L1 engagement to reinvigorate T cell activity is supposed to be a potential therapeutic scheme. Studies have verified the participation of PD-1/PD-L1 in hepatitis C virus (HCV) core protein-regulated immune response. To determine the roles of PD-1/PD-L1 signal pathway in HCV F protein-induced immunoreaction in chronic HCV infection, variations in T cells were examined. The results showed that PD-1 expression on CD8(+) and CD4(+) T cells was increased with HCV F stimulation in both chronic HCV patients and healthy controls, and could be reduced partly by PD-1/PD-L1 blocking. Additionally, by PD-1/PD-L1 blocking, HCV F-induced inhibition of T cell proliferation and promotion of cellular apoptosis were partly or even totally recovered. Furthermore, levels of both Th1 and Th2 cytokines were elevated in the presence of anti-PD-L1 antibody. All these results indicated that PD-1/PD-L1 signal pathway also participates in HCV F protein-induced immunoregulation. PD-1/PD-L1 blocking plays important roles in the restoration of effective functionality of the impaired T cells in chronic HCV patients.

HCV F protein amplifies the predictions of IL-28B and CTLA-4 polymorphisms about the susceptibility and outcomes of HCV infection in Southeast China.

Cytotoxic T lymphocyte associated antigen-4(CTLA-4) is an inhibitory receptor with great value in the progression of hepatitis C virus (HCV) infection related diseases. To determine the potential associations of IL-28B rs12979860 and CTLA-4 rs231775, rs3087243 and rs5742909 polymorphisms with the generation of HCV F protein, susceptibility and outcomes of HCV infection, a total of 375 healthy controls, 219 HCV spontaneous recovered patients and 600 chronic HCV patients from Southeast China were recruited and genotyped in this study. And the relative mRNA levels of CTLA-4 in T cells were detected. Logistic regression analysis showed that rs231775 A allele was associated with significantly higher rate of spontaneous viral clearance in anti-HCV F antibody negative patients (adjusted OR=0.512, P=0.008), but allele A was related to higher mRNA level of CTLA-4 with the generation of HCV F protein. And rs5742909 T allele added up to the risk of HCV infection chronicity significantly in patients with the presence of HCV F protein (adjusted OR=2.698, P=0.003). Also, the rs5742909 CC genotype, along with the presence of HCV F protein, indicated a significantly higher CTLA-4 level than that in anti-HCV F antibody negative patients. The AG+AA genotype of rs3087243 significantly increased the susceptibility to HCV infection in subjects over 56 years old (adjusted OR=1.595, P=0.011). Genotype-genotype interaction between IL-28B rs12979860 and CTLA-4 rs3087243 was found to be significantly associated with increased susceptibility to HCV infection (adjusted OR=1.509, P=0.005). Haplotype analysis in CTLA-4 also showed significant association with the generation of HCV F protein. All these results indicated the importance of IL-28B and CTLA-4 polymorphisms and their associations with HCV F protein in the risk and chronicity of HCV infection in Chinese Han population in Southeast China.

Potential Role of Hepatitis C Virus Alternate Reading Frame Protein in Negative Regulation of T-Bet Gene Expression.

Hepatitis C virus (HCV) is a major cause of chronic liver disease and has led to cirrhosis or hepatocellular carcinoma in a majority of infected individuals. We have previously demonstrated that the HCV alternate reading frame protein (F protein) is related to Th1/Th2 bias in chronic hepatitis C (CHC) patients, and we aimed to explore the relative molecular mechanisms here. A total of 104 cases including CHC patients and healthy donors were enrolled. T-bet and GATA-3 expression levels were analyzed in peripheral blood mononuclear cells (PBMCs). The levels of signal transducer and activator of transcription-1/-6(STAT1/6) and phosphorylated STAT1/6(pSTAT1/6) in PBMCs were measured by Western blotting. Our results showed that the levels of T-bet in PBMCs, as well as the levels of gamma interferon (IFN-γ) in sera, were decreased in anti-F protein antibody seropositive patients compared with anti-F protein antibody seronegative patients, whereas the levels of GATA-3 did not show difference between the two groups. Moreover, the decreased pSTAT1 and increased pSTAT6 were observed in PBMCs by HCV core/F protein stimulation with constant STAT1/6 expression. Taken together, it suggested that T-bet may be involved in Th1/Th2 bias induced by HCV F protein, and the disruption of STAT phosphorylation may participate in this mediation.

