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The current outbreak of Zika virus (ZIKV) poses a severe threat to human health. Two ZIKV strains were isolated from mosquitoes collected from the Dejiang prefecture in China in 2016, which was the first isolation of ZIKV in nature in China.In this study, serum samples were collected from 366 healthy individuals and 104 animals from Dejiang prefecture in 2017, and the plaque reduction neutralization test (PRNT) was used to evaluate the seroprevalence of ZIKV.None of the 366 residents from whom the samples were collected were seropositive for ZIKV. None of the 11 pigs from whom the samples were collected were seropositive for ZIKV, while 1 of 63 (1.59%) chickens and 2 of 30 (6.67%) sheep were seropositive for ZIKV.The extremely low seropositivity rate of ZIKV antibodies in animals in the Dejiang prefecture, Guizhou province in this study indicates that ZIKV can infect animals; however, there is a low risk of ZIKV circulating in the local population.

To detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed.By aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all2.8%. The amplification efficiency of this method was between 90% and 103%.A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.

Transcription of the subgenomic mRNA of Sindbis virus (SINV) is initiated at a subgenomic promoter (SP). Alignment of SINV sequences identified a 68-nucleotide conserved domain spanning -19 to +49 relative to the subgenomic mRNA start site. Nucleotide T or C is present at -18 or +49 in all known SINVs while a Sindbis-like virus XJ-160 has an A or T at a corresponding position. Our results indicate that deletion or substitution of the T at +49 decreased the activity of SP, while substituting T for A at -18 did not decrease the activity of SP or genetic stability of recombinant SINV.

In this study, we isolated a virus strain (YN12031) from specimens of Armigeres subalbatus collected in the China-Laos border. BHK-21 cells infected with YN12031 exhibited an evident cytopathic effect (CPE) 32 h post-infection. The virus particles were spherical, 70 nm in diameter, and enveloped; they also featured surface fibers. Molecular genetic analysis revealed that YN12031 was closely related to alpha viruses such as Chikungunya virus and Sindbis virus, and located in the same clade as MM2021, the prototype of Getahvirus (GETV) isolated in Malaysia in 1955. Phylogenetic analysis of the E2 and capsid genes further revealed that YN12031 was located in the same clade as the Russian isolate LEIV/16275/Mag. Analysis of the homology of nucleotides and amino acids in the coding area and E2 gene demonstrated that the YN12031 isolated from the China-Laos border (tropical region) was related closest to the LEIV/16275/Mag isolate obtained in Russia (North frigid zone area) among other isolates studied. These results suggest that GETV can adapt to different geographical environments to propagate and evolve. Thus, strengthening the detection and monitoring of GETV and its related diseases is very crucial.

Prior to 1976 only Japanese encephalitis virus (JEV) genotype III could be detected in China. Recently, numerous genotype I JEV strains have been isolated from JE patients, mosquitoes and pigs while genotype III strains remain present. Two kinds of JEV vaccines are currently used in China for the prevention disease: the JE live attenuated vaccine (LAV) SA14-14-2 virus and the inactivated P3 strain (IPV) vaccine. The SA14-14-2 and P3 viral strains were isolated in the year of 1953 and 1949 respectively and both belonged to the JEV genotype III. In order to evaluate the protective efficacy of both vaccines against the JEV genotype I isolates we conducted vaccination-challenge protection assays in mice. These data demonstrated that both LAV (≥ 234 pfu virus) and IPV (1:5 dilution) vaccines effectively conferred protection against all 16 isolates tested following intraperitoneal (i.p.) challenge. However, when vaccinated mice were challenged via intracerebral (i.c.) injection, ≥ 60% LAV vaccinated animals were protected against challenge with most JEV isolates but only ≤ 40% protection was observed following vaccination with IPV. These results indicated that JE vaccines used in China still protected effectively against both JEV genotypes now prevalent in China and that the LAV formulation conferred higher levels of protection compared to the protection conferred by IPV.

In 2006, Tahyna virus was isolated from Culex spp. mosquitoes collected in Xinjiang, People’s Republic of China. In 2007, to determine whether this virus was infecting humans, we tested serum from febrile patients. We found immunoglobulin (Ig) M and IgG against the virus, which suggests human infection in this region.

Sixty-two new Japanese encephalitis virus (JEV) isolates were obtained from mosquitoes, biting midges, human cerebrospinal fluid and human blood samples in China during 2002–2005. The E and prM genes were sequenced and phylogenetic analyses were performed with 38 JEV other isolates from China and 36 JEV strains from other countries. Phylogenetic trees based on the E and prM gene sequences were similar. The results indicate that: (i) recent JEV isolates from China are divided into two genotypes, genotype 1 and genotype 3; (ii) recent JEV isolates from China are grouped into the same clusters within genotypes 1 and 3; and (iii) genotype 1 JEV strains have been isolated in China since 1979, whilst genotype 3 JEV strains were isolated before the 1970s. The results suggest that genotype 1 JEV was introduced to China around 1979 and that JEV strains belonging to genotypes 1 and 3 circulate in China.

This study was conducted to investigate the influence of superoxide dismutase (SOD) on the quality of boar semen during liquid preservation at 17°C. Semen samples from 10 Duroc boars were collected and pooled, divided into five equal parts and diluted with Modena containing different concentrations (0, 100, 200, 300 and 400 U/mL) of SOD. During the process of liquid preservation at 17°C, sperm motility, acrosome integrity, membrane integrity, total antioxidant capacity (T-AOC) activity, malondialdehyde (MDA) content and hydrogen peroxide (H

Background There have been four strains on Manzanilla virus (MANV) identified to date. Here, we identify a novel MANV strain (DHL10M107) isolated from Culex tritaeniorhynchus Giles mosquitoes from Ruili city, Dehong prefecture, Yunnan Province, in the People’s Republic of China. Results The DHL10M107 L, M and S genes were sequenced at the nucleotide and deduced amino acid levels. The L, M and S gene sequences of DHL10M107 clustered with the MANV strains VN04-2108, TRVL3587, SA An 4165, and AV 782. DHL10M107 was most closely related to VN04-2108. Nucleotide homology ranged between 96 and 99% between DHL10M107 and VN04-2108. In terms of amino acid homology, all of the amino acid differences were in the L (96.3% homologous) and M (97.7% homologous) fragments. Conclusions DHL10M107 is likely a MANV isolated from mosquitos in the Yunnan Province. This is the first reported isolation of MANV in mainland China. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-1198-5) contains supplementary material, which is available to authorized users.

