Search

To understand the rabies virus (RABV) evolutionary relationship between the strains of China and Asia and to know the evolution and transmission characteristics of RABV in Asia.The G sequences of representative strains from China were selected and combined the sequences of other countries in Asia to analyze using BEAST and MigraPhyla software.The phylogenetic analysis showed that six groups (China I-VI) of China had different epidemic range: China I , II and V groups just cycled in our country; China VI group, from Guangxi and Yunnan provinces, crossed with Southeast Asian strains; China III group and IV group also have closer genetic relationship with Asian country strains.Geographic migration in Asia showed that Thailand and India may be two rabies transmission centers in Asia.

To construct and characterize EGFP reporter gene labeled Sindbis virus (SINV).The reporter gene EGFP was inserted into the genome of infectious clone pBR-XJ160 by using multi-fusion long fragment PCR method. Then apply reverse genetic manipulation technique to rescue and obtain EGFP labeled SINV.We successively obtained labeled SINV, which has good fluorescent expression characteristics and genetic stability.The labeled virus can be seen in living cells and living body, and this serves as a good tool for cell and tissue tropism and biological function study of viruses. This study laid a foundation for further studying the cell tropism, biological functions and infection mechanism of SINV.

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 investigate the molecular basis of pathogenicity of Japanese encephalitis virus (JEV) by sequencing of complete nucleotide sequence and analyze the characteristics of full-length genome of genotype I Japanese encephalitis virus strains (GZ56) which was isolated from the first cerebrospinal fluid (CSF) of Japanese encephalitis patients.The complete nucleotide sequence was obtained by RT-PCR and sequencing was performed directly. Bioinformatics was used to analyze the nucleic acid data, deduced amino acid sequence and phylogenetic trees.The result of sequence analysis showed that the genome of GZ56 strains had 10 965 nucleotides, which coded for a 3432-amino acid polyprotein. Phyolngenetic analysis based on full-length genome showed that GZ56 strains and M-28 strains which were the first isolated from mosquitoes in Yunnan in 1977 were in the same evolutionary branch. GZ56 strains belongs to genotype I of Japanese encephalitis virus, the homology of genome ranged from 96.2% to 98.6% in nucleotide and from 98.2% to 99.7% in amino acid sequences respectively when compared with selected genotype I of JEV strains in GenBank. There were 11 amino acid divergences in E protein when compared with the JEV inactivated P3 strain but they are not the key virulence sites. However, there were 14 amino acid divergences in E protein when compared with the JEV live attenuated vaccine SA14-14-2 strain and 8 amino acid divergences were the key virulence sites.This study indicated that the full length of genome GZ56 strains had no ignificant change. It can be hypothesized from genomic level that the currently available JEV vaccines(inactivated and live attenuated) can protect against GZ56 strains infection, meanwhile, the JEV live attenuated vaccine (SA14-14-2) formulation conferred higher levels of protection.

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.

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 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.

For constructing the high-throughput platform of sequencing the JEV whole genome, the two systems of multiplex primers for genotype I and III should be designed and used for detected the whole genome of genotype I and III JEV in the research.The two systems of JEV genotype-specific primers was designed based on the reference sequence of all the available genotype I and III JEV genome sequence on GenBank, then, they were used to amply and sequence the 121 JEV strains isolated in China contains 63 GIII JEV and 58 GI JEV.The self-designed genotype-specific primers for genotype I and genotype III JEV were 16 pairs and 17 pairs respectively, which were used for detecting the whole genome of 121 JEV. The average quality value for GI JEV is 40.037. The average quality value for GIII JEV is 40.857.The two systems of JEV genotype-specific primers could sequenced the genotype I and III JEV qualified and specific. It is the basis of the high throughput platform of sequencing the JEV whole genome.