Genetic variations of IL-28B and PD-1 are in association with the susceptibility and outcomes of HCV infection in Southeast China.

Programmed cell death-1 (PD-1) is an important co-inhibitory molecule involved in the progression of chronic viral infections. To investigate the associations of three single nucleotide polymorphisms (SNPs) (rs10204525, rs2227982 and rs36084323) in PD-1 and a previously well-inquired SNP rs12979860 in IL-28B with the outcomes of hepatitis C virus (HCV) infection in Southeast China, a total of 375 healthy controls, 219 spontaneous resolved HCV patients and 600 chronic HCV patients were enrolled in this study. The generation of HCV F protein and PD-1 expression on T cells was determined. Multivariate logistic regression analysis showed no association of rs12979860 CC genotype with spontaneous clearance of HCV infection in our subjects. The generation of HCV F protein was significantly related to HCV infection chronicity, but no significant relationship was found between HCV F protein and SNPs in PD-1. The rs10204525 TT genotype was associated with an increased risk of HCV infection chronicity in age ⩽56years subgroup (adjusted OR=0.390, P=3.8×10(-4)). The C allele of rs10204525 played protective roles in females infected with HCV (adjusted OR=0.608, P=0.008). A significant higher percentage of PD-1 expression on T cells was observed in rs10204525 TT genotype when compared to CC genotype (P=0.047). Moreover, a significant genotype-genotype interaction between IL-28B rs12979860 CC and PD-1 rs10204525 TC+CC was found to be associated with higher rates of spontaneous clearance (adjusted OR=0.689, P=0.032). The combined effect of rs12979860 and rs10204525 was of great value in predicting the outcomes of HCV infection. These analyses showed the importance of IL-28B and PD-1 polymorphisms and their interactions in the outcomes of HCV infection in Chinese Han population in Southeast China.

Toll-like receptor 7 variations are associated with the susceptibility to HCV infection among Chinese females.

Toll-like receptors 7 (TLR7) play a crucial role in provoking an immune response in HCV infection. We aimed to investigate whether single nucleotide polymorphisms (SNPs) of TLR7, including rs179009, rs179010 and rs179012, affect the outcomes of HCV infection among the Chinese population. A total of 1767 Chinese Han individuals were enrolled. The distribution of SNP frequencies among three groups with different outcomes of HCV infection was assessed, including healthy controls, cases with spontaneous clearance and cases with viral persistence. Then TLR7 mRNA expression and the production of IFN-α and IL-6 after TLR7 agonist Imiquimod stimulation in vitro were determined. Our results suggested that rs179009 GG genotype was significantly associated with a higher risk of the susceptibility to HCV infection among female subjects (OR=2.42, 95% CI=1.24-4.71, P=0.01). Haplotype GCG was significantly associated with a high risk for HCV susceptibility (OR=1.50, 95% CI=1.11-2.03, P=0.01) as compared with the reference haplotype ACG among females. In the functional research of rs179009, a lower IFN-α level was observed in GG genotype than in AA genotype (P=0.032). Our data indicate that TLR7 rs179009 GG genotype and haplotype GCG were associated with an increased risk of the susceptibility to HCV infection among Chinese females, which may be due to the impaired IFN-α response.

Sex-specific association between X-linked Toll-like receptor 7 with the outcomes of hepatitis C virus infection.