Nucleocapsid protein plays a critical role in SARS-CoV pathogenesis, and high-level anti-nucleocapsid antibodies are detected in the patients infected by severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Several studies have shown that there exists an interaction between nucleocapsid (N) and membrane (M) protein. In this paper, we investigate whether the expression of membrane protein can affect the immune responses induced by nucleocapsid DNA immunization. Two recombinant plasmids containing M and N coding sequence were constructed. Moreover, in order to get the antigen for ELISA and in vitro stimulation assay, N protein were expressed and purified from E. coli bacteria. Injection of 20mug of the mixture of pVAX1-M and pVAX1-N into the Balb/c mice could elicit the humoral and cellular responses. The ELISA analysis using the N antigen or inactivated SARS-CoV particles as capture antigen showed that co-injection of SARS-M could enhance N-induced antibody production, especially IgG2a subclass. After lymphocytes were stimulated with 10mug/ml purified N antigen, The CD4+ and CD8+ T cells of N and M plus N group were increased compared with those of control groups, and the M protein could augment the activation of lymphocytes induced by N DNA vaccine. Cytokine ELISA analysis revealed that co-injection of M could enhance the levels of IFN-gamma, IL-2 release induced by N antigen. Further experiments in field mouse also support the claim that membrane protein can augment the N-specific immune responses. Virus challenge test was conducted in BSL3 bio safety laboratory with Brandt's vole SARS-CoV model, and the results indicated that co-immunization of M and N antigens could reduce the mortality and pathological changes in lung from the virus infection.

The expression of the hemagglutinins of Avian influenza virus H5 H7and H9 subtypes was studied in this article by Pichia pastoris, one of the eukaryotis expression systems. Three reconstructed expression plasmids and engineering strains, named pPIC9K-H5HA, pPIC9K-H7HA, pPIC9K-H9HA and GS115/pPIC9K-H5HA, GS115/pPIC9K-H7HA, GS115/pPIC9K-H9HA repectively, were obtained. The reconstructed yeast engineering strains were identified by MD and MM plate selecting and PCR. The induced interests proteins were examined by SDS-PAGE and Western-bloting,the results showed that the interest genes were expressed exactly. And this will be helpful in the future study of antigen detection and antibody detection kit, as well in the subunit vaccines developing.

To study the B cell linear epitopes of rabies virus CVS-11 nucleoprotein, peptides were synthesized according to the amino acid sequences of B cell linear epitopes. Linear epitopes predicted by bioinformatics analysis were evaluated with immunological techniques. Indirect enzyme-linked immunosorbent assay showed that titers of antibodies to peptides (355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein) were above 1:12 800 in mouse sera. The antibodies recognized denatured rabies virus CVS-11 nucleoprotein in Western blot analysis. Purified anti-peptide antibodies recognized natural rabies virus CVS-11 nucleoprotein in BHK-21 cells in indirect fluorescent antibody test. The 355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein were validated as B cell linear epitopes.

This study aims to investigate the distribution patterns of mosquito-borne viruses in Menghai County, Xishuangbanna Prefecture, Yunnan Province, China and to provide evidence for the prevention and control of mosquito-borne diseases. Mosquito samples were collected using mosquito lamps. Viruses were isolated from the samples by cell culture, and the isolates were identified by RT-PCR. The genomes of isolates were sequenced for phylogenetic analysis. In July 2012, a total of 1468 mosquitoes were captured in Daluo Town of Menghai County; they were divided into 32 pools, including Culex tritaeniorhynchus (28 pools, 1383 mosquitoes), Culex quinquefasciatus (2 pools, 66 mosquitoes), and Anopheles (2 pools, 19 mosquitoes). Golden hamster kidney cells (BHK-21) and Aedes albopictus cells (C6/36) were used for virus isolation. The results showed that C6/36 cells were susceptible to two isolates recovered from Culex tritaeniorhynchus (BNDL1205 and BNDL1227), with marked cytopathic effect (CPE) of cell fusion. By contrast, the two isolates could not cause CPE in BHK-21 cells. RT-PCR was performed for the two isolates using the flavivirus-specific primers FU2/cFD3, and a 800-bp amplicon was obtained from both of them. Phylogenetic analysis showed that the two isolates shared the same evolutionary branch with the Quang Binh virus (QBV) strain VN180, which had been isolated from Vietnam, with nucleotide sequence homologies of 83.4% and 82.9%, respectively. However, there existed relatively large differences in nucleotide sequence between them and other Culex flavivirus strains previously isolated in China and other regions. In light of the similarity between the two isolates and QBV, BNDL1205 and BNDL122 were referred to as Quang Binh-like virus, which were first reported in China.

A series of phthalazine ketone compounds were synthesized and the structures were confirmed by H NMR and HR-MS spectrum. All target compounds were obtained through 7 steps, including selective reduction, nitration, bromination, ring enlargement, reduction, Knoevenagel and acylated reaction. The compounds were evaluated for their immunosuppressive effects of T-cell proliferation and inhibitory activity of IMPDH type II in vitro, as well as their structure-activity relationship were assessed. Several compounds exhibited strong immunosuppressive properties, especially compounds 7f and 7h, with IC50 values of 0.093 micromol x L(-1) and 0.14 micromol x L(-1) respectively, which were superior to mycophenolic acid. The information obtained from the studies may be useful for further research on the immunosuppressive agents.

