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The novel coronavirus pneumonia (COVID-19) pandemic is a great threat to human society and now is still spreading. Although several vaccines have been authorized for emergency use, only one recombinant subunit vaccine has been permitted for widespread use. More subunit vaccines for COVID-19 should be developed in the future. The receptor binding domain (RBD), located at the S protein of SARS-CoV-2, contains most of the neutralizing epitopes. However, the immunogenicity of RBD monomers is not strong enough. In this study, we fused the RBD-monomer with a modified Fc fragment of human IgG1 to form an RBD-Fc fusion protein. The recombinant vaccine candidate based on the RBD-Fc protein could induce high levels of IgG and neutralizing antibody in mice, and these could last for at least three months. The secretion of IFN-γ, IL-2 and IL-10 in the RBD-stimulated splenocytes of immunized mice also increased significantly. Our results first showed that the RBD-Fc vaccine could induce both humoral and cellular immune responses and might be an optional strategy to control COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing and has become an important public health threat. This disease is caused by a new coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, and so far, little is known about this virus. In this study, by using plaque purification, we purified two SARS-CoV-2 virus strains from the same specimen, one named F8 containing a 12-bp deletion in the E gene and the other named 8X containing the wild-type E gene. There was no significant difference in the viral titer and infectivity of these two strains. The S protein content of the F8 viral culture was 0.39 μg/ml, much higher than that of 8X. An inactivated vaccine made from the F8 strain could trigger high levels of the IgG titer and neutralizing antibody titer, which could last for at least 6 weeks and were significantly higher than those from the 8X strain at 1 and 3 weeks post vaccination, respectively. In conclusion, we reported that both the E gene mutant and wild-type SARS-CoV-2 strains were isolated from the same clinical sample by plaque purification. A 12-bp deletion in the E gene was important for SARS-CoV-2 replication and immunogenicity.

Breast cancer is the second leading cause of cancer deaths among women. The development of breast cancer is a multi-step process involving multiple cell types, and its prevention remains challenging in the world. Early diagnosis of breast cancer is one of the best approaches to prevent this disease. In some developed countries, the 5-year relative survival rate of breast cancer patients is above 80% due to early prevention. In the recent decade, great progress has been made in the understanding of breast cancer as well as in the development of preventative methods. The pathogenesis and tumor drug-resistant mechanisms are revealed by discovering breast cancer stem cells, and many genes are found related to breast cancer. Currently, people have more drug options for the chemoprevention of breast cancer, while biological prevention has been recently developed to improve patients' quality of life. In this review, we will summarize key studies of pathogenesis, related genes, risk factors and preventative methods on breast cancer over the past years. These findings represent a small step in the long fight against breast cancer.

Penemuan sasaran dadah antikoksidia baharu merupakan antara usaha yang diperlukan untuk mengawal penyakit koksidiosis ayam yang disebabkan oleh spesies Eimeria. Dalam kajian ini, serpihan yang mengekodkan glikogen sintase kinase-3 (GSK-3) Eimeria tenella putatif telah diamplifikasi daripada cDNA E. tenella. Hasil pemadanan homologi menunjukkan jujukan GSK-3 E. tenella yang terjana mempunyai padanan yang tinggi dengan jujukan GSK-3 organisma lain. Domain terpulihara GSK-3 dan residu yang penting untuk aktiviti GSK-3 juga diramalkan hadir dalam jujukan GSK-3 E. tenella. Analisis struktur sekunder serta pemodelan homologi menunjukkan pembahagian struktur protein kepada domain bebenang beta pada hujung N dan domain heliks alfa pada hujung C, yang merupakan ciri enzim GSK-3. Kesemua hasil analisis ini menyokong bahawa jujukan yang dikaji mengekodkan protein GSK-3 dalam E. tenella. Walaupun darjah keterpuliharaan adalah tinggi, namun terdapat perbezaan yang bermakna diperhatikan antara GSK-3 E. tenella dan perumahnya. Residu Ser 9 yang dilaporkan penting untuk perencatan aktiviti GSK-3 didapati tidak terpulihara dalam GSK-3 E. tenella. Memandangkan Ser 9 merupakan tapak pemfosfatan bagi GSK-3β dalam haiwan vertebrata, ketiadaan residu ini dalam jujukan GSK-3 E. tenella mencadangkan bahawa pengawalaturan GSK-3 E. tenella melibatkan tapak pemfosfatan dan mekanisme yang berbeza. Tambahan pula, hasil analisis filogenetik menunjukkan bahawa GSK-3 E. tenella mempunyai pertalian yang rapat dengan protein GSK-3 tumbuh-tumbuhan. Analisis superposisi GSK-3 E. tenella dengan GSK-3β Homo sapiens pula menunjukkan bahawa perencat GSK-3 mampu berinteraksi dengan protein GSK-3 E. tenella. Keputusan kajian ini mencadangkan bahawa GSK-3 E. tenella mempunyai potensi untuk diperkembangkan sebagai sasaran dadah antikoksidia.