Toll-like receptor 7 (TLR7) senses hepatitis C virus (HCV) infection and drives the host specific innate and adaptive immune response. The aim of this study was to estimate the distributions of TLR7 single nucleotide polymorphisms (SNPs), including rs179019 and rs3853839, as well as the effect of TLR7 gene variants on TLR7 mRNA expression and cytokine production in response to TLR7 agonist in vitro. TLR7 SNP genotyping was performed among a Chinese sample population of 418 patients with persistent HCV infection, 317 patients with HCV spontaneous clearance, and 989 healthy controls. TLR7 mRNA expression and TLR7-specific IFN-α and IL-6 secretion in peripheral blood mononuclear cells, derived from 60 healthy individuals in vitro, were then quantified. We identified the association of TLR7 rs3853839C allele, haplotype CC and haplotype AC (rs179019/rs3853839) with protection against HCV persistence in Chinese females (OR=0.49, 95% CI=0.29-0.81, P=0.01 for rs3853839 GC; OR=0.29, 95% CI=0.11-0.75, P=0.01 for rs3853839 CC; OR=0.51, 95% CI=0.38-0.77, P<0.01 for haplotype CC; OR=0.29, 95% CI=0.10-0.88, P=0.03 for haplotype AC). In addition, the rs3853839 CC genotype among female carriers had significantly low TLR7 mRNA expression (P=0.006 for GG vs. CC, P=0.021 for GC vs. CC), along with decreased IFN-α (P=0.002 for GG vs. CC, P=0.021 for GC vs. CC) and increased antiviral IL-6 production (P=0.002 for GG vs. CC, P=0.030 for GC vs. CC), after treatment with Imiquimod in vitro. The cytokine profile among rs3853839 CC genotype female carriers may indicate a pronounced protective effect against persistent HCV infection. The functional polymorphism of TLR7 rs3853839C allele was found to be sex-specific and associated with protection against HCV persistence among Chinese females, which may be due to specific IFN-α and IL-6 secretion profiles.

Meta-analysis on the associations of TLR2 gene polymorphisms with pulmonary tuberculosis susceptibility among Asian populations.

BACKGROUND: Publications regarding the associations of toll-like receptor 2 (TLR2) G2258A and T597C polymorphisms with pulmonary tuberculosis (PTB) susceptibility are inconsistent. A meta-analysis was conducted to investigate the relationship between TLR2 G2258A and T597C polymorphisms with PTB susceptibility. METHODS: A systematic search was performed for published studies on the relationship between TLR2 polymorphisms and PTB susceptibility. Information was gathered from each eligible study, and statistically analyzed. RESULTS: 6 eligible studies, totaling 1301 cases and 1217 controls on G2258A genotypes, and 8 studies, totaling 2175 cases and 2069 controls on T597C genotypes, were included in the analysis. TLR2 2258G allele and 2258GG genotype were found to be associated with decreased PTB susceptibility (A vs. G: OR  = 3.02, 95% CI: 2.22-4.12, P<0.001, GA+AA vs. GG: OR  = 2.69, 95% CI = 1.49-4.87, P = 0.001). In the subgroup analyses, the 2258G allele and 2258GG genotype also exhibited a protective effect of PTB risk in Asians (A vs. G: OR  = 2.95, 95% CI: 1.91-4.55, P<0.001; GA+AA vs. GG: OR  = 3.59, 95% CI: 2.23-5.78, P<0.001), while no associations were observed in Caucasians. No significant associations between T597C polymorphism and PTB were found in the allele model (C vs. T: OR  = 0.95, 95% CI: 0.86-1.04, P = 0.28), co-dominant model (CC vs. TT: OR  = 0.88, 95% CI = 0.92-1.40, P = 0.25; CT vs. TT: OR  = 0.92, 95% CI = 0.80-1.06, P = 0.28), recessive model (CC vs. TT+TC: OR  = 0.96, 95% CI: 0.80-1.16, P = 0.69), or dominant model (TC+CC vs. TT: OR  = 0.93, 95% CI = 0.76-1.15, P = 0.51). The associations of T597C polymorphism with PTB susceptibility, in the ethnic-specific analyses, were still not significant. CONCLUSION: TLR2 2258G allele may provide protective effects against PTB susceptibility, particularly among Asians, whereas TLR2 T597C polymorphism might not be associated with PTB susceptibility.

