[Mechanism of chlorogenic acid on apoptosis of rat nucleus pulposus cells induced by oxidative stress].

OBJECTIVE: To investigate the mechanism of chlorogenic acid (CGA) on H2O2-induced apoptosis in the rat nucleus pulposus cells (NPCs). METHODS: NPCs were isolated from SD rats and cultured in vitro. Cultured cells (P3) were randomly divided into normal control group, H2O2 group, CGA + H2O2 group, CGA group and LY294002 pretreatment group. The apoptosis and ROS production of rNPCs was detected by flow cytometry. The expressions of p-Akt, BCL-2 and Akt were analyzed by Western blot. RESULTS: Compared with normal control group, in the H2O2 group, the production of ROS and the apoptosis rate significantly increased in rNPCs; CGA treatment inhibited ROS production and cell apoptosis, while increased the expression of p-Akt and BCL-2; LY294002, a PI3Kinse inhibitor, not only decreased the expression of p-Akt and BCL-2, but also obviously increased ROS production and cell apoptosis. CONCLUSION: Chlorogenic acid can protect NPCs against apoptosis by oxidative stress through decreasing reactive oxygen species production and increasing anti-apoptotic protein BCL-2 expression in NPCs by activation of PI3K-Akt signaling pathways.

Effect of mesenchymal stromal cells transplantation on the outcomes of patients with knee osteoarthritis: A systematic review and meta-analysis.

Cell therapy has been explored as a new regenerative treatment for osteoarthritis in the field of regenerative medicine. However, the efficacy of stem cell transplantation from different sources for the treatment of knee osteoarthritis (KOA) remains controversial. This study integrates and evaluates the previously published data of stem cell transplantation for KOA to explore the curative effect of different stem cells. We conducted a meta-analysis of randomized controlled trials on stem cell therapy for KOA. Measures of efficacy included Visual Analog Scale (VAS), Lequesne index, Lysholm Knee Scoring Scale (LKSS), and Western Ontario and McMaster University Osteoarthritis Index (WOMAC). Joint injury was evaluated through the Whole-Organ Magnetic Resonance Imaging Score (WORMS) system. We analyzed 16 studies involving 875 KOA patients. The stem cell treatment showed significant VAS reduction from the third month onwards. Subgroup analysis suggested the most significant pain relief at different postoperative months came from adipose-derived and umbilical cord-derived stem cells. Autologous adipose tissue resulted in better pain alleviation compared with allogenic. However, autologous bone marrow stem cells did not show increased pain relief over allogeneic ones. Combination therapy (HA and/or PRP) showed no effect. Autologous adipose-derived stem cells demonstrate the most effective recovery of knee joint function. In WORMS assessment, there was no significant difference between the stem cell group and control. Stem cell transplantation proved safe and effective for KOA treatment. Different sources stem cells have a good effect on alleviating knee joint pain, restoring knee joint function, and minimizing patient trauma.

Antigenic and immunogenic properties of truncated VP28 protein of white spot syndrome virus in Procambarus clarkii.

Previous studies identify VP28 envelope protein of white spot syndrome virus (WSSV) as its main antigenic protein. Although implicated in viral infectivity, its functional role remains unclear. In the current study, we described the production of polyclonal antibodies to recombinant truncated VP28 proteins including deleted N-terminal (rVP28ΔN), C-terminal (rVP28ΔC) and middle (rVP28ΔM). In antigenicity assays, antibodies developed from VP28 truncations lacking the N-terminal or middle regions showed significantly lowered neutralization of WSSV in crayfish, Procambarus clarkii. Further immunogenicity analysis showed reduced relative percent survival (RPS) in crayfish vaccinating with these truncations before challenge with WSSV. These results indicated that N-terminal (residues 1-27) and middle region (residues 35-95) were essential to maintain the neutralizing linear epitopes of VP28 and responsible in eliciting immune response. Thus, it is most likely that these regions are exposed on VP28, and will be useful for rational design of effective vaccines targeting VP28 of WSSV.

Protective efficacy of Toxoplasma gondii GRA12 or GRA7 recombinant proteins encapsulated in PLGA nanoparticles against acute Toxoplasma gondii infection in mice.