To understand the epidemic situation and factors influencing rabies cases in children in China, we obtained an overview of the current epidemic based on individual data of rabies cases in children and a descriptive analysis was carried on the prevalence and related factors. The results showed that the rabies cases in children accounted for 21.3% of the total number of rabies cases in China, 97.0% of these cases occurred in rural areas, they were mainly caused by dogs (81.5%), and were primarily level III exposure (47.7%). More than half of the cases were not treated with wound care, vaccination rate was extremely low (15.7%), and only 5.9% of cases were injected with antibodies. Furthermore, 25.4% of cases adopted incorrect treatments such as extruding bleed and wound closure, cases vaccinated with 5 injections accounted for only 22.5%. In conclusion, the prevalence of rabies cases in children in China remains a serious concern, the number and immune status of dogs in rural areas, and knowledge of rabies by risk populations should be considered in future rabies prevention and control programs.

To characterize two strains of street rabies virus (RABV) isolated from the brain tissue of cattle from Inner Mongolia. Differences in the histopathological and ultrastructural changes in the brain tissue of infected mice were determined to reveal variation in the pathogenesis of infection between street rabies virus strains.Ten-day-old mice were intracranially inoculated with one of three virus strains and brain tissue harvested when the mice were moribund. Various histopathological and ultrastructural markers of disease were then compared between the groups.Infection with the street virus strain CNM1101C resulted in severe neuronal dendrites damage, but only mild cell apoptosis, T lymphocyte infiltration and microglial activation. Infection with the other street virus strain, CNM1103C, was characterized by cell apoptosis, T lymphocyte infiltration and microglial activation as well as dendrites damage. However, in comparison, infection with the attenuated virus strain CTN caused severe T lymphocyte infiltration, microglial activation and cell apoptosis, but left the neuronal dendrites intact.The two street rabies virus strains isolated from cattle from Inner Mongolia had different levels of virulence and caused distinct pathological changes in infected mice. Therefore, we concluded that different pathogenic mechanisms exist between different RABV strains.

To perform pathological observation and etiological identification of specimens collected from dairy cows, beef cattle and dogs which were suspected of rabies in Inner Mongolia in 2011, and analyze their etiological characteristics.Pathological observation was conducted on the brain specimens of three infected animals with Hematoxylin-Eosin staining, followed by confirmation using immunofluorescence and nested RT-PCR methods. Finally, phylogenetic analysis was conducted using the virus N gene sequence amplified from three specimens.Eosinophilic and cytoplasmic inclusion bodies were seen in neuronal cells of the CNS; and rabies non-characteristic histopathological changes were also detected in the CNS. The three brain specimens were detected positive. N gene nucleotide sequence of these three isolates showed distinct sequence identity, therefore they fell into different groups in the phylogenetic analysis. N gene in the cow and dog had higher homology with that in Hebei isolate, but that in the beef cattle had higher homology with that in Mongolian lupine isolate and Russian red fox isolate.Rabies were observed in the dairy cow, beef cattle and canine in the farm in Inner Mongolia, in 2011, which led to a different etiologic characteristics of the epidemic situation.

To understand molecular characteristics of Japanese encephalitis virus (JEV) isolated from the major Japanese encephalitis epidemic areas in Sichuan Province, and to provide the foundation for JEV prevention.13 JEV strains were isolated from mosquitoes in Sichuan during 2007-2010, E genes and preM genes were sequenced and phylogenetic analyses were performed using MEGA5 molecular software.Phylogenetic analysis indicated that all 13 JEV strains from Sichuan belonged to genotype I, homologies at nucleotide level and deduced amino acid level in PreM gene were 97%-100% and 98.7%-100%, and 97.8%-99.9% and 99.6%-100% in E gene, respectively. Homologies at nucleotide level and deduced amino acid level in PreM gene between 13 JEV strains and JEV isolated in 2004 in Sichuan were 96.2%-99.1% and 97.5%-98.7%, and were 97.7%-99.6% and 98. 6%-100% in E gene, respectively. By comparison with vaacine strains P3 and SA14-14-2, homologies at nucleotide level and deduced amino acid level were 84.1%-85.8% and 93.7%-96.2% in PreM gene, and were 87.6%-88.3% and 97%-97.8% in E gene, respectively. The neurovirulence-related 8 amino acid sites encode by E gene remained unchanged in 13 JEV strains.JEV with genotype I predominated in Sichuan, nucleotide sequences and deduced amino acid sequences in PreM gene and E gene were highly conserved, key neurovirulence-rerlated sites remained unchanged. It suggested currently used vaccine is still capable of preventing JEV infection.

To grasp the infection rate and genotypes of Japanese encephalitis virus (JEV) in mosquito in Fujian province.Mosquito specimens in Sanming city, Jianyang city and Fuzhou city in Fujian province were collected in 2010. RT-PCR was used to detect the JEV sequence from the mosquitoes by specific primers. The sequence splicing and the differentiation analysis for nucleotides, deduced amino acid sequence and phylogenetic tree were performed by the software of ATGC, Clustal X (1.83), MegAlign, GeneDoc 3.2 and Mega (4.0).Totally 6987 mosquitoes were collected and main species was Culex tritaeniorhynchus and Anopheles sinensis. The infection rate of JEV in mosquitoes in Sanming, Jianyang and Fuzhou were 1.25%, 1.76% and 0.65%, respectively. One full genome in the positive specimens was sequenced. And further study showed that the positive JEV sequences belonged to genotype I.Genotype I Japanese encephalitis virus is the main genotype in mosquitos in Fujian province.