Association of interleukin-18 gene polymorphisms with the outcomes of hepatitis C virus infection in high-risk Chinese Han population.

Interleukin 18 (IL-18) gene polymorphisms have been reported to be associated with the outcomes of hepatitis C virus (HCV) infection in Americans, Indians and Europeans. We aimed to investigate whether the association can be replicated in Chinese Han population. Three IL-18 variants, -656G>T, -137G>C and +105A>C, were genotyped in three independent Han cohorts consisting of 552 cases and 784 controls. By using logistic regression analysis and multiple testing, haplotype GCC were associated with a protection from susceptibility to HCV. After stratified analysis, both the carriage of -137C allele in the older or hemodialysis subgroup and the carriage of +105C allele in the younger subgroup were found to be significantly associated with a decreased risk of HCV susceptibility. By using logistic regression analysis and multiple testing for the resolution of HCV infection, our study showed that +105C allele and haplotype GGC displayed a negative effect on HCV persistence. After stratified analysis, a significantly decreased risk for HCV persistence was found in +105C allele in the subgroups of young, male or female, drug user or hemodialysis and HCV-1 or HCV mixed genotype. No significant association was observed between -656G>T and the outcomes of HCV infection. Our results demonstrated that the carriage of -137C allele, +105C allele, haplotype -656G/-137C/+105C and haplotype -656G/-137G/+105C of IL-18 gene may contribute to better outcomes of HCV infection in high-risk Chinese Han population.

Pathogenicity of a natural reassortant hantavirus CGRn9415 in newborn rats and newborn mice.

To better understand the pathogenicity and infectivity of a natural reassortant CGRn9415 generated from Hantaan virus (HTNV) and Seoul virus (SEOV), CGRn9415, HTNV 76-118 and SEOV L99 were used to infect newborn Kunming (KM) mice and newborn Wistar rats. In KM mice, there was no statistical difference between the death rate with CGRn9415 and that of L99, while 76-118 killed all mice even at low dosage; CGRn9415 killed all infected rats similar to L99 at the dosage of 10(5) f.f.u., while no death occurred in rats infected with 76-118 even as high as 2 × 10(5) f.f.u., suggesting that the reassortant CGRn9415 possesses similar pathogenicity as L99. Furthermore, the reassortant CGRn9415 could establish a persistent infection in both KM mice and Wistar rats more easily than 76-118 or L99. These data suggest that the reassorted hantavirus behaves more like SEOV as far as the pathogenicity is concerned.

[Association between IL-10 gene polymorphisms and the outcomes of hepatitis C virus infection among high risk populations in Jiangsu province].

OBJECTIVE: To explore the relationship between interleukin (IL)-10 gene polymorphisms and the susceptibility or the outcomes of HCV infection among high-risk populations in Jiangsu province. METHODS: IL-10 gene SNPs were detected in 1555 subjects including 264 self-limited HCV infections. 371 persistent HCV infections and 920 healthy controls were selected through Taqman-MGB. RESULTS: After adjusted for cofounders as sex, age and high-risk population, data from logistic regression analysis showed that the distribution of IL-10 genotypes among the controls, spontaneous clearances and those with persistent infections did not show much differences. RESULTS: from further stratified analysis showed that, at the position of -819T/C, when compared with TT genotype, TC genotype had a significantly increasing chance of self-limited HCV infection among middle-aged, females and paid blood doners (adjusted OR values and 95%CI were: 2.160, 1.163 - 4.011; 1.693, 1.066 - 2.688 and 4.084, 1.743 - 9.570). It also had a lower risk of progressing to persistent HCV infection among those paid blood doners (the adjusted OR values and 95%CI were: 0.312, 0.130 - 0.747). CC genotype had a higher chance of self-limited HCV infection among people underwent blood dialysis (the adjusted OR values and 95%CI were: 2.120, 1.071 - 4.197). RESULTS: also showed a decreased risk of progressing to persistent infection among paid blood doners (the adjusted OR values and 95%CI were: 0.156, 0.043 - 0.566). At the position of -592A/C, when compared to AA genotype, the AC genotype had a significantly increasing chance of self-limited HCV infection among middle-aged, females and paid blood doners (the adjusted OR values and 95%CI were: 2.176, 1.173 - 4.037; 1.659, 1.055 - 2.607; 3.704, 1.625 - 8.443) but had an increased risk of persistent HCV infection among females (the adjusted OR values and 95%CI were: 1.525, 1.017 - 2.286). AC genotype showed an increased opportunity to progress to HCV persistent infection among drug users (the adjusted OR values and 95%CI were: 1.845, 1.122 - 3.034) but had a reduced risk of progressing to HCV persistent infection among paid blood doners (the adjusted OR values and 95%CI were: 0.361, 0.155 - 0.841). CC genotype had an increased opportunity to self-limited HCV infection as well as having a decreased risk of progressing to persistent infection among paid blood doners (the adjusted OR values and 95%CI were: 3.125, 1.016 - 9.605; 0.218, 0.063 - 0.748). At the position of -1082A/G, AG/GG genotypes had an increased chance of self-limited infection among blood doners (the adjusted OR values and 95%CI were: 3.780, 1.620 - 8.820). CONCLUSION: IL-10-819T/C, -592A/C, -1082A/G SNPs might be related with the susceptibility and the outcomes of HCV infection among populations at high risk.