The data of valuation of forest ecosystem service function (FESF) in 101 primary case studies of China were collected and obtained based on Specifications for Assessment of Forest Ecosystem Services in China (LY/T 1721-2008). FESF was then analyzed synthetically in terms of value coefficient. The results showed that the average value per unit area (VPUA) of FESF in China was 6.11×10基于《森林生态系统服务功能评估规范》(LY/T 1721—2008),搜集得到全国101个案例点的森林生态系统服务功能价值评估数据,并依据价值系数对森林生态系统服务功能进行综合分析.结果表明:中国森林生态系统服务功能单位面积总价值为6.11万元·hm

In this thesis, the two 3D computer games were designed by Java 3D. In the first game, by using 3DMax, one aircraft carrier, one helicopter, submarines, warships, and destroyer escorts etc were designed. By using Loader3DS of MicrocrowLoader in Loader3DS1_2.jar or ModelLoader of Java 3D, these 3D models are loaded into the Java 3D. The second 3D game displays the 3D fighting and collision of tanks. This function is very important for the 3D game design. By using textures, the different appearances of 3D models were designed. By using Java 3D classes such as Alpha and PositionPathInterpolator and Sounds etc, the motions of 3D models and interactions and the sounds were defined.

The microstructures and tribological properties of materials for friction pairs used to space docking were investigated by optical metallographic microscope and a special home-made tribo-tester, respectively. The results demonstrate that friction material appears homogeneous and compact microstructure. Counterpart material presents temper sorbite which keeps martensite morphology and residues a small amount of blocky undissolved ferrite; the porosity of friction material is about 3.7%. Materials for space docking show favorable rigidity; during running-in, friction torque of friction pairs can be enhanced obviously; under different operation conditions, friction pairs for space docking possesses different friction coefficient.

The microstructure and tribological properties of space copper-based friction material fabricated by Powder Metallurgy technology were studied using optical metallographic microscope and MM-1000-type tribo-tester, respectively. The results are shown as follows: The lubricant MoS2 was resolved during sintering, resulting in the loss of S element. MoS2 and its resolvents reacted with other components into some new compounds which contribute to the tribological properties of friction material. The lubrication mechanism of these new compounds showed essential difference in comparison to that for MoS2; the friction coefficient was higher under atmospheric condition than that under vacuum conditions, but almost the same under low and high vacuum conditions. It decreased under both atmospheric and low vacuum conditions with the increase of load. The environmental temperature had insignificant effect on the friction coefficient; under atmospheric condition as load increased, the mass loss of material decreased linearly, then increased. With regard to low vacuum condition as load increased, the mass loss of material increased, then decreased; the stable coefficient of friction material under vacuum condition was higher than that under atmospheric condition.

In this work, a visible photocatalytic system containing Eosin Y, multiwalled carbon nanotubes and CuO was prepared. The photocatalytic hydrogen evolution from water over this system was studied using triethanolamine (TEA) as a sacrificial reagent. The results indicated that it was an efficient photocatalyst with an average rate of H2 evolution of 373 mmol·h-1·g-1 during first three hours under visible irradiation.

In the presence of Cu2+ ions, the photocatalytic hydrogen evolution from a methanol aqueous solution was achieved when potassium hexaniobate nanotubes were used as the catalyst. It was found that there existed a photo-induced period in the initial reaction stage. Furthermore, the photo-induced period was prolonged by increasing the amount of Cu2+ ions. After that, the rate of hydrogen evolution was dramatically improved. Combined with the reaction phenomena and the result of the photocatalytic hydrogen evolution, it was deduced that the Cu2+ ions captured the photo-generated electrons of potassium hexaniobate nanotubes in the photo-induced period. Consequently, the Cu2+ ions were reduced and deposited on the potassium hexaniobate nanotubes. When the mass ratio of Cu to potassium hexaniobate nanotubes was 3 wt%, the rate of hydrogen evolution over the catalyst reached 21.9 mmol·g-1·h-1, which could almost match with Pt as a co-catalyst. These results showed that Cu was an effective alternative to Pt as a co-catalyst of potassium hexaniobate nanotubes for the photocatalytic hydrogen evolution from the methanol aqueous solution.