Background: Toxoplasma gondii is an apicomplexan parasite that affects the health of humans and livestock, and an effective vaccine is urgently required. Nanoparticles can modulate and improve cellular and humoral immune responses. Methods: In the current study, poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles were used as a delivery system for the T. gondii dense granule antigens GRA12 and GRA7. BALB/c mice were injected with the vaccines and protective efficacy was evaluated. Results: Mice immunized with PLGA+GRA12 exhibited significantly higher IgG, and a noticeable predominance of IgG2a over IgG1 was also observed. There was a 1.5-fold higher level of lymphocyte proliferation in PLGA+GRA12-injected mice compared to Alum+GRA12-immunized mice. Higher levels of IFN-g and IL-10 and a lower level of IL-4 were detected, indicating that Th1 and Th2 immune responses were induced but the predominant response was Th1. There were no significant differences between Alum+GRA7-immunized and PLGA+GRA7-immunized groups. Immunization with these four vaccines resulted in significantly reduced parasite loads, but they were lowest in PLGA+GRA12-immunized mice. The survival times of mice immunized with PLGA+GRA12 were also significantly longer than those of mice in the other vaccinated groups. Conclusion: The current study indicated that T. gondii GRA12 recombinant protein encapsulated in PLGA nanoparticles is a promising vaccine against acute toxoplasmosis, but PLGA is almost useless for enhancing the immune response induced by T. gondii GRA7 recombinant protein.

High prevalence of sexually transmitted infections and risk factors among HIV-positive individuals in Yunnan, China.

BACKGROUND: Yunnan has the highest rates of HIV in China. Other treatable sexually transmitted infections (STIs) are associated with accelerated HIV transmission and poor ART outcomes, but are only diagnosed by syndromic algorithms. METHODS: We recruited 406 HIV-positive participants for a cross-sectional study (204 ART-naive and 202 receiving ART). Blood samples and first-voided urine samples were collected. Real-time polymerase chain reaction methods were used for diagnosing Chlamydia trachomatis (CT), Neisseria gonorrhea (NG) and Mycoplasma genitalium (MG). Syphilis and herpes simplex virus type 2 (HSV-2) tests were also performed. RESULTS: Among the 406 participants, the overall prevalence of STIs was 47.0% and 45.1% in ART-naive individuals and 49.0% in individuals receiving ART, respectively. The testing frequencies were 11.6% (11.8% vs. 11.4%), 33.2% (29.4% vs. 37.1%), 3.2% (3.4% vs. 3.0%), 2.0% (3.4% vs. 0.5%) and 4.7% (6.4% vs. 3.0%) for active syphilis, HSV-2, CT, NG and MG, respectively. The percentage of multiple infections in both groups was 10.8% (22/204) in ART-naive participants and 9.9% (20/202) in participants receiving ART. Female sex, an age between 18 and 35 years, ever injecting drugs, homosexual or bisexual status, HIV/HBV coinfection, and not receiving ART were identified as risk factors. Self-reported asymptomatic patients were not eliminated from having a laboratory-diagnosed STI. CONCLUSIONS: The STI prevalence was 47.0% (45.1% vs. 49.0%), and HSV-2, syphilis and MG were the most common STIs in HIV-infected individuals. We found a high prevalence (6.4%) of MG in ART-naive individuals. HIV-positive individuals tend to neglect or hide their genital tract discomfort; thus, we suggest strengthening STI joint screening and treatment services among HIV-infected individuals regardless of whether they describe genital tract discomfort.

[Expression of B7-H1 on peripheral myeloid dendritic cells in patients with HIV infection and its correlation with diseases progression].