To sequence and analyze the whole genome of Japanese encephalitis virus (JEV) isolated from mosquitoes in Liaoning province in 2008.Using RT-PCR to amplify fragments with genome sequencing primer. The full-length genome was obtained by sequencing and splicing. The differentiation analysis for nucleotides, deduced amino acid sequence and phylogenetic tree was performed by the software of Clustal X (1.83), ATGC (V4), DNAStar, GENEDOC (3.2) and Mega (4.0).The whole genome of strain LN0828 possesses 10 965 nucleotides. An open reading frame from 97 to 10 392 including 10 296 nucleotides is capable of coding for a 3432 amino acid polyprotein. Comparison of strain LN0828 genomic sequence with those of 32 JEV isolates in GenBank showed that nucleotide sequence divergence ranges from 1.6% to 16.4%, which resulted in amino acid sequence divergence from 0.3% to 5.1%. In comparison with live attenuated vaccine stain SA14-14-2 in open reading frame, strain LN0828 has a total of 1186 nucleotide substitutions, 86 amino acid divergences. Based on phylogenetic analysis, the strain LN0828 belongs to the genotype I JEV.The whole genome of strain LN0828 is close to those of isolates from Liaoning in 2002 and 2007, which were grouped into genotype I JEV.

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.

Sequence and analysis the complete nucleotide of the Japanese encephalitis virus (JEV) newly strain SD08-10, isolated in 2008 in Shandong, China in order to understand the characterization of the virus.Overlapping primers were designed according to the full-length genomes in GenBank. RT-PCR was used to amplify the fragments and the full-length genome was obtained by sequencing and splicing. Using the computer software to analysis the nucleic acid data, deduced amino acid sequence and phylogenetic tree, including Clustal X (1.8), DNASTAR, GENEDOC (3.2).The result of sequence analysis shows that the genome of SD08-10 strain was 10 965 nucleotides long. An open reading frame from 96 to 10 392 including 10 296 nucleotides is capable of coding for a 3432 amino acid polyprotein. Compared with the live attenuated vaccine strain SA-14-14-2 in China, there was 1253 nucleotide difference and 82 amino acid divergence. Comparison of the complete genome sequences with 59 different JEV isolates showed a 0.7%-18.9% nucleotide sequence divergence among them, which resulted in 0.1%-5.2% amino acid sequence divergence. Phylogenetic analysis of full-length genome showed that the SD08-10 strain was belonging to genotype I.Analysis based on the complete genome sequences of different JEV isolates showed that the SD08-10 strain isolated in 2008 in Shandong was belonging to genotype I and close to SH17M-07 isolated in 2007 in China.

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.

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 RdRp gene (L) of rabies virus aG and CTN181 strain in China.Overlapped fragments were amplified and assembled. Then characterization and phylogenetic analyses as well as prediction of functional regions were performed using biologic softwares.L gene of CTN181 and aG strains were composed with 6387nt and 6384nt respectively and there were two repeated ATG in the start of L gene in CTN181 strain. In addition, some mutations and new functional regions were discovered and presumed to be crucial to the role of these regions in replication of rabies virus. Analyses of L gene provide new insight into the functional regions, and phylogenetic analyses showed that human vaccine strain aG were independent to others recommended by WHO.The characterization of complete L genes will be helpful for research on new functional regions, antiviral drugs targeted at RdRp and phylogenetic analyses.

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.

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 study the clinical and laboratory characteristics of adult Japanese encephalitis (JE) patients in a JE outbreak in Yuncheng, Shanxi Province in 2006.All the clinical data from the Second People's Hospital in Yuncheng city were analyzed, part of patients' sera and cerebrospinal fluid were tested by serology and RT-PCR.The majority of patients were middle-aged and elderly, 77.8% (35/45) of the total cases were more than 40 years old. Severe and fulminating type cases accounted for 60.0% (27/45). Most patients had underlying diseases. IgM antibody to JE virus (JEV) in serum was positive in each of the 45 patients analyzed and 4-fold or greater rise in sera neutralization antibody titer were found in convalescent serum. JEV nucleic acid was positive in part of cerebrospinal fluid specimens.Viral encephalitis emerged in Yuncheng city, Shanxi Province was Japanese encephalitis B, and most of the cases belonged to elderly group.