To construct the recombinant virus-like particles containing HCV envelope glycoprotein E1E2 based on sindbis virus vector.The gene encoding HCV envelope glycoprotein E1E2 was cloned into sindbis virus vector to construct recombinant plasmids pBR-XJE1E2 and pVA-XJE1E2, and transfect them into BHK-21 cells to obtain recombinant virus-like particles. The expression of E1 and E2 protein were verified by Western Blot and indirect immunofluorescent assay (IFA).The results of restriction enzyme digestion, PCR and sequencing analysis showed that the recombinant plasmids were constructed successfully. And the results of RT-PCR, Western blotting and IFA detection showed that the transfect cells could package HCV-like particles of expressing structural proteins E1E2.The recombinant expression plasmids pBR-XJE1E2 and pVA-XJE1E2 based on sindbis virus vector could package HCV-like particles in eukaryotic cell, which provides a foundation for further study of its in vivo animal immune response.

To investigate the virulence characteristics of two fixed strains (CTN and aG) and a street strain (HN10) of rabies viruses isolated in China.ICR mice of different age groups were inoculated with CTN, aG and HN10 rabies virus strains via the intracracerebral (i.c.) or intramuscular (i.m.) routes, and observed for 20 days.The CTN strain was pathogenic to 7-day-old suckling mice that received i.c. inoculations and 3-day-old suckling mice that received i.m. inoculations. The aG strain was pathogenic to 4-week-old mice that received i.c. inoculations and 7-day-old suckling mice that received i.m. inoculations. The HN10 strain was pathogenic to mice of all age groups via both inoculation routes. In moribund mice, the viruses had spread to most regions of the brain. The CTN and HN10 strains had similar dissemination patterns in the brain; both viral antigens could be found in the dentate gyrus (DG), whereas few viral antigens were present in the DG from specimens that had been infected with the aG strain.A comprehensive sequence analysis of the G protein suggested that differences in gene sequences may be responsible for producing strain-specific differences in pathogenicity and distribution in the brain.

The number of human rabies cases acquired from dog bites constitutes a high proportion of the total rabies cases in China, although the number of human rabies cases has gradually decreased in recent years. The pivotal role of dogs in the spread of rabies indicates that controlling and preventing canine rabies could be a key step in eradicating human rabies in China. The primary aims of this review are to discuss the properties and pathogenesis of the rabies virus, the clinical signs and diagnosis of canine rabies, threshold host density and vaccination of dogs, and the prevention and control of canine rabies in China.

Construction and recovery of chimeric rabies virus expressing HCV envelop proteins E1E2.On the basis of the previously established reverse genetic system CTN-GFP, HCV E1E2 genes were cloned to both replication competent and replication constrained viral vectors based on CTN181 strain and the chimeric viruses CTN-HCV E1E2 and CTNdeltaG-HCV E1E2 were recovered.The result demonstrated that both the chimeric viruses were rescued successfully, had the ability to re-infect normal sensitive cell lines and express HCV E1E2 genes detected in the level of mRNA.The establishment of chimeric RVs expressing HCV E1E2 genes provides the evidence that it is feasible to develop novel HCV vaccines based on viral vectors in theory and in practice.

To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test.Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate (FITC) after purification and used in fluorescent antibody test (FAT).Two positive hybridoma cell lines, RVNP-mAb1-CL and RVNP-mAb2-CL, were obtained. RVNP- mAb1-CL produced a higher concentration of monoclonal antibody RVNP-mAb1 in Balb/c ascites. FITC-labeled RVNP-mAb1 showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line (BSR).FITC-labeled RVNP-mAb1 has potential application for laboratory diagnosis of rabies.

In 2006, the first Chinese Tahyna virus isolate (XJ0625) was obtained in Xinjiang province and human infection were found in the same region. In this study, cell culture, animal experiments, electron microscopy, immunofluorescence assay and cross neutralization tests were performed to see the cell susceptibility, animal pathogenicity, morphology and antigenic and other biological characteristics of XJ0625. In addition, molecular biology software was used to analyze the characteristics of molecular evolution. The results showed that BHK-21 cell line was susceptible to XJ0625 and the virus was lethal to suckling mice when injected by intracranial ways. Similar to the other Bunyavirus, Tahyna virus is spherical enveloped virus under electron microscopy. XJ0625 infected cells showed strong fluorescent signal and could be neutralized by immune asities fluid with immnity to protype Tahyna virus Bardos 92. The sequence of the S and M segments showed 91.8% and 81.9% homology with Bardos 92.

To study the viremia formation in guinea-pigs infected with wild type and attenuated Japanese encephalitis virus (JEV).Guniea pigs were inoculated intraperitoneally with different wild JEV strains and the attenuated vaccine strain and its parent virulent strain. Viremia was detected on different days following virus inoculation.All the guinea-pigs inoculated with the wild JEV strains induced different levels of viremia (1.00-3.40 Lg pfu) on the 1st and 3rd day post inoculation. Using a virus titer of 10(4) pfu for inoculation, the animals inoculated with the SA14 parent strain induced relatively high viremia (10(2.4)-10(3.4) pfu), however no viremia coulds be detected on any tested days.The degree of viremia in guinea pigs can be used as a new method to evaluate the attenuation of JEV.

To sequence and analyze the complete genome of two new Japanese encephalitis virus (JEV) strains isolated from mosquitoes collected in Hubei province in 2008, and to understand the molecular biological characteristics of JEV in this area.RT-PCR was used to amplify the fragments of HBZG08-09 strain and HBZG08-55 strain with 16 pairs overlapping primers after they had been recovered and identified, then the full-length genome was obtained by sequencing and splicing. Biological sequence alignment, nucleotide and amino acid sequence analysis, phylogenetic analysis and analysis of amino acid differences were performed by the software of Clustal X (1.83), MegAlign, Mega (4.0) and Genedoc (3.2).The genome of two new strains were both 10 965 nucleotides in length with a single open reading frame from 96 to 10 392 coding for a 3432 amino acid poly-protein, the homology of nucleotide and amino acid sequence between two isolates were 98.2% and 99.7% respectively. Further study showed that the new strains were both belonging to genotype I. Two new strains were most closely related to isolates obtained from Henan and Zhejiang province in recent years. Compared with the live attenuated vaccine strain SA-14-14-2 in China, HBZG08-09 strain had 82 amino acid divergence; HBZG08-55 had 84 amino acid divergences. But the amino acid difference occurred in sites were not the key ones affecting the toxicity or antigenic of JEV.Two new JEV isolates were both belonging to genotype I, and the key sites of amino acid were not changed.

