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1.
Article in English | WPRIM | ID: wpr-828995

ABSTRACT

Pseudorabies virus (PRV), a veterinary pathogen that infects domestic animals as well as wild animals such as wild boar and feral swine, was recently reported to infect human and led to endophthalmitis and encephalitis. A retrospective seroepidemiologic survey was conducted using 1,335 serum samples collected from patients with encephalitis and ELISA positive rates were 12.16%, 14.25%, and 6.52% in 2012, 2013, and 2017, respectively. The virus neutralizing antibody titers of positive samples correlated well with ELISA results. The pseudorabies virus antibody positive rate of patients with encephalitis were higher than that of healthy people in 2017. The above results suggest that some undefined human encephalitis cases may be caused by PRV infection.


Subject(s)
Adult , Animals , Antibodies, Viral , Blood , China , Encephalitis , Allergy and Immunology , Virology , Enzyme-Linked Immunosorbent Assay , Female , Herpesvirus 1, Suid , Allergy and Immunology , Humans , Male , Middle Aged , Prevalence , Pseudorabies , Blood , Allergy and Immunology , Virology , Retrospective Studies , Seroepidemiologic Studies , Young Adult
2.
Article in English | WPRIM | ID: wpr-781430

ABSTRACT

OBJECTIVE@#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.@*METHODS@#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.@*RESULTS@#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.@*CONCLUSION@#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.

3.
Article in English | WPRIM | ID: wpr-773407

ABSTRACT

OBJECTIVE@#Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease.@*METHODS@#A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay.@*RESULTS@#The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay.@*CONCLUSION@#A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.


Subject(s)
Encephalitis Viruses, Tick-Borne , Genetics , Nucleic Acid Amplification Techniques , RNA, Viral
4.
Article in English | WPRIM | ID: wpr-690669

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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 all < 2.8%. The amplification efficiency of this method was between 90% and 103%.</p><p><b>CONCLUSION</b>A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.</p>


Subject(s)
Animals , Culicidae , Virology , Encephalitis Virus, Japanese , Genetics , Polymerase Chain Reaction , Methods , Reproducibility of Results , Sensitivity and Specificity
5.
Article in English | WPRIM | ID: wpr-690667

ABSTRACT

Japanese encephalitis (JE) is a serious public health issue. This study was undertaken to better understand the relationship between JE distribution and environmental factors in China. JE data from 2005 to 2010 were retrieved from National Notifiable Disease Report System. ArcGIS, remote sensing techniques, and R software was used to exhibit and explore the relationship between JE distribution and environmental factors. Our results indicated that JE cases were mostly concentrated in warm-temperate, semitropical and tropical zones with annual precipitation > 400 mm; Broad-leaved evergreen forest, shrubs, paddy field, irrigated land, dryland, evergreen coniferous forest, and shrubland were risk factors for JE occurrence, and the former five were risk factors for counties with high JE incidence. These findings will inform the effective allocation of limited health resources such as intensive vaccination, surveillance and training in areas with high environmental risk factors.


Subject(s)
China , Epidemiology , Encephalitis, Japanese , Epidemiology , Virology , Environment , Epidemiological Monitoring , Humans , Incidence , Risk Factors
6.
Article in English | WPRIM | ID: wpr-296524

ABSTRACT

Fifteen pediatric cases of suspected Japanese encephalitis (JE) were reported in Beijing Children's Hospital during the late summer of 2013. The clinical manifestations in most cases included high fever, seizures, and abnormal magnetic resonance imaging findings. Twelve of 15 cases were laboratory-confirmed as JE cases by pathogen identification. Epidemiological investigations showed that five of the 12 laboratory-confirmed patients had an incomplete JE vaccination history. Follow-up investigations after discharge indicated that seven laboratory-confirmed JE patients without JE vaccinations had relatively poor prognoses, with an average Modified Rankin Scale (MRS) score of 2.6 when compared with the other five laboratory-confirmed, JE-vaccinated patients with an average MRS score of 0.5. The observation of pediatric JE cases among those with a history of JE vaccination warrants further attention.


Subject(s)
Beijing , Epidemiology , Child , Child, Preschool , Encephalitis Virus, Japanese , Physiology , Encephalitis, Japanese , Diagnosis , Epidemiology , Virology , Female , Humans , Japanese Encephalitis Vaccines , Male , Prognosis
7.
Article in English | WPRIM | ID: wpr-264574

ABSTRACT

A real-time RT-PCR (RT-qPCR) assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.