To understand the viral etiology of viral encephalitis in China by detecting IgM antibody and viral RNA in the clinical samples of patients from some provinces of China by enzyme-linked immunosorbent assay and polymerase chain reaction.Serum and cerebrospinal fluid samples of 771 patients with viral encephalitis or meningitis were collected from six provinces of China and were stored at -20 degrees C or -70 degrees C. Enzyme-linked immunosorbent assays were used for detection of IgM antibody to Japanese encephalitis virus, coxsackievirus, echovirus, herpes simplex virus, measles virus, varicella-zoster virus, mumps virus, cytomegalovirus and Epstein-Barr virus. Polymerase chain reaction was applied for the detection of viral RNA of enteroviruses and seadornavirus.IgM antibody was detected in 567 of 771 (73.5%) cases. The most common pathogen was Japanese encephalitis virus (47.0%, 362/771), followed by mumps virus (10.6%, 82/771), enteroviruses (8.8%, 68/771), herpes simplex virus (5.7%, 44/771), measles virus (0.4%, 3/771), varicella-zoster virus (0.4%, 3/771), Epstein-Barr virus (0.4%, 3/771), and cytomegalovirus (0.3%, 2/771). Enterovirus was positive in 8 cases, seadornavirus was negative in all the cases by PCR.According to the study, Japanese encephalitis was the most important encephalitis in China. Mumps virus was another important pathogen. Enteroviruses and herpes simplex virus were also important pathogens.

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.

To analyze the difference of nucleotides and deduced amino acids sequences E gene between the newly isolated Japanese encephalitis (JE) virus strains from mosquitoes or patients and P3 strain.The E gene sequences of corresponding strains of JE virus were obtained from GenBank. Computer analyze of nucleic acid data and deduced amino acid sequence were accomplished using the Clustal X (1.8), DNASTAR, GENEDOC (3.2) programs.The result showed that compared with the Fujian strains and P3 strain the nucleotide sequence homology was up to 98.3%, and the amino acid sequence homology was up to 98.2%, respectively. Compared with the Shanghai strains and P3 strain, the nucleotide sequence differences were 12%, and the amino acid sequence homology was up to 98.2%, respectively. Compared with P3 strain, there were nineteen amino acid variations in E gene of all the newly isolated strains. Between P3 and all the newly isolated JE virus strains, there are three common variations at E-129, E-222, E-366. And two common variations E-160 and E-487 were found only in Fujian strains, common variations at E-129, E-222, E-227, E-366 in Shanghai strains.There are some differences between P3 strain and JE viruses which were isolated from mosquitoes belonging to genotype I in Shanghai and from patients belonging to genotype III from Fujian province. But these variations are not in the important locations affecting the biological characteristic of the viruses.

To study arboviruses carried by mosquitoes in Liaoning Province in 2002.Totally 4927 mosquitoes were collected from Liaoning Province in July 2002. Virus strains were isolated by inoculating BHK-21, C6/36 and Vero cells. The newly isolated strains were identified by serological (ELISA and IFA) and molecular methods (Real-Time PCR and RT-PCR).Two strains were isolated from mosquitoes causing cytopathogenic effect (CPE) on cells and were fatal to suckling mice. Serological tests showed that both were positive for the antibody to JEV. The PrM and E gene were cloned and sequenced. The phylogenetic analysis showed that the new isolates belonged to genotype I, JEV. Sequence analysis showed that the homology of nucleotide sequences and amino acid (AA) sequences between the two strains was 100%. Compared with the nucleotide sequences between the two strains and the standard JEV vaccine strain SA14-14-2, the difference was up to 4.11%, and the difference of AA was 0.6%.Two strains of JEV were isolated and identified in Liaoning province, both belonged to genotype I JEV.

Mosquitoes were collected in Heilongjiang province in 2002, four virus strains were isolated by inoculation of homogenates onto BHK cell lines. The viruses were identified. Multiple alignment and phylogenetic analysis were carried out by Clustal X (1.8) program.Amino acid (AA) analysis was carried out by GENEDOS (3.2).Biological characters of four newly isolated strains were examined and it was found that all of them could produce cytopathogenic effect (CPE) in BHK cells, killing sucking mice. Serological tests showed that all of these stains reacted positively to JEV antibodies. PrM and E gene regions were amplified and sequenced. Phylogenic analysis showed that all the newly isolated JEV strains belong to genotype III. Using the vaccine strains (SA14-14-2) as control, analysis of the E gene of the new strains and two JEV strains (47, Ha-3) isolated previously from Heilongjiang province showed that these new strains' nucleotide sequence had a homology of up to 99.9% and the amino acid sequence homology up to 99.8%, respectively. Compared with the standard JE vaccine strain SA-14-14-2 and the four new strains, the nucleotide sequence homology was 97.3% and amino acid sequence homology was between 96.8% and 97.0%, respectively. Compared with vaccine strain, there were seven common variations in all the four newly isolated strains.Four JE virus strains were isolated in Heilongjiang province. As compared to the vaccine strain, six variations were found in the newly isolated strains at the eight sites relevant to the virulence of the virus.