The pompon-like microspheres of AgIn5S8 with a cubic phase were prepared in a low temperature aqueous solution using mercaptoacetic acid as capping agent and thioacetamide as sulfur source and characterized with XRD, UV-vis absorption spectra, SEM and BET analysis. It is interesting that the microspheres are quite open and puffy. More importantly,the obtained microspheres showed remarkable photocatalytic activity for the degradation of methyl orange under visible light irradiation, which is probably due to the pompon-like morphology for providing reactive sites and facilitating the charge transfer between the target pollutant and the photocatalyst. This method could be extended to the synthesis of other functional micro/nano materials.

A novel Cu-based P/M aircraft brake material was prepared and the effects of sintering pressure and temperature on microstructure and tribological characteristic were investigated. For the constant sintering temperature, when the sintering pressure increases from 0.5 MPa to 1.5 MPa, the porosity, wear loss and friction coefficient decrease remarkably. When the sintering pressure increases from 1.5 MPa to 2.5 MPa, the porosity further decreases but in a little degree and wear behaviors are improved slightly. However, once the sintering pressure is larger than 2.5 MPa, it has no obvious effect on microstructure and tribological characteristic. For the constant sintering pressure, when the sintering temperature increases from 900 °C to 930 °C, the sintered density remarkably-increases, and wear behaviors are obviously improved. For further increasing sintering temperature to 1000 °C, the density keeps on increasing, but wear behaviors change slightly.

Cu-based P/M friction materials were prepared and the effects of Fe and SiO 2 friction components on the friction and wear behaviors of the materials were studied. The results show that Fe and SiO 2 have different wear mechanisms. Fe can decrease wear loss of friction materials and enhance friction coefficient, while SiO 2 can remarkably enhance friction coefficient and wear properties of friction materials under the condition of high rotating rate, and increase wear loss under the condition of low rotating rate. The wear behaviors and mechanisms of Fe and SiO 2 in Cu-based friction materials have been discussed.

Cu-based powder metallurgy brake materials are used for aircraft widely and successfully. The characteristics of worn surface of Cu-based powder metallurgy brake materials for aircraft after working under service condition were studied, and two main wear mechanisms were discussed. The results show that the main components of worn surface are graphite. SiO2, Fe, Cu and oxide of Fe (Fe3O4 and FeO); the worn surface can be divided into three zones: severe wear zone, mild wear zone, and low wear zone; fatigue wear and grain wear are the main wear mechanisms of Cu-based materials. Some debris kept between brake discs reduce the wear rate to a certain extent by taking part in the regeneration of friction film.

The laccase and ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) have been assembled inside the tubular mesoporous silica via co-adsorption technology to prepare host/guest-type immobilized laccase, which is applied to degrade methoxychlor (MXC) in aqueous and reverse micelle environments. The effects of various parameters on degradation of MXC were studied. Under the optimum conditions, the degradation rate could reach maximum value of 45.6 % and remain at 20.8 % after seven cycles. Moreover, the addition of small molecular compound 2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) to the system could greatly improve the degradation efficiency. The MXC degradation process is a first-order reaction, and the activation energy of MXC degradation catalyzed by immobilized laccase (41.46 kJ mol(-1)) is relatively lower than that catalyzed by free laccase (44.91 kJ mol(-1)). Based on the degradation products measured by gas chromatograph-mass spectrometer (GC-MS) and nuclear magnetic resonance (NMR), the degradation mechanism of MXC has also been proposed.