Subject(s)
Animals , Culicidae , Virology , Encephalitis Virus, California , Real-Time Polymerase Chain Reaction , Methods , Reverse Transcriptase Polymerase Chain Reaction , Methods , Sensitivity and Specificity
8.
Article in English | WPRIM | ID: wpr-270609

ABSTRACT

<p><b>OBJECTIVE</b>To determine the molecular characterization of full-length genome of Japanese encephalitis virus (JEV) genotype V.</p><p><b>METHODS</b>The full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis.</p><p><b>RESULTS</b>The full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% (G I, KV1899) to 79.7% (G III, JaGAr01), for the nucleotide sequences, and from 90.0% (G I, KV1899) to 91.8% (G III, JaGAr01) for the amino acid sequences. The open reading frame (ORF) of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides (encoded with a serine residue) were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3'-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V.</p><p><b>CONCLUSION</b>The molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.</p>


Subject(s)
Amino Acid Sequence , Animals , Base Sequence , Culex , Virology , Encephalitis Virus, Japanese , Genetics , Genome, Viral , Genotype , Humans , Male , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Tibet , Young Adult
9.
Chinese Journal of Epidemiology ; (12): 428-432, 2013.
Article in Chinese | WPRIM | ID: wpr-318382

ABSTRACT

<p><b>OBJECTIVE</b>To understand the epidemiologic characteristics of dengue fever, imported from Myanmar to the border of Yunnan province, China. Viral molecular epidemiologic features were also studied.</p><p><b>METHODS</b>Questionnaires were used on each diagnosed, suspected dengue fever, case or unknown cases with fever when coming from Myanmar entering the port and hospitals in Ruili city of Yunnan province. Serum samples of these patients were collected to detect IgM antibody against dengue virus and RT-PCR assay. Homology and phylogenetic tree based on the whole nucleotide sequence of PrM-C and NS5 gene of dengue virus were further analyzed.</p><p><b>RESULTS</b>A total of 103 sera were collected from patients at acute stage in Ruili city in July to November 2008. Among them, 49 cases were confirmed for dengue fever according to IgM and nucleic acid testings. Except one, other 48 cases were all imported into Ruili, from Myanmar. Of those, 18 patients were residents from Mujie city of Myanmar and hospitalized in Ruili and the rest 30 patients were Chinese citizens who had finished business and returned from Myanmar. Two isolates of serum samples from the imported cases were identified and both homology and phylogenetic analysis were performed, using the nucleotide sequences of PrM and NS5 genes. They were divided into dengue type 1 (RLB61) and dengue type 3 (RLC31) and were closer to the dengue virus strains isolated from Southeast Asia countries.</p><p><b>CONCLUSION</b>It is confirmed that an epidemic of dengue fever which was imported from Myanmar to Ruili city of Yunnan province, China. Evidence also showed that both type I and III epidemic strains of dengue virus did exist in Mujie city of Myanmar in 2008.</p>


Subject(s)
Adolescent , Adult , Child , Child, Preschool , China , Epidemiology , Dengue , Epidemiology , Dengue Virus , Genetics , Female , Genotype , Humans , Infant , Male , Middle Aged , Molecular Epidemiology , Myanmar , Epidemiology , Phylogeny , RNA, Viral , Genetics
10.
Article in Chinese | WPRIM | ID: wpr-305070

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>


Subject(s)
Amino Acid Sequence , Animals , China , Epidemiology , Culicidae , Virology , Encephalitis Virus, Japanese , Chemistry , Classification , Genetics , Encephalitis, Japanese , Epidemiology , Virology , Genotype , Humans , Molecular Sequence Data , Phylogeny , Sequence Alignment , Viral Proteins , Chemistry , Genetics
11.
Article in Chinese | WPRIM | ID: wpr-246175

ABSTRACT

<p><b>OBJECTIVE</b>To grasp the infection rate and genotypes of Japanese encephalitis virus (JEV) in mosquito in Fujian province.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Genotype I Japanese encephalitis virus is the main genotype in mosquitos in Fujian province.</p>


Subject(s)
Animals , Culicidae , Virology , Encephalitis Virus, Japanese , Classification , Genetics , Genotype , Phylogeny , Time Factors
12.
Article in Chinese | WPRIM | ID: wpr-246174

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>


Subject(s)
Computational Biology , Encephalitis Virus, Japanese , Classification , Genetics , Encephalitis, Japanese , Virology , Enzyme-Linked Immunosorbent Assay , Genome, Viral , Genotype , Japanese Encephalitis Vaccines , Allergy and Immunology , Phylogeny , Sequence Analysis, DNA
13.
Chinese Journal of Virology ; (6): 571-579, 2011.
Article in Chinese | WPRIM | ID: wpr-354789

ABSTRACT

To conduct sequencing of full-length genomes of two Japanese encephalitis virus strains (JEV) newly isolated in 2009 in China and analyze the characteristics of complete nucleotide sequences. The complete genomic sequences were obtained by RT-PCR and sequencing 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 genomes of YN0911 and YN0967 strains were both 10965nt in length, which coded 3432 amino acid polyprotein. The homology of genome ranged from 83.3% to 98.9% in nt and from 94.8% to 99.7% in aa, respectively, when compared with selected JEV strains in GenBank. There were 13 amino acid divergences which were not the key virulence sites in E protein when compared with vaccine strain SA14-14-2. There were 11nt deletions in the 3' UTR region. Phylogenetic analyses based on C/ PrM, E gene and full-length genome all showed that YN0911 and YN0967 strains belonged to genotype I. The result also showed that two new JEVs had close phylogenetic relationship with the strains from Viet Nam, Sichuan Province, Guizhou Province, Guangxi Province, China. This study indicated that JEV strains newly isolated in 2009 in China were the members of JEV genotype I. The key virulence sites in E protein did not change.