To survey arboviruses in Yunnan province.Mosquitoes were collected from Yunnan Province in 2002 and 2004. Virus strains were isolated by the inoculation of homogenates of the mosquitoes onto BHK cell line. The isolated strains and their molecular biological characteristics were identified by real-time PCR, reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent antibody technique.Twelve strains of viruses producing CPE in BHK cells were isolated from 4810 mosquitoes. All the 12 isolates were identified to be Japanese encephalitis viruses. Genotype analysis showed the new virus (DL-0437 strain) belonged to genotype III.Twelve strains of Japanese encephalitis viruses were isolated from mosquito pools collected in Yunnan. It was the first isolation of genotype III Japanese encephalitis viruses in Yunnan Province in recent years.

To study the molecular characteristics of YN92-4 strain isolated from mosquitoes in Yunnan Province and define its classification.The S segment of YN92-4 strain was amplified and sequenced by 2 different sets of primers. The phylogenic tree of S fragment was constructed by Phylip bio-software. The amino acid sequences of N and NSs proteins were also studied.YN92-4 strain could be amplified by 2 sets of primers respectively, S segment showed a highest homology with Batai virus (X73464), reached 96.4%, the homology of protein N and NSs amio-acid sequence with Batai virus was 99.1% and 98% respectively.The YN92-4 strain belongs to Batai virus, this is the first report of molecular biological identification of Batai virus in China.

To study the molecular biology of Xinjiang hemorrhagic fever (XHF) viruses, to explore its relationship with other Crimean-Congo hemorrhagic fever viruses, analyzing the epidemic origin and the tendency of geographic distribution of XHF.The S partial segment from the patient and tick samples collected in 2001 and 2002 was tested by RT-PCR, the positive samples were sequenced directly. The nucleotide homology of S partial segment as well as the whole segments were analyzed and the phylogenetic tree of S and M gene segments was drawn by computer.All compared sequences of S partial segments from the patient and tick samples showed a high homology of nucleotide sequences. Phylogenetic tree divided all the analyzed viruses into three groups; Europe, African and Asian group. The Asian group can be divided further into another two branches: the middle Asian branch and the Chinese branch. All the Chinese isolates were clustered into one single group and was easy to be discriminated from the other isolates. The dividing of M segments seemed not completely related to the geographic origin of the viruses.M segment classification was not consistent to the geographic distribution of the viruses. S segments analysis showed the close relationship of genetic background between the patient isolates and the tick isolates. Besides, all the Chinese isolates have the common evolution route and the gene structure characteristics displayed the regional distribution pattern.

To develop a rapid, specific and sensitive method for detecting Sindbis virus (SINV) with SYBR GREEN I real time PCR.Total RNA of strains of Sindbis virus and a related virus were extracted and reverse transcribed to cDNAs. With the cDNAs as template, the SYBR GREEN I real time PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Sindbis virus, and the sensitivity, specificity and reproducibility were evaluated.For the PCR, 55 degrees C was chosen as the optimal anneal temperature and 0.5 micromol/L as the optimal primer concentration. Using this method, all the selected SINV were detected as positive, while the results of control arboviruses such as Geta virus, Japanese encephalitis virus (JEV), Batai virus, Seadornavirus Orbiviruses and synthesized WEEV cDNA were negative. With this system, 0.1 PFU/ml SINV cDNA could be detected; The sensitivity of this assay was about 100 times higher than standard RT-PCR. All the results were reproducible within two compatible tests, and the stability of the detection system was very good. The test results of simulated infection human serum samples showed that human serum had no obvious interference with this system. With this system, 6 of 151 clinical samples with unknown fever or encephalitis were determined as positive.The developed SYBR GREEN I real time PCR assay for detecting Sindbis virus was highly sensitive, specific and showed a good reproducibility and stability. It is our belief that the present method can be further used in clinic sample to verify its stringency.