A novel high performance timing discrimination circuit architecture for a pulsed laser proximity fuze based on constant fraction discrimination technique is constructed and tested. A LC resonant circuit is designed to replace the traditional transmission delay-line to accommodate the special requirements of laser proximity fuze for low size, power consumption, weight and cost. The walk error of the improved constant fraction discriminator is analyzed with lots of detailed experiments. The experiment results indicate that the discriminator eliminates the dependency of the echo pulse amplitude from the timing result, the walk error caused by the amplitude variation and the noise variation of the received echo pulse is less than 125ps, when the SNR is more than 20 and the echo pulse is 0.2V to 2.134V (20.12dB dynamic range). The error is less than 100ps when the SNR more than 60. Furthermore, Detection on the whole system integrating the timing discrimination circuit verifies that, the laser repetition frequency can reach 10 KHz, the accuracy of the system with a measurement time of 0.1ms is 1.45cm in the case of a noncooperative target at a measurement range from 2m to 24m at room temperature, which improve greatly the fixed distance precision of pulsed laser proximity fuze.

In order to develop an animal model and an assay method to evaluate protective immune response to H5N1 avian influenza vaccination, H5N1 avian influenza vaccine was prepared. New Zealand rabbits were assigned to receive two doses of vaccine with different hemagglutinin (HA) dosage. The sera from vaccinated rabbits was evaluated to determine antibody titer and specificity using different tested methods including hemagglutination inhibition assay (HI), neutralizing assay (NT), cross-HI assay, cross-single immunodiffusion assay and cross-neutralization assay. The titer of HI antibody from rabbits immunized with different doses of HA were no less than 1:40 among groups 14 days after the first immunization. Whereas the NT antibody titer was less than 1:10 among groups 14 days after the first immunization. NT antibodies can be detected 14 days after the second immunization in rabbits immunized at HA doses higher than 6 μg, and the NT antibody titers were equal to or higher than 1:40. A good concentration-dependent NT antibody response can be detected in the vaccinated rabbits 14 days after the second immunization, and in contrast, no concentration-dependent relationship can be seen for HA antibody. The cross-HI test showed sera from vaccinated rabbits could cross react with influenza A H5N1 virus with the titers higher than 1:40. No cross reaction among different types (influenza A/H1N1 virus, influenza A/H3N2 virus, influenza B virus and influenza A/H5N1 virus) can be detected in the sera using the single immunodiffusion assay and using NT antibody test. This showed NT antibody test was demonstrated as a more accurate assay method for evaluating vaccination and quality of the vaccine than HI antibody test.

To identify the genotype and clades of hantavirus (HV) in Zhejiang province.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.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.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.

To develop a new method to detect anti-Hantavirus IgG antibodies (HV IgG) based on quantum dots (QDs) and indirect immune technique.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.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.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.

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.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.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.[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.

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.

The objective of this research is to use item response theory (IRT) to validate a 14-item peer relations scale for use in the adolescent treatment population. Subjects are 509 adolescents discharged from substance abuse treatment from 2004–2009. The person reliability is 0.76 and the Cronbach's alpha person raw score reliability is 0.93 both indicating the scale is a strong metric. The item reliability of 0.99 is high showing the model is reliable. The real separation (8.49) meaning items are placed on the Rasch “ruler” with about eight levels of importance identified. The mean-square statistics of the infit and outfit values were between 0.5 and 1.5 for the items indicating a low level of randomness and thus unidimensionality of the scale. Inspection of a Wright Item Map shows the hierarchical structure of the scale with a moderate degree of inter-item spread. The analysis shows the scale is a reliable unidimensional metric.

The purpose of this study is to express partial S gene of Hantavirus Z10.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.The recombination Pichia were cultivate, and expressed the SP300 or S300 gene induced in Pichia by methanol.The nucleocapsid secreted from the Pichia can be detected by ELISA and WesternBlot.

To investigate whether Leptotrombidium scutellare could be naturally infected by both Hantaan virus (HV) and Orientia tsutsugamushi (OT) and transmission status by stinging.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.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.Both HV and OT could be coexisted in wild Leptotrombidium scutellare and transmitted by stinging.

To develop a loop-mediated isothermal amplification (LAMP) method for rapidly diagnosing of Escherichia coli (EHEC) O157:H7 in pathogen detection department or small-scale laboratories.Primers for LAMP test were designed by targeting the antigen coding rfbE of EHEC O157:H7, the Shiga-like toxin stx2 and the fliC encoding gene of H7 flagella antigen, respectively. The reaction condition and reaction system of LAMP were optimized. 2 EHEC O157:H7 type strains, 17 local strains and 33 other enterobacteria were analyzed to evaluate the LAMP's specificity and sensitivity. The results of the LAMP reaction were also compared with routine PCR method.The amplification products of O157 which had the corresponding target genes turned green by visual inspection and had ladder-like pattern on the gel, but products of other enterobacteria remained orange by visual examination and had no band on the gel. The detection results of LAMP were the same as of routine PCR method. The reaction time of the LAMP method was only 1.5 hours and the detection limit of LAMP assay was 26 CFU/reaction. In addition, the LAMP results could be determined only by visual inspection.LAMP assay is rapid, specific, and sensitive for the detection of EHEC O157:H7. This method might not only reduce the dependence of complicated equipments but also be a potential method for wider use in pathogen detection department, small-scale laboratory, emergency motor vehicle or field survey.