[A study of immunoassay by using quantum dots to detect Hantavirus infection].

OBJECTIVE: To develop a new method to detect anti-Hantavirus IgG antibodies (HV IgG) based on quantum dots (QDs) and indirect immune technique. METHODS: The carbodiimide crosslinking method was used to couple protein G and goat antihuman IgG on the surface of water-solubility QDs. The coverglass covered HV antigen was used as carrier, and QDs-PG-IgG conjugates was used as labeled second antibody to detect the HV-IgG in the serum samples. The detecting conditions were optimized. RESULTS: The optimum reaction time, pH and goat antihuman IgG concentration for conjugating the QDs with goat antihuman IgG were 6.0, 2h, and 20 microg/ml, respectively. The optimum working dilution of QDs-PG-IgG conjugates was 1: 200. The detection limit of the serum samples was about 1: 1280 dilution. CONCLUSION: The method established in this study has been demonstrated to be a specific, sensitive, rapid test for detecting HV antibodies, laying the foundation of single molecule detection. The anti-fluorescence quenching ability of this method was significant improved.

[HCV DF protein inhibits expression of p16 and p21 in HepG2 cells].

AIM: To investigate the effect of HCV DF (Double-shift F) protein on the expression p16 and p21 in HepG(2); cells. METHODS: DF gene was amplificated from the whole HCV 1b genome, and cloned into pCDNA3.0 vecter. The recombinant plasmid (pCDNA3.0/HCV-DF) and empty vector were transfected into HepG(2); cells. Screening was performed with G418. p16 and p21 mRNA were detected by semi-quantitative RT-PCR, and protein by Western blot. RESULTS: Stable expression of the recombinant plasmid was found in HCV DF protein. The expression of p16 and p21 in HepG();2 cells transfected with pCDNA3.0/HCV-DF were lower than those with blank plasmid. CONCLUSION: HCV DF protein inhibits expression of p16 and p21 in HepG(2); cells. This suggested that HCV DF protein may participate in the progress of hepatocellular carcinoma.

[Study on the transmission of Hantaan virus and Orientia tsutsugamushi by naturally dual infected Leptotrombidium scutellare through stinging].