The purpose of this study is to elucidate the molecular mechanism of one-way serological reaction between XJ-160 virus and SINV by recombinant viruses which exchanged the glycoprotein genes individually or simultaneously. Three recombinant viruses were obtained based on the whole-length infectious cDNA clone of XJ-160 virus. The infectivity and pathogenesis to BHK-21 cells and animals were studied and the gene which controlled this one-way serological reaction phenomenon was searched by MCPENT. The results showed that the E2 glycoprotein was the main factor which influenced the growth rate, plaque morphology and pathogenicity of BHK-21 cells and suckling mice. The results of MCPENT showed that the E2 glycoprotein of SINV played a major role in this one-way serological reaction phenomenon. Our study identified the SINE2 gene was the determined gene for one way serological reaction between XJ-160 virus and SINV, and this research laid the foundation for further analysis of the genomic structure and function of SINV.

To investigate the effects of site-directed mutagenesis at nsP2-726Pro on the characteristics of replicon vector derived from XJ-160 virus, a Sindbis virus (SINV) isolated in China. The mutant vector pBRep-726L, pBRep-726S, pBRep-726V or pBRep-726A was constructed by introducing nsP2-726Pro --Leu, nsP2-726Pro --Ser, nsP2-726Pro --Val or nsP2-726Pro --Ala into XJ-160 viral replicon vector pBRepXJ respectively. To quantitatively and qualitatively determine the site-directed mutagenesis on the replicon, the recombinant plasmids expressing Neomycinr (Neo(r)), enhanced green fluorescent protein (EGFP) or Renilla luciferase (R. luc) were constructed by cloning report genes into pBRepXJ or mutant XJ-160 vector respectively. And in vitro-synthesized RNA from expression vectors were electroporated into BHK-21 cells. Compared with the wild-type replicon, the mutation nsP2-726Pro --Val or nsP2-726Pro --Ala accelerated the processing of CPE on BHK-21 cells and simultaneously enhanced its self-replicating capacity. The mutant vector pBRep-726L with Leu substitution exhibited similar packaging capacity to that of pBRepXJ. In contrast, pBRep-726S exhibited a medium phenotype, including the process of CPE and the activity of R. luc expression in BHK-21 cells. The site-directed mutagenesis at nsP2-726Pro not only regulates directly XJ-160 virus vector-host cell interactions, but also plays an important role in its packaging capacity. All of these results lay a basis for researching the relation between the structure and function of alphavirus genome and developing alphavirus vector system with Chinese intellectual property.

To investigate arboviruses in Wenshan and Hekou county which are the Sino-Vietnam frontier regions of Wenshan, Yunnan province, China.In September 2007, 6091 Culicines, 1334 Anophelines, 848 Aedes vexans and 53 Armigeres obturbans were collected from 5 field sites. Mosquitoes were collected and stored in liquid nitrogen after classification. The mosquito pools were homogenized, and centrifuged, then the supematant was inoculated onto C6/36 and BHK-21 cells, and the viral isolates were identified by serological and molecular biological methods.Sequence alignment and phylogenetic analysis on the viral isolates were carried out using Clustal X 1.85, GENEDOC and MEGA4 software.A total of 4 pairs of virus isolated with C6/36 cells cytopathic effect were observed, and other mosquito species have not cytopathic effect.Strain WS0704-2 was Banna virus which identified by antibody response and PCR. Strain WS0704-1, WS0708-1, WS0708-2 were Culex pipens pallens densovirus (CppDNV) which identified by PCR. The phylogenetic analysis the 12th segment showed significant difference between the new Banna virus and other strains isolated in China.There are many mosquito vectors in frontier regions (China and Vietnam) of Wenshan in Yunnan province of China, and mosquito-borne arbovirus, such as BAV were isolated here.

To sequence PrM and E gene of the Japanese encephalitis virus isolated from Gansu province in 2008 and analysis the genotype of new JEV isolates and the molecular characterization of E gene.Computer software was used to analyze nucleic acid sequence and deduced amino acid sequence, and draw phylogenetic trees, including ClustalX2.09, MegAlign and Mega4.The six JEV strains were clustered in genotype I. 87.5%-87.9% identity in nucleotide sequence and 96.8%-97.2% identity in amino acid sequence were found in E gene when compared with the vaccine strain SA14-14-2. Eleven common amino acid differences were observed in E protein between new isolates and the vaccine strain.Genotype I JEVs were isolated from mosquitoes collected in Gansu province. The amino acid difference occurred in sites that were not the key ones affecting the antigenic of JEV.

To identify the virus isolated from a mosquito Culex modestus collected from Tongliao city of Inner Mongolia Autonomous Region.A strain of virus isolated from mosquito in Tongliao city was identified by serological and molecular biological methods. The nucleotides of the virus isolate were amplified by RT-PCR, and the products were purified and sequenced. Multiple alignment, phylogenetic and amino acid (AA) analysis were carried out by software Clustal X, MEGA4 and MegAlign (DNAStar).The new isolate was identified to be Banna virus by serological and molecular biological methods. Phylogenetic analysis showed that the Chinese isolates were distributed within one cluster. The homologue of nucleotide and amino acid of 12 segments between the new isolate and other strains isolated from China were 89.6%-98.4% and 90.4%-98.6%.The virus isolated from Culex modestus in Inner Mongolia belonged to Banna virus, and it is the first time that Banna virus was isolated in this region.