To study the complete genome sequence of Japanese encephalitis virus (JEV) strain XJ69 isolated in ZheJiang province and explore its evolution.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.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.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.

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.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.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.ZLS6-11 and ZLS-12 Strains were believed to belong to HTN-type and phylogenetically different from the HTNV.

In order to understand the molecular characters of Hantavirus ZJ5 strain, its complete M and S genome were sequenced and compared with that of other hantavirus strains.We prepared the total RNA from ZJ5. Infected cells and the raw or purified RT-PCR product was cloned and sequenced.With sequence compation, we found ZJ5 strain complete M and S segment had higher homology with SEO-type strains than other type of HV, but differential genes were 11.7%-19.2% and 6.7%-14.5% from SEOV. The phylogenetic trees constructed by complete M ind S segment showed that ZJ5 strain was located in SEOV group, and structured alone embranchment.The ZJ5 strain was believed to belong to SEO-type virus,and suggest that ZJ5 strain is a new subtype S SEOV group,and structured alone embranchment.The ZJ5 strain was believed to belong to SEO-type virus, and suggest that ZJ5 strain is a new subtype from other SEO viruses.

To investigate the natural infection and genotype of Japanese encephalitis virus in mosquitoes and swine in some areas of Zhejiang province.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.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.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.

To learn about the complete genomic sequence of the Seoul virus strain ZT10 isolated from M. fartis.The total RNA was extracted from the infected Vero E6 cells and amplified by RT-PCR. The purified PCR products were cloned into T-vector and sequenced.The results demonstrated that the complete genome of ZT10 was comprised of L(6530), M(3651) and S(1753) segments which encoded 2151-1133 and 429 amino acids respectively.Analysis of sequence revealed that the ZT10 belonged to Seoul virus. The nucleotide sequence identity of the M gene with Seoul virus was 84.0%-96.3%. The identity with Hantan vrisu (Prospect Hill virus, Tula virus) isolated from M. fartis was 57.5%-60.9%. The sequence identity of the S gene with Seoul virus was 87.9%-96.0% at nucleotide level and 96.9%-97.9% at amino acid level.

To clone the gene encoding nucleocapsid protein (NP) of hantavirus strain Z10 (HV-Z10), to construct its prokaryotic expression system as well as to establish a rNP-IgM direct capture ELISA based on HRP-labeled recombinant NP (rNP), in order to detect serum samples of patients suffering from hemorrhagic fever with renal syndrome (HFRS) and to evaluate the effects of detection.Gene encoding NP of strain HV-Z10 was amplified by PCR and then its prokaryotic expression system pET28a-Z10N-E. coli BL21DE3 was constructed, using routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP and ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Western blot assay was used to determine the specific immuno-reactivity of rNP while HRP-labeled rNP-IgM direct capture ELISA was established to detect the serum samples from 95 cases of confirmed HFRS patients. The detection effect was compared with that by routine HV-IgM indirect capture ELISA method.pET28a-Z10N-E. coli BL21DE3 was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV-IgG could efficiently recognize rNP and hybridize with the recombinant protein. 94.73% (90/95) of HFRS patients' serum samples were positively confirmed by rNP-IgM direct capture ELISA, while a positive rate of 92.63% (88/95) in the same samples was confirmed by HV-IgM indirect capture ELISA. The distributions of A450 values of the serum samples detected by the two IgM capture ELISAs as well as the changes of the A450 mean values from several serum samples with different dilutions were similar.We successfully constructed a high efficient prokaryotic expression system of NP encoding gene of hantavirus strain HV-Z10. The rNP-IgM direct capture ELISA that established in this study could be used as a new serological test for HFRS diagnosis because of its simplicity, safety, with high sensitivity and specificity.