OBJECTIVE: To investigate whether Leptotrombidium scutellare could be naturally infected by both Hantaan virus (HV) and Orientia tsutsugamushi (OT) and transmission status by stinging. METHODS: 3459 Leptotrombidium scutellares from mice bodies and 3265 which were free were collected in the epidemic area of hemorrhagic fever with renal syndrome (HFRS) and tsutsugamushi disease.15 days later, the suspensions of lung and spleen of mice with 6 in a group stung by 1, 5 or 10 infected mites were injected intra-cerebrally into other mice for the detection of HV and OT in the next 6 generations of the mice, with immunofluorescent antibody technique (IFAT) and Giemsa staining technique. The passages of Vero-E6 cells inoculated on the aseptic filtrations from different number of infected mites were used to detect HV and OT pathogens. HV-RNA and OT-DNA were detected by PCR. RESULTS: After passage, HV positive mouse body mite group out of both 5 and 10 mites in the sixth generation, OT positive mouse body mite group out of the 10 mites in the sixth generation, both HV and OT positive mouse body mite group out of 1 mite in the fifth and sixth generation, both HV and OT positive mouse body mite group out of 5 and 10 mites in the sixth generation, and free mites group out of 1, 5 and 10 mites in the sixth generation, were found one mouse infected by both HV and OT, respectively. Out of the fourth generation of Vero-E6 cells, one sample was found both HV and OT positive out of 5 and 10 HV and OT mouse body mite group, respectively. In the sixth generation, both HV and OT positive cells were detected in one mouse mite group and the 1, 5, 10 free mite groups, respectively. HV-RNA and OT-DNA were all detected by PCR. CONCLUSION: Both HV and OT could be coexisted in wild Leptotrombidium scutellare and transmitted by stinging.

[Molecular evolution analysis of hantaviruses in Zhejiang Province].

In order to analyze the molecular epidemiology of Hantavirus (HV) in Zhejiang Province, the complete M and S genome sequences of 12 HV strains from different hosts and locations in Zhejiang Province of China during the period of 1981-2007 were analyzed on genetic evolution by DNAstar and MEGA 4.0 software in this research. Phylogenetic analyses revealed that HTN and SEO strains were co-circulating in Zhejiang Province, and the difference in sequence similarity and the phylogeny was closely related to the isolated regions, but had no distinct relationship with the isolate year and the host, indicating a relationship between epidemiology of HFRS and the distribution region, especially in HTNV. The isolates in the same region could be assigned in same or near phylogenetic clade sharing high sequence similarity. Interestingly, the Gou3 strain and ZJ5 strain isolated from Jiande region in Zhejiang Province formed a distinct phylogenetic lineage in SEOV clade, and different from the other SEOV variants outside China. We believed that the special SEOV variants were distributed in Jiande region.

[Influence of F protein of hepatitis C virus subtype 1b inhibits on human hepatocellular carcinoma HepG2 cell apoptosis].

OBJECTIVE: To investigate the effects of F protein of hepatitis C virus subtype 1b on the apoptosis of human hepatocellular carcinoma HepG2 cells. METHODS: HepG2 cells were transfected with recombinant plasmid pcDNA3.0-F-EGFP and pcDNA3.0-F-EGFP-HepG2 strain was exposed to Act-D and tumor necrosis factor alpha (TNF alpha) treatment in order to induce cell apoptosis with positive control pcDNA3.0-C-EGFP-HepG2, negative control pcDNA3.0-C-EGFP-HepG2 and blank control HepG2. Annexin V-FITC/PI of flow cytometry was performed to determine the number of apoptotic cells. DNA Ladder was used to observe the isolation of apoptotic DNA fragments in the apoptotic cells. RESULTS: pcDNA3.0-F-EGFP- HepG2 cell strain showed a much delayed apoptosis as well as obviously lowering the apoptotic rate when compared with the pcDNA3.0-HepG2 strain and HepG2 strain (P < 0.001). CONCLUSION: The introduction and expression of extraneous gene (the F gene of hepatitis C virus subtype 1b) could significantly inhibit the apoptosis of HepG2 cells.

[Study on the difference of genes and the type identification of hantavirus from Lishui, Zhejiang province].