OBJECTIVE: To investigate the features of B7-H1 expression on peripheral myeloid dendritic cells (mDCs) in patients with HIV infection and evaluate the correlations between B7-H1 expression and disease progression. METHODS: Peripheral blood samples from 82 treatment-naïve patients with HIV infection, 28 viral complete responders (CRs) under antiretroviral therapy (ART) and 14 healthy controls (HCs) were collected. Flow cytometry was applied to investigate the expression of B7-H1 on mDCs and CD4 cell counts. Plasma HIV-1 viral load was detected by bDNA. RESULTS: The frequency of B7-H1 expression on mDCs were 14.15% +/- 2.63%, 3.31% +/- 0.51% and 0.52% +/- 0.10% in AIDS patients, asymptomatic HIV infected individuals and HCs respectively. As compared with HCs, B7-H1 was significantly up-regulated on mDCs in HIV/AIDS patients. The order was as follows: AIDS patients > asymptomatic HIV infected individuals > HCs (all P < 0.05). Interestingly, the expression of B7-H1 on mDCs in long-term nonprogressors (LTNPs) was 3.12% +/- 1.14%. And it was lower than that in typical progressors (TPs) [8.12% +/- 1.37% (P = 0.001)]. Moreover, the expression of B7-H1 was negatively correlated with CD4 cell counts and positively correlated with plasma viral load in these patients (r = -0.631, P < 0.01 and r = 0.482, P < 0.01 respectively). The expression of B7-H1 on mDCs was significantly lower in ART complete responders than that in AIDS patients (6.59% +/- 1.43% vs 14.15% +/- 2.63%) (P < 0.01). Expression of B7-H1 on mDCs decreased markedly in patients whose CD4 cell counts greatly elevated after a successful antiretroviral treatment. CONCLUSION: The expression of B7-H1 on mDCs is significantly up-regulated in HIV/AIDS patients. With a close correlation with disease status, it acts as a marker of disease progression.

Regulatory Patterns of Crp on Monensin Biosynthesis in Streptomyces cinnamonensis.

Monensin, produced by Streptomyces cinnamonensis, is a polyether ionophore antibiotic widely used as a coccidiostat and a growth-promoting agent in agricultural industry. In this study, cyclic AMP receptor protein (Crp), the global transcription factor for regulation of monensin biosynthesis, was deciphered. The overexpression and antisense RNA silencing of crp revealed that Crp plays a positive role in monensin biosynthesis. RNA sequencing analysis indicated that Crp exhibited extensive regulatory effects on genes involved in both primary metabolic pathways and the monensin biosynthetic gene cluster (mon). The primary metabolic genes, including acs, pckA, accB, acdH, atoB, mutB, epi and ccr, which are pivotal in the biosynthesis of monensin precursors malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA, are transcriptionally upregulated by Crp. Furthermore, Crp upregulates the expression of most mon genes, including all PKS genes (monAI to monAVIII), tailoring genes (monBI-monBII-monCI, monD and monAX) and a pathway-specific regulatory gene (monRI). Enhanced precursor supply and the upregulated expression of mon cluser by Crp would allow the higher production of monensin in S. cinnamonensis. This study gives a more comprehensive understanding of the global regulator Crp and extends the knowledge of Crp regulatory mechanism in Streptomyces.

[The distributions of mosquito vectors carrying Japanese encephalitis virus in Zhejiang province].

OBJECTIVE: To investigate the natural infection and genotype of Japanese encephalitis virus in mosquitoes and swine in some areas of Zhejiang province. METHODS: Samples of mosquitoes and sera of swine were collected in three counties (Xianju, Longquan and Cixi) of Zhejiang province from May to October in 2007. 10 662 mosquitoes were collected during 2007, of which, C.pipiens pallens and C.quinquefasciatus were the most dominant species and 204 pig serum samples were detected. Japanese encephalitis virus in mosquitoes were detected by virus isolation and real time RT-PCR. The antibody against Japanese encephalitis virus in swine was detected by ELISA. The isolated strain were identified by real time RT-PCR. The PrM gene of the isolated strain was amplified by RT-PCR. Three strains were typed by the gene of PrM. RESULTS: Seven positive mosquitoe samples were identified by real time RT-PCR. Three strains were isolated and identified by real time RT-PCR. The PrM gene was cloned and sequenced. The phylogenetic analysis showed that three isolates belong to genotype I of Japanese encephalitis virus. Of 204 swine serum samples, 121 positive samples were identified positives. Above 50% sera samples from swine were positive in June. CONCLUSION: The vector of Japanese encephalitis virus existed and carried the Japanese encephalitis virus in these areas of Zhejiang province. Three strains of Japanese encephalitis virus were isolated from mosquito pools collected in Zhejiang province. It should be the first isolation of genotype I Japanese encephalitis virus in Zhejiang province in recent years.

Genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus 3 (MRV3) from a diarrheic piglet and seroepidemiological survey of MRV3 in diarrheic pigs from east China.

Mammalian orthoreoviruses (MRVs), which cause gastrointestinal and respiratory illness, have been isolated from a wide variety of mammalian species including bats, minks, pigs and humans. Here we report the isolation and genetic and pathogenic characterization of a novel MRV type 3 (MRV3), named MRV-ZJ2013, from the diarrheic feces of piglets in Zhejiang province, China. Genomic and phylogenetic analysis shows that MRV-ZJ2013 may have originated from reassortments among mink, bat, and pig MRVs, suggesting the hypothesis that interspecies transmission has occurred in pig herds. Neonatal piglets infected with MRV-ZJ2013 displayed mild clinical signs such as poor appetite and soft feces, but vomiting and diarrhea were not observed. Fecal virus shedding was detected only in three out of six piglets, each for one- or two-day post-infection. In contrast, piglets inoculated with a virulent porcine epidemic diarrhea virus (PEDV) strain as the control group had severe signs characterized by acute vomiting and watery diarrhea. These findings suggest that the virulence of MRV-ZJ2013, if any, was likely not significant compared to that of PEDV. A seroepidemiological survey of MRV by means of an indirect enzyme-linked immune-sorbent assay (ELISA) based on a recombinant MRV3 capsid protein sigma1 as antigen revealed a high seroprevalence (77%) in 1037 samples from diarrheic pigs of different ages from 24 herds in seven provinces of east China between 2015 and 2016, indicating that MRV3 is endemic in pig herds in China, and may contribute collectively to enteric disease along with other porcine pathogens.

Sero-Molecular Epidemiology of Japanese Encephalitis in Zhejiang, an Eastern Province of China.

BACKGROUND: Sporadic Japanese encephalitis (JE) cases still have been reported in Zhejiang Province in recent years, and concerns about vaccine cross-protection and population-level immunity have been raised off and on within the public health sphere. Genotype I (GI) has replaced GIII as the dominant genotype in Asian countries during the past few decades, which caused considerable concerns about the potential change of epidemiology characteristics and the vaccine effectiveness. The aim of this study was to investigate the prevalence of JE neutralizing antibody and its waning antibody trend after live attenuated JE vaccine immunization. Additionally, this study analyzed the molecular characteristics of the E gene of Zhejiang Japanese encephalitis virus (JEV) strains, and established genetic relationships with other JEV strains. METHODOLOGY/PRINCIPAL FINDINGS: A total of 570 serum specimens were sampled from community population aged from 0 to 92 years old in Xianju county of Zhejiang Province in 2013-2014. Microseroneutralization test results were analyzed to estimate the population immunity and to observe antibody dynamics in vaccinated children. E genes of 28 JEV strains isolated in Zhejiang Province were sequenced for phylogenetic tree construction and molecular characteristics analysis with other selected strains. Positive JE neutralizing antibody rates were higher in residents ≥35 years old (81%~98%) and lower in residents <35 years old (0~57%). 7 or 8 years after the 2nd live attenuated vaccine dose, the antibodies against for 4 different strains with microseroneutralization test were decreased by 55%~73% on seropositive rates and by 25%~38% on GMTs respectively. JEV strains isolated in recent years were all grouped into GI, while those isolated in the 1980s belonged to GIII. On important amino acid sites related to antigenicity, there was no divergence between the Zhejiang JE virus strains and the vaccine strain (SA14-14-2). CONCLUSION/SIGNIFICANCES: JE neutralizing antibody positive rates increase in age ≥10 years old population, likely reflecting natural infection or natural boosting of immunity through exposure to wild virus. JE seropositivity rates were quite low in <35 years old age groups in Zhejiang Province. Waning of neutralizing antibody after live attenuated vaccine immunization was observed, but the clinical significance should be further investigated. Both the peripheral antibody response and genetic characterization indicate that current live attenuated JE vaccine conferred equal neutralizing potency against GI or GIII of wild strains. GI has replaced GIII as the dominant genotype in Zhejiang in the past few decades. Although the chance of exposure to wild JE virus has reduced, the virus still circulates in nature; therefore, it is necessary to implement immunization program for children continually and to conduct surveillance activity periodically.