Subject(s)
Amino Acid Sequence , Base Sequence , China , Encephalitis Virus, Japanese , Classification , Genetics , Encephalitis, Japanese , Virology , Genome, Viral , Genetics , Humans , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA
14.
Chinese Journal of Virology ; (6): 71-74, 2011.
Article in Chinese | WPRIM | ID: wpr-286076

ABSTRACT

To investigate the infection status and the spatial distribution of Tahyna virus infection among unknown fever cases in Xinjiang, China. Sera samples of unknown fever cases from Kashi in southern Xin-jiang and Yili in northern Xinjiang were tested against Tahyna virus by IFA. Partial positive cases were tested against Tahyna virus/Snowshoe hare virus/Inkoo virus parrelled. Finally, 742 sera samples of unknown fever cases were collected from Kashi, Southern Xinjiang in 2007-2008, the positive rate of IgM antibody against Tahyna virus was 5.3%, the positive rate of IgG antibody against Tahyna virus was 18.3%. 222 sera samples of unknown fever cases were collected from Yili, Northern Xinjiang in 2008, no positive case of IgM antibody against Tahyna was found. 10 cases showed antibody neutralization against Tahyna virus by plaque reduction neutralization test. Our results demonstrate that there is current infection and past infection of Tahyna virus among Southern Xinjiang residents.


Subject(s)
Adolescent , Adult , Antibodies, Viral , Blood , Child , Child, Preschool , China , Epidemiology , Encephalitis Virus, California , Allergy and Immunology , Physiology , Encephalitis, California , Blood , Epidemiology , Virology , Female , Fever , Blood , Epidemiology , Virology , Humans , Immunoglobulin M , Blood , Infant , Male , Middle Aged , Young Adult
15.
Chinese Journal of Virology ; (6): 97-102, 2011.
Article in Chinese | WPRIM | ID: wpr-286070

ABSTRACT

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.


Subject(s)
Animals , Cell Line , China , Encephalitis Virus, California , Genetics , Physiology , Evolution, Molecular , Female , Fluorescent Antibody Technique , Mice , Nervous System , Virology , Phylogeny , Polymerase Chain Reaction , Sequence Analysis, DNA
16.
Article in Chinese | WPRIM | ID: wpr-291518

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Two new JEV isolates were both belonging to genotype I, and the key sites of amino acid were not changed.</p>


Subject(s)
Animals , China , Culicidae , Virology , Encephalitis Virus, Japanese , Classification , Genetics , Genome, Viral , Genotype , Molecular Sequence Data , Phylogeny , RNA, Viral , Sequence Alignment
17.
Chinese Journal of Virology ; (6): 121-127, 2010.
Article in Chinese | WPRIM | ID: wpr-297896

ABSTRACT

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.


Subject(s)
Amino Acid Substitution , Animals , Cell Line , China , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Metabolism , Luciferases , Genetics , Metabolism , Microscopy, Fluorescence , Mutagenesis, Site-Directed , Methods , Mutation , Plasmids , Genetics , Proline , Genetics , Recombinant Fusion Proteins , Genetics , Metabolism , Replicon , Genetics , Sindbis Virus , Genetics , Transfection
18.
Chinese Journal of Virology ; (6): 228-233, 2010.
Article in Chinese | WPRIM | ID: wpr-297879

ABSTRACT

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.


Subject(s)
Alphavirus , Genetics , Allergy and Immunology , Physiology , Amino Acid Sequence , Animals , Cell Line , DNA, Recombinant , Genetics , Female , Genetic Engineering , Glycoproteins , Chemistry , Metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Neutralization Tests , Sindbis Virus , Allergy and Immunology , Viral Load , Viral Proteins , Chemistry , Metabolism
19.
Article in Chinese | WPRIM | ID: wpr-329537

ABSTRACT

Objective To isolate and identify arboviruses from mosquito pools in some regions of Liaoning province.Methods 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.Results 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 BI-IK-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 fi'om Russia,mainland of China and Taiwan region.Conehmion 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.

20.
Article in Chinese | WPRIM | ID: wpr-316055

ABSTRACT

<p><b>OBJECTIVE</b>To investigate arboviruses in Wenshan and Hekou county which are the Sino-Vietnam frontier regions of Wenshan, Yunnan province, China.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>


Subject(s)
Animals , Arboviruses , Classification , Genetics , China , Culicidae , Virology , Densovirus , Genetics , Phylogeny , Sequence Alignment , Sequence Analysis, DNA
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