For clarifying the situation of the natural infection of rodents having hemorrhagic fever with renal syndrome (HFRS) virus and to type Hantavirus (HV) using molecular technique in Shenzhen city in 2005, and offering guidance for prevention and control of HFRS.Data on the host animals was collected from the city of Shenzhen. ELISA and indirect immunofluorscent antibody(IFA) test were applied to the specific antibodies against HV in the sera of captured rats. Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues of the HV infected rats were inoculated into Meriones unguiculata to isolate HV. The whole viral RNA was extracted from the lung tissues of the HV infected rats and amplified the partial M fragments with RT-nested-PCR, using the HV genotype specific primers. The amplified genes were then sequenced, and subjected to genotyping and homology analysis.472 rodents were captured from Shenzhen in 2005. Surveillance on rats demonstrated 9.96% rats carrying HV (with a density of 8.25%) and the main host was Rattus norvegicus. In the blood samples of rats, anti-HV IgG antibodies were detectable in 56 cases by IFA, and proved to be positive in 76 cases by ELISA. We successfully isolated a HV strain designated as SZ2083 from Rattus norvegicus for the first time in Shenzhen and was identified to SEO type by RT-nested-PCR. Compared with the coding region of the M gene of HV L99 virus strain, the homologies of nucleotide among them were 97%, but the homology was 76% of the SZ2083 with HTN 76-118 virus strain.Results showed the existence of natural epidemic areas of HFRS in Shenzhen city. Based on the results of sequencing, it is possible that the Seoul strain of HV might be the predominant serotype of virus harbored.

To establish a new technique for SARS-CoV antibody test to detect infection of severer acute respiratory syndrome (SARS).Nucleocapsid gene was obtained by reverse transcription and polymerase chain reaction from a SARS patient and inserted into the vector pFastBacHTa expressing baculovirus. Insect Sf9 cells were transfected with the recombinant baculovirus expressing SARS nucleocapsid antigen and then cultured, fixed by acetone so as to make SARS-specific antigen. Immunofluorescence assay (IFA) technique and plaque reduction neutralization test (PRNT) were used to detect 7 samples of sera of 4 newly diagnosed SARS patients collected in different days, 48 samples of convalescent sera of SARS patients, 24 serum samples of healthy person undergoing physical examination, and 40 serum samples from non-SARS patients with fever by double blind test.The recombinant SARS-specific antigen reacted only with SARS positive sera but not with normal sera. Double blind test showed that 45 of the 46 PRNT positive sera were IFA positive with an accordance rate of 97.8%. 7 samples of sera from 4 SARS patients in acute progressive stage in Guangdong province were all IFA positive. SARS antibody could be detected since the sixth day after onset, and the titer increased from 1:40 to 1:600 on the ninth day.Immunofluorescence assay is highly specific and sensitive in detection of SARS. This reagent is safe and easy to prepare.

Background The erythrocyte membrane lesion is a serious diabetic complication. A number of studies suggested that n-3 fatty acid could reduce lipid peroxidation and elevate α- or γ-tocopherol contents in membrane of erythrocytes. However, evidence regarding the protective effects of flaxseed oil, a natural product rich in n-3 fatty acid, on lipid peroxidation, antioxidative capacity and membrane deformation of erythrocytes exposed to high glucose is limited. Methods Human peripheral blood erythrocytes were isolated and treated with 50 mM glucose to mimic hyperglycemia in the absence or presence of three different doses of flaxseed oil (50, 100 or 200 μM) in the culture medium for 24 h. The malondialdehyde (MDA) and L-glutathione (GSH) were measured by HPLC and LC/MS respectively. The phospholipids symmetry and membrane fatty acid composition of human erythrocytes were detected by flow cytometry and gas chromatograph (GC). The morphology of human erythrocyte was illuminated by ultra scanning electron microscopy. Results Flaxseed oil attenuated hyperglycemia-induced increase of MDA and decrease of GSH in human erythrocytes. Human erythrocytes treated with flaxseed oil contained higher C22:5 and C22:6 than those in the 50 mM glucose control group, indicating that flaxseed oil could reduce lipid asymmetric distribution and membrane perturbation. The ultra scanning electron microscopy and flow cytometer have also indicated that flaxseed oil could protect the membrane of human erythrocytes from deformation at high glucose level. Conclusion The flaxseed oil supplementation may prevent lipid peroxidation and membrane dysfunction of human erythrocytes in hyperglycemia.