Enterovirus 71 infection causes severe pulmonary lesions in gerbils, meriones unguiculatus, which can be prevented by passive immunization with specific antisera.

Neurogenic pulmonary edema caused by severe brainstem encephalitis is the leading cause of death in young children infected by Enterovirus 71 (EV71). However, no pulmonary lesions have been found in EV71-infected transgenic or non-transgenic mouse models. Development of a suitable animal model is important for studying EV71 pathogenesis and assessing effect of therapeutic approaches. We had found neurological disorders in EV71-induced young gerbils previously. Here, we report severe pulmonary lesions characterized with pulmonary congestion and hemorrhage in a gerbil model for EV71 infection. In the EV71-infected gerbils, six 21-day-old or younger gerbils presented with a sudden onset of symptoms and rapid illness progression after inoculation with 1×105.5 TCID50 of EV71 via intraperitoneal (IP) or intramuscular (IM) route. Respiratory symptoms were observed along with interstitial pneumonia, pulmonary congestion and extensive lung hemorrhage could be detected in the lung tissues by histopathological examination. EV71 viral titer was found to be peak at late stages of infection. EV71-induced pulmonary lesions, together with severe neurological disorders were also observed in gerbils, accurately mimicking the disease process in EV71-infected patients. Passive transfer with immune sera from EV71 infected adult gerbils with a neutralizing antibody (GMT=89) prevented severe pulmonary lesion formation after lethal EV71 challenge. These results establish this gerbil model as a useful platform for studying the pathogenesis of EV71-induced pulmonary lesions, immunotherapy and antiviral drugs.

Enterovirus 71-induced neurological disorders in young gerbils, Meriones unguiculatus: development and application of a neurological disease model.

A reliable disease model mimicking Enterovirus 71 (EV71) infection in humans is essential for understanding pathogenesis and for developing a safe and effective vaccine. Commonly used rodent models including mouse or rat models are not suitable for vaccine evaluation because the rodents are resistant to EV71 infection after they reach the age of 6 days. In this study, 21-day-old gerbils inoculated intraperitoneally (IP) with a non mouse-adapted EV71 strain developed neurological lesion-related signs including hind limb paralysis, slowness, ataxia and lethargy similar to those of central nervous system (CNS) infection of EV71 in humans. The infected gerbils eventually died of the neurological lesions and EV71 could be isolated from lung, liver, spleen, kidney, heart, spinal cord, brain cortex, brainstem and skeletal muscle. Significantly high virus replication was detected in spinal cord, brainstem and skeletal muscle by cellular analysis, real-time quantitative PCR (RT-PCR) and immunohistochemical staining. Histopathologic changes such as neuronal degeneration, neuronal loss and neuronophagia were observed in spinal cord, brain cortex, brainstem, and skeletal muscle along with necrotizing myositis and splenic atrophy. Gerbils that received two doses of inactive whole-virus vaccine showed no EV71-specific symptoms after challenged with EV71. In contrast, gerbils that received mock vaccination died of EV71-induced neuropathology after challenged with EV71. The result indicates that gerbils can serve as a reliable disease model for evaluating safety and efficacy of EV71 vaccine.

[Genotype of hantavirus in Zhejiang province].

OBJECTIVE: To identify the genotype and clades of hantavirus (HV) in Zhejiang province. METHODS: The partial S and M segment of the HV in Zhejiang province were amplified with RT-PCR using genotype-specific primers, and then were sequenced and compared with other known hantaviruses. RESULTS: The genotype of 11 strains were HTNV and other 7 strains were SEOV by homology and phylogenesis analysis, yet the clade distribution was significantly different among foci of Zhejiang with 5 clades of HTNV and 3 clades of SEOV. There also existed special clade of HTNV named ZNB-1, ZNB-2, A3 and of SEOV named Gou3, ZJ5. The homology of M segments of ZNB-1 and ZNB-2 with other HTNV clades were 69.7%-74.0% except Nc167, A3 with other HTNV clades were 73.6%-76.3% except B78. CONCLUSION: Zhejiang province is co-circulating with HTN and SEO. Say the least of the clades are 5 of HTNV and 3 of SEOV and there also existed special clade of HTNV and SEOV.