To investigate arboviruses in some regions of Shanxi province, isolation and identification for arbovirus activity from mosquitoes was conducted.Mosquitoes were collected from these area in 2007 and then used for virus isolation by cell culture. The virus isolates were identified by molecular biology and the sequences were analyzed by bioinformatics.Ten Banna virus (strains SX0765, SX0766, SX0767, SX0771, SX0789, SX0790, SX0793, SX0794, SX0795, SX0796) were isolated, and two Liaoning virus were also isolated from isolates SX0771, SX0794. Phylogenetic tree of the Banna virus isolates showed that ten strains are located in a distinct branch from all of the other Chinese Banna virus isolates. The homology is between 89.7% and 94.1%.Ten Banna virus and two Liaoning virus were isolated during this arbovirus investigation in Shanxi province. New Banna virus isolates showed a distinct phylogenetic relationship with the other Chinese Banna virus strains.

By RT-PCR and TAIL-PCR, the full coding region of Batai virus isolated in China (YN92-4 strain)was sequenced for the first time. According to the results, the genome of the virus contained three segments S, M and L of 947, 4,371 and 6,860 nucleotides, respectively. The S segment coded a nucleoprotein of 234 amino acids and a nonstructural protein of 102 amino acids, the M and L segments coded a precursor protein of 1 ,435 amino acids and RNA polymerase of 2,239 amino acids, respectively. Compared with the full coding sequence of Batai viruses isolated out of China, the S and M segments of YN92-4 and ON-7/B/01 showed the highest homology in nucleotide and amino acid sequenes with similarity of 97.7% (100%) and 95.7% (98%), respectively. Since there was no full coding sequence information on the L segment in GenBank for the reference, the L segment of YN92-4 was compared with that of Bunyamwera virus and the homology of nucleotide and amino acid was 73.5%and 81.6%, respectively. Phylogenetic analysis showed YN92-4 strain was clustered into one group with the prototype of Batai virus (MM2222). The results suggested that the YN92-4 strain had no occurrance of genetic reassortment (like Ngari virus) and was close to the Batai virus (ON-7/B/01 strain) isolated from cattle serum in Japan.

5 strains of virus isolated from Culex tritaeniorhynchus, Anopheles sinensis and Armigeres subalbatus, which caused cytopathic effect in C6/36 cells, had been obtained in the survey of arboviruses in Northwestern Yunnan Province. China. The virus particles displayed 70 nanometers diameter (n=7) with no envelope but spikes on the surfaces. RNA-PAGE of the genomes of the isolates showed 6-5-1 profile. A fragment of the 12th segment sequence was amplified by a pair of specific primers for Kadipiro virus strain JKT-7075 in RT-PCR. The full length of the 12th segment was 758 nucleotides, BLAST analysis revealed the highest identity was 90% to JKT-7075. Phylogenetic analysis demonstrated that the isolates appeared to be Kadipiro viruses (Family Reoviridae). It was the first report of kadipiro virus isolation in China.

To isolate and identify arboviruses from mosquito pools in some regions of Liaoning province.Mosquitoes were collected from Shenyang, Yingkou, Panjin, Jinzhou and Dandong cities of Liaoning province in 2006. Viruses were isolated by inoculating the specimens onto C6/ 36 and BHK-21cells. The new isolates were identified using serological and molecular biological methods.5410 mosquitoes were collected from the five cities in total. Three isolates produced CPE in C6/ 36 cell and five isolates produced CPE in both C6/36 and BHK-21 cell. Three isolates (LN0684, LN0688 and LN0689) were identified as Banna virus and one isolate (LN0636) was identified as Getah virus. Phylogenetic analysis showed that the three Banna virus strains were clustered into the same evolution branch as the other Chinese isolates. The identity of nucleotide sequence was between 91.2% and 94.7%, compared with other Banna virus strains. The new isolated Getah virus was clustered into the same branch with the strain of South Korea (swine). The identity of nucleotide sequence was 99.2%, when comparing with the strain of South Korea and was 95% to 99% with the strains from Russia, mainland of China and Taiwan region. Conclusion Eight virus isolates, including three Banna virus, one Getah virus and four unknown virus strains were isolated from mosquitoes in Liaoning province. Banna virus and Getah virus were reported for the first time in Liaoning province, while Getah virus showed the highest nucleotide homology with the South Korea strains.

To probe the primary characteristic of 0507JS11 virus isolated from Culex sp. and determine the classification of 0507JS11 virus in taxonomy.0507JS11 virus was cultured in Aedes albopictus C6/36 cells and cytopathic effects (CPEs) were recorded. Electro-microscopic morphology of 0507JS11 virus was observed. Total DNA extract of 0507JS11 virus was detected by 1% Agarose Gel Electrophoresis. Complete genomic sequence of 0507JS11 virus was sequenced and then made phylogenetic analysis.0507JS11 virus could cause CPEs in Aedes albopictus C6/36 cells. Viral particles have no envelope and appear icosahedron symmetry with diameter of 20 nm. The genome of 0507JS11 virus was positive single strand DNA (ssDNA) with full length of 3977 nt. However, a DNA band about 4 kbp was observed in the electrophoresis of total DNA extract of 0507JS11 virus. The coding region of the genome included three ORFs, ORF1 and ORF2 code NSP1 and NSP2, ORF3 codes VP. Phylogenetic analysis of the complete genomic sequence of 0507JS11 virus indicated an independent linear in Brevidensovirus.0507JS11 virus is a new member in Brevidensovirus.