OBJECTIVE: To isolate hantavirus from Lishui county--one of the epidemic regions for hemorrhagic fever with renal syndrome (HFRS), in Zhejiang province, and to identify the serotype and molecular/biological characteristics of a new HTN subtype hantavirus (HV) strains, hopefully to provide evidence for HFRS prevention and therapy. METHODS: Data on the host animals was collected from Lishui, Zhejiang province in 2007. Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues from HV infected Vero-E6 cells for HV isolation, then total RNA was extracted from Hantavirus Lishui strains and amplified by RT-PCR M, S segments of strains genome were also cloned and sequenced and compared with those of other strains of HV. RESULTS: 2 strains virus (ZLS6-11 and ZLS-12) were successfully isolated from 7 positive lung samples of mice and were identified as HTNV by anti-McAb and phylogenetic analysis. With sequence compation,we found that 2 strains with complete M and S segment had higher homology with HTN-type strains than with other types of HV, but 13.4%-20.7% and 10.3%-16.1% of the genes were found which were different from HTNV. The phylogenetic trees constructed by complete S and M segment showed that ZLS6-11 and ZLS-12 strains were located in HTNV group,and structured independent embranchment. CONCLUSION: ZLS6-11 and ZLS-12 Strains were believed to belong to HTN-type and phylogenetically different from the HTNV.

[Expression of partial S gene of Hantavirus Z10 in Pichia pastoris].

OBJECTIVE: The purpose of this study is to express partial S gene of Hantavirus Z10. METHODS: The 300 bp S gene of Z10 strain was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris and subcloned into pMD19-T. The SP300 gene was constructed into pPICZaA and sequenced. The recombinant pPICZaA-SP300 and pPICZaA-S300 was transformed into Pichia with LiCI. RESULTS: The recombination Pichia were cultivate, and expressed the SP300 or S300 gene induced in Pichia by methanol. CONCLUSION: The nucleocapsid secreted from the Pichia can be detected by ELISA and WesternBlot.

[Study on the molecular epidemiology of hantavirus carried by rodent hosts in Cixi, Zhejiang province].

OBJECTIVE: To collect more data on the epidemiology of hantavirus in rodents in Cixi, Zhejiang province. METHODS: Rodents were captured in Cixi, where hemorrhagic fever with renal syndrome (HFRS) appeared endemic. Hantavirus antigens in the rat lungs were detected by immunofluorescence assay. Partial S segment sequences were amplified by reverse transciption-polymerase chain reaction (RT-PCR) and then sequenced. The phyologenetic trees were constructed by maximum likelihood method to detect the genetic characteristics of hantavirus. RESULTS: A total of 243 rodents were trapped in the epidemic areas, and hantavirus antigens were identified in 7 out of these lung samples (2.88%). Partial S segment sequences (620-999 nt) were recovered from 6 samples and sequenced. Data from phylogenetic analysis of these S segment sequences indicated that all viruses belonged to Seoul virus (SEOV), despite the origins of sources were either from Rattus norvegicus or from R. flabipectus. These viruses could further be divided into two distinct lineages but the viruses carried by R. norvegicus were different from those carried by R. flabipectus. CONCLUSION: Two distinct lineages of SEOV had been cocirculating in rodents in Cixi.

[Mass spectrometric analysis of peripheral blood mononuclear cells in HCV infected patients].

AIM: To investigate the diversity of proteins' pattern of the peripheral blood mononuclear cells (PBMCs) in patients with hepatitis C virus (HCV) infection, identify the differentially expressed proteins and analyze their roles in the mechanism of chronic hepatitis C. METHODS: The total proteins from PBMCs in HCV infected patients (28) and healthy persons (10) were separated by the immobilized pH gradient-based 2-DE. The differentially expressed proteins were screened by PDQuest analysis software, and then identified by peptide mass fingerprint (PMF) based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and database searching. RESULTS: 2-DE results showed that the mean number of protein spots in HCV infected patients and healthy persons was 625 and 614. Twelve differential proteins were tested by MALDI-TOF-MS, and 10 of them were identified. Some of the proteins participated in protein synthesis and degradation, signal transduction, metabolism, or cytoskeleton construction while some of them were proteins of virus. CONCLUSION: 2-DE pattern of PBMCs from HCV infected patients or healthy persons has been established and ten differentially expressed proteins has been characterized. Our study is helpful for clinical detection of HCV infection and further research into the mechanisms of chronic hepatitis C.