To disclose the species and distribution of tick-borne arboviruses in the southern part of Xinjiang.Totally 5045 ticks were collected from 36 collecting sites of 23 places in the southern Xinjiang, which were made into cDNA pools with pd(N)6 primer through RT-PCR method. Then PCR was used to detect viral nucleotide sequence from cDNA.All 34 cDNAs showed negative to flavivirus and California serogroup virus primers; but nairovirus and primers derived from Xinjiang hemorrhagic fever virus had amplified and yielded some obvious bands corresponding to the nucleotide sequences of Xinjiang hemorrhagic fever virus. A phylogenetic analysis was done to the obtained partial sequences of L and S segments.Nucleotide sequences of Neither flaviviruses nor California serogroup viruses were detected from the samples. However partial L segment sequence was first reported in China. Phylogenetic analysis of partial L and S segments disclosed the molecular characteristic of Xinjiang hemorrhagic fever virus.

To analyze the genotype of newly isolated Japanese encephalitis viruses in Fujian province and the characteristics of amino acid sequence in the E gene.PrM and E segments of newly isolated Japanese encephalitis viruses were amplified by RT-PCR, the PCR products were cloned into T vector for sequencing. Phylogenetic analysis was carried out by PHYLIP program.Newly isolated Japanese encephalitis viruses belonged to genotype III, the nucleotide and amino acid of E gene showed high homology to vaccine strain SA14-14-2, but had some difference in some domains.Newly isolated viruses in Fujian province belonged to Japanese encephalitis virus genotype III. E protein of the isolates showed some differences as compared with vaccine strains.

To study the levels of SARS-associated coronavirus IgG antibody of SARS patients, people who closely contacted SARS patients and normal subjects in Gansu province.The levels of SARS-associated coronavirus IgG antibody were measured by ELISA. The material included acute and (or) recovery period sera of 9 SARS patients, sera from 1,109 doctors and nurses closely contacted with SARS patients, laboratory workers, personnel for disease control and prevention, persons who contacted SARS patients, and sera from 978 normal subjects.SARS coronavirus IgG antibody was detected positive in 6 of the 9 patients, it was still positive in the sera twelve months after recovery; 1 of the closely contacted persons and 3 normal subjects were found positive.The positive rate of SARS coronavirus IgG antibody of patients was consistent with the clinical diagnosis. The low positive rate of the persons who closely contacted SARS patients and normal subjects suggests that SARS probably had no subclinical infection.

To find out the timing of serologic responses after illness onset and distribution of IgG antibody to SARS-CoV in SARS cases of transmission chain or non-transmission chain.The IgG and IgM antibodies to SARS-CoV were tested by indirect ELISA in serum samples from 301 clinically diagnosed SARS cases.Totally 158 SARS cases were involved in 15 chains of transmission. The positive rates of SARS-CoV IgG in those chains were 85.70%-100.00% and the overall rate was 94.30% (149/158). The chain of transmission could spread to four generations, but the SARS cases were reduced with increase of generations. There was no significant difference among positive rates of SARS-CoV IgG for generations, Chi square=5.11, P greater than 0.05. The positive rate of SARS-CoV IgG in cases who were not in chain of transmission was 12.59%(18/143) which was statistically significantly different from that of cases in chain of transmission, Chi square=199.64, P less than 0.001. During days 0-7,8-14,15-21,22-30 after onset, the cumulated positive rate of SARS-CoV IgG was 16.67%, 40.00%, 70.00% and 93.10%, respectively, then was kept at the level above 90% and lasted for 217 days. The cumulated positive rate of SARS-CoV IgM during days 0-7 after onset was the same to that of IgG. During days 8-14, 55.17% of cases had seroconversion for IgM which reached a peak (86.96%) during days 21-30. Then the rate rapidly declined.More than 94% of cases with SARS could produce IgG antibody when they were infected by SARS-CoV. Detecting SARS-CoV IgG could provide a diagnostic evidence for case confirmation. SARS-CoV IgG appeared as early as 7 days after onset and reached the peak at about weeks 4. Then the high rate of antibody was maintained for more than 6 months.