To isolate viruses from mosquitoes in the south of Xinjiang and identify these viruses primarily.A total of 13 491 mosquitoes were collected in the south of Xinjiang from Jul to Aug, 2005. These mosquitoes were divided into 130 groups and grinded respectively. The supernates were inoculated in C6/36 and Vero cells. Viruses isolated were detected, the genomic nucleic types by electrophoresis of viral genomes and the morphologies observed under electronmicroscope.All 42 viruses were isolated, which caused CPEs on C6/36 but not on Vero cells. 27 viruses showed similar genomic profiles with 12 dsRNA segments. 1 virus displayed genomic profile with 10 dsRNA segments. 5 viruses took on similar genomic profiles with about 4 kbp DNA band. 9 viruses did not get any taxonomy information. Electromicroscopic pictures of these viruses revealed that above four types of viruses had distinguished morphologies indicating different virus species.There should be several virus species in the mosquitoes in the south of Xinjiang. dsRNA virus with 12 genomic segments should play analysis a predominant role in the south of Xinjiang.

0507JS60 virus was isolated from a pool of Culex sp. collected in Kashi, Xinjiang, which could be propagated stably on C6/36 cells and caused cytopathic effects continuously. Viral particles had no envelope and appeared round with diameter of about 55nm (n = 10). Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 12 double stranded RNA (dsRNA) segments. Sequencing of the twelfth segment revealed the length of 760bp (GenBank ID: FJ157354). A single open reading frame (ORF) was found and encoded a protein of 174 amino acids with a molecular mass of 18.9kD. The nucleotide sequence had similarity over 89% with that of LNV, but the deduced amino acid sequence had similarity over 91% with that of LNV. A phylogenetic tree was constructed to compare the corresponding genetic sequences in Seadornavirus. The tree demonstrated that 0507JS60 virus lied in the same branch with LNV and more closely related to LNV-NE9712. 0507JS60 virus was identified as LNV, which was firstly isolated outside the Northeast of China.

In order to reveal the phenotypic characteristics of 17 JE virus strains isolated from different years, plaque sizes, mice neurovirulence and mice neuroinvasiveness of the isolates were studied and compared. BHK21 cell monolayers were used for testing the plaque sizes. The virus neurovirulence was tested in 9-11g mice inoculated intracerebrally and the virus neuroinvasiveness was tested in 9-11g and 14-16g by subcutaneous inoculation. Results showed that all the viruses produced clear plaques on the BHK21 cell monolayers with different sizes and all the virus strains appeared high neurovirulence in the mice with higher than lg8. 0/0.03 mL virus titers, while no apparent difference among them. The neuroinvasiveness (subcutaneous virulence) tested in the 9-11g mice had shown a little difference, but when tested in the 12-14 g mice,the difference was apparent. The results demonstrated that JEV in nature were highly neurovirulent with no apparent difference. However the neuroinvasiveness of the JEV in nature was greatly different, which didn't relate to the years of isolation and genotypes, but most of the viruses isolated from patients showed higher neuroinvasiveness.

0507BS3 virus was isolated from a mixed pool of Culex sp. and Anopheles sp. collected in Kashi, Xinjiang, China. 0507BS3 virus could cause cytopathic effects on C6/36 cells but not on Vero and BHK-21 cells. Viral particles had no envelope and appeared round with diameter of about 60 nm (n = 20). Viral capsid was composed of a single layer and a central core. Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 10 double stranded RNA (dsRNA) segments. Sequencing of the tenth segment revealed the length of 964bp (GenBank ID: FJ150869). A single open reading frame (ORF) was found and encoded a protein of 275 amino acids with a molecular mass of 30.8kDa. The nucleotide sequence had no similarity with any other viral genomic sequences, but the deduced amino acid sequence significantly matched the polyhedrin genes of cytoplasmic polyhedrosis virus (CPV) in some sections. A phylogenetic tree was constructed to compare the polyhedrin gene sequences of all CPV types in GenBank. The tree demonstrated that 0507BS3 virus was only distantly related to the other CPV types and belonged to a new CPV type.

Evaluation of the direct rapid immumohistochemical test (DRIT) for laboratory surveillance of rabies.72 brain specimens of domestic dogs or patients collected from Guizhou, Guangxi, Hunan, Anhui, Jiangsu and Yunnan provinces were detected by conventional methods including Direct Fluorescent-antibody Assay (DFA) and Reverse Transcription Polymerase Chain Reaction (RT-PCR), and by DRIT which was newly developed in the Rabies Section of the Centers for Disease Control and Prevention in the United States. The sensitivity and specificity of DRIT were evaluated by compare of the three results. By analysis of the index including cost of experiment, technique requirement and so on, the advancement and applicability of DRIT were discussed.Compared with DFA and RT-PCR, DRIT will be more applicable for laboratories with limited funds and weak techniques because of its lower cost needed and simpler techniques required while its sensitivity and specificity are equal to the other two methods.DRIT is more valuable in rabies diagnosis and more applicable for extension and popularization in rabies laboratory surveillance in local CDC.

Characterization of rabies virus phosphoprotein through analyzation of genetic variations about rabies virus phosphoproteins in high-incidence regions in China.The nucleotide sequence of the P gene of Guangxi, Guizhou and Hunan provinces positive sample's were sequenced, and the P region's similarity and phylogenetic analyses were completed by using softer wares.The similarity of P region's nucleotide sequence is 82.1%-100%, while, the similarity of amino acid sequence is 87.5%-100%. A little variation in phosphoprotein cannot influence its biological functions.All rabies viruses isolated from Guangxi, Guizhou and Hunan provinces belong to genotype 1 and share same phylogenesis and same genome characteristic; Virus distribution presents unique Characterization; Some virus isolates from Hunan province and Thailand may come from the same virus.

To investigate the situation of dog rabies and analyze it's relationship with human rabies.In Guizhou, Guangxi and Hunan provinces which suffered from rabies most heavily, one or two cities were selected respectively from regions with high-, middle-, low-incidence rate of human rabies as investigation spots where Dogs' brain specimens were collected and detected with both direct immunofluorescence assay (IFA) and RT-PCR.A total of 2887 specimens were collected and 66 of these were positive for IFA and RT-PCR. Therefore, the rate of positive specimens was 2.3%. However, there was not a close relationship in the incidence rate of dog rabies and human rabies.Dog's infection situation of rabies contributed to the severe epidemic of human rabies.