Characterization and diagnostic use of a monoclonal antibody for VP28 envelope protein of white spot syndrome virus.

The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21. After induction, the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production. It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection. Competitive PCR showed that the viral level was approximately 10(4) copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay. Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.

[The expression and bioactivity of S gene in Vero-E6 cells using baculovirus vector].

OBJECTIVE: The S gene of a Hanta Virus (HV) Z10 strain was cloned into a baculovirus shuttle bacmid pDual-CMV contained a CMV promoter to generated a recombinant baculovirus BAC-pDual-CMV-HVS, then the recombinant baculovirus was transfected into Vero-E6 cell. The cells with recombinant baculovirus were applied to the detection of HV antiserum. METHODS: To generate the recombinant baculovirus BAC-pDual-CMV-HVS, the sequence of CMV promoter was obtained from the plasmid pEGFP-N1 by PCR, and subsequently cloned to the baculovirus shuttle bacmid pFastBacDUAL resulting the recombinant plasmid pDual-CMV. Then the sequence of HV-S gene was inserted to the plasmid pDual-CMV, to generate the plasmid pDual-CMV-HVS. Plasmid pDual-CMV-HVS was transformed into the DH10BAC competent cells to get the recombinant baculovirus BAC-pDual-CMV-HVS. The antigen substrate slides were made by transfecting the recombinant virus into Vero-E6 cells. RESULTS: The plasmid pDual-CMV-HVS was verified by sequencing. The recombinant virus BAC-pDual-CMV-HVS was generated according to the protocol of the baculovirus and transfected into Vero-E6 cells. The expression of the HV-S gene was verified by positive HV antiserum. CONCLUSION: [corrected] The recombinant virus were successfully generated and applied to prepare the antigen substrate slides. The antigen substrate slides was conveniently prepared without special equipments, and can be used to detect the antiserum of HV virus.

[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.

[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.

[Complete genome sequence analysis of Japanese encephalitis virus newly isolated in China].

OBJECTIVE: To study the complete genome sequence of Japanese encephalitis virus (JEV) strain XJ69 isolated in ZheJiang province and explore its evolution. METHODS: Overlapping primers were designed according to the full-length genomes from GenBank. RT-PCR was used to amplify the fragments and RT-PCR products were cloned T vector, sequenced and analyzed. RESULTS: The genome of strain XJ69 and XJP613 were 10 964 nucleotides in length with a single open reading frame encoding 3432 amino acids. Comparison of the complete genome sequences of different JEV isolates showed XJ69 and XJP613 were 83.5%-99.2% and 83.4%-99.4% nucleotide sequence homology among them respectively, which resulted in 94.8%-99.7% amino acid sequence homology. Phylogenetic analysis through PrM/C,E and full-length genome showed that the XJ69 and XJP613 strain belonged to genotype I. CONCLUSION: The nucleotitede sequence and deduced amino acid sequence of XJ69 and XJP613 strain were similar to that of those of genotype I of Japanese encephalitis virus. It belonged to genotype I and were close to the isolates SH17M-07.

[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.

[Development and evaluation of TaqMan-based one-step reverse transcription-polymerase chain reaction assay for the detection of Japanese encephalitis virus].

OBJECTIVE: To establish a TaqMan based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of Japanese encephalitis virus. METHODS: The gene sequences of Japanese encephalitis virus downloaded from the GenBank was aligned, using the biologic software. Specific primers and probes were designed in the conserved region of the C gene for Japanese encephalitis virus. The real-time RT-PCR reactive condition was optimized and the sensitivity, specificity and the stability of the assay were evaluated. Mosquitoes collected from Zhejiang province were detected by this assay. RESULTS: Mg2+, primer and probe were optimized at 5 mmol/L, 0.2 micromol/L and 0.1 micromol/L respectively. The specificity of the assay was high and there were no cross reactions with dengue virus, rabies virus, seoul virus or hantan virus. The detection limits of the assay was 0.1 TCID50. Results from preliminary application showed that TaqMan RT-PCR for Japanese encephalitis virus was sensitive, easier and faster to perform the process of traditional virus isolation and identification. It took only three hours to extract viral RNA and perform the real-time RT-PCR. CONCLUSION: This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.