Objectives: XJ-160 virus is a mosquito-derived Sindbis-like virus isolated in China. Based on its infectious clone (pBR-XJ-160) we have developed an RNA-based vector system. In this work, we constructed packaging cell lines (PCLs) for XJ-160 virus. Methods: Firstly, XJ-160 virus structural protein expression cassette pcE or pICH was constructed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pcDNA3.1(+) or pIRES, respectively. Then the PCLs (BHK-21E+Capsid cells) for XJ-160 virus were obtained by two selections with G418 and hygromycin. Results: The results indicate that BHK-21E+Capsid cells, stably expressing E3E26KE1 protein and capsid protein of XJ-160 virus, not only highly increased packaging efficiency of the vector from XJ-160 virus, but also provided packaging function for the vector from Semliki Forest virus. Conclusion: These results suggest potential utility of the PCLs of XJ-160 virus for large-scale vector production and facilitating broad alphavirus applications. Also, the construction of PCLs for XJ-160 virus lays a basis for developing alphavirus-derived vector systems.

To isolate Japanese encephalitis virus (JEV) from mosquitoes collected in Liaoning province and analysis the genotype of new isolated JEV strains and the characters of nucleotide and amino acid in the E gene.Collected mosquitoes in Dandong Liaoning Province in August, 2006. Virus isolation was using issue culture cells. Isolated viruses were identified by using serological and molecular methods.Two new JEV strains, LNDG07-02 and LNDG07-16, were isolated from 1500 mosquitoes were belonging to genotype 1. The identity of nucleotide and amino acid of E gene between new JEV strains and live attenuated vaccine strain SA14-14-2 were 87.8-88% and 97.2%, respectively. Total 11 amino acid sites were differences in E gene between new isolates and SA14-14-2. However, there were no differentiation between the new JEV strains and the isolates in Donggang 2002.Genotype 1 JEV was isolated again from Donggang, since the first isolation of this genotype in 2002. Genotype 1 JEV continues in existence in Donggang Liaoning Province.

Explore the genotype of the epidemic Japanese encephalitis virus (JEV) in Yunnan Province from molecular level and to understand the molecular differences of the virus isolated from Yunnan at different time, locality and host.Three-day suckling mice were inoculated with viruses continuously and when the disease developed and the mice were dying, the brain was taken and DNA was extracted from the supernatants of the brain after grinding; then the gene fragments of Prm-C region were amplified by RT-PCR. The viral gene sequences were compared with those of other 72 strains of JEV originated from both China and abroad at different times. Finally the genotypes were analyzed with the method which was established by Woan-Ru Chen.All the 3-day suckling mice which were inoculated with the virus died within 78 h. The results of the nucleic acid-sequence analysis showed that 17 strains of the experimental virus belonged to genotype 1 and 2 strains belonged to genotype 3. The difference between genotype 1 and type 3 were more than 15%. While the difference between 17 strains of genotype 1 which were separated at different time, location and hosts were only 3.8%-5.2%.The above results suggest that the genotype of the epidemic JEV in Yunnan Province are type 1 and 3 and the latter is the main type.

To isolate Japanese encephalitis virus (JEV) from mosquitoes collected in Tanghe County, Henan province and analyze the genotype of the newly isolated JEV strains and the characteristics of amino acid in the E gene.Viruses were isolated from mosquitoes collected in 2004 and identified by biological, serological and molecular biologic methods. PrM and E segments of the newly isolated JEV were amplified by RT-PCR, the PCR products were purified and sequenced. Multiple alignment, phylogenetic and amino acid (AA) analysis were carried out by Clustal X (1.8) program, MEGA 3.1 and GENEDOS (3.2).Totally 3722 mosquitoes were collected including Culex, Armigeres, Aedes, Anopheline. Three new JEV strains isolated from Culex belonged to genotype 1. The homologue of nucleotide and amino acid of E gene between new JEV strains and live attenuated vaccine strain SA14-14-2 was 86.9-87.7% and 95.2%-97.0%, respectively. Totally there were 12 common sites of amino acid differences in E gene between them.Newly isolated viruses in Henan province belonged to JEV genotype 1. It suggests that the vaccine strain SA14-14-2 currently used for preventing JE is able to protect people from JEV infection, although there are some amino acid differences between them.

To develop a rapid and more sensitive TaqMan RT-PCR assay specific for Banna virus.Total RNA of strains of Banna virus and other arboviruses were extracted and reverse transcribed to cDNAs. With the cDNAs as templates, the TaqMan RT-PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Banna virus. The sensitivity, specificity and the possibility of this assay to detect Banna virus in pools of mosquitoes. Meanwhile, human sera samples added with different dilutions of Banna virus culture medium supernatant were evaluated.All the collected 12 Banna virus strains in our country were detected as positive, while the results of other arboviruses such as Japanese encephalitis virus (JEV), Batai virus, Sindbis virus and Orbiviruses were negative. This assay was more than 100 times sensitive than that of traditional PCR. About 0.5 CCID-50 of Banna virus in 50 microl samples could be detected with this assay. The sensitivity decreased by about 10 times when the dsRNA was denatured in 65 degrees C instead of 99 degrees C, and also when the virus seeds were kept at room temperature for 1 day or 1 week at 4 degrees C. There was no significant difference in the results of Banna virus detection between artificial human serum samples and positive control virus. Six were detected as positive for Banna virus-specific nucleic acids in 112 pools of mosquitoes, while 3 was positive with virus isolation.The Banna virus-specific TaqMan RT-PCR assay was proved to be highly sensitive, specific and showed a good reproducibility; the assay may be applied for surveillance of this virus in clinical samples and pools of mosquitoes screening.