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1.
Article in English | WPRIM | ID: wpr-927643

ABSTRACT

OBJECTIVE@#To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene.@*METHODS@#A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay.@*RESULTS@#The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses.@*CONCLUSION@#A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.


Subject(s)
African Swine Fever/virology , African Swine Fever Virus/isolation & purification , Animals , Nucleic Acid Amplification Techniques/methods , Recombinases/chemistry , Sensitivity and Specificity , Swine , Viral Proteins/genetics
2.
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
3.
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
4.
Article in English | WPRIM | ID: wpr-690598

ABSTRACT

<p><b>OBJECTIVE</b>In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice.</p><p><b>METHODS</b>A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test.</p><p><b>RESULTS</b>Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1:105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies.</p><p><b>CONCLUSION</b>An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.</p>

5.
Article in English | WPRIM | ID: wpr-311395

ABSTRACT

<p><b>OBJECTIVE</b>To develop a rapid, highly sensitive, and quantitative method for the detection of NT-proBNP levels based on a near-infrared point-of-care diagnostic (POCT) device with wide scope.</p><p><b>METHODS</b>The lateral flow assay (LFA) strip of NT-proBNP was first prepared to achieve rapid detection. Then, the antibody pairs for NT-proBNP were screened and labeled with the near-infrared fluorescent dye Dylight-800. The capture antibody was fixed on a nitrocellulose membrane by a scribing device. Serial dilutions of serum samples were prepared using NT-proBNP-free serum series. The prepared test strips, combined with a near-infrared POCT device, were validated by known concentrations of clinical samples. The POCT device gave the output of the ratio of the intensity of the fluorescence signal of the detection line to that of the quality control line. The relationship between the ratio value and the concentration of the specimen was plotted as a work curve. The results of 62 clinical specimens obtained from our method were compared in parallel with those obtained from the Roche E411 kit.</p><p><b>RESULTS</b>Based on the log-log plot, the new method demonstrated that there was a good linear relationship between the ratio value and NT-proBNP concentrations ranging from 20 pg/mL to 10 ng/mL. The results of the 62 clinical specimens measured by our method showed a good linear correlation with those measured by the Roche E411 kit.</p><p><b>CONCLUSION</b>The new LFA detection method of NT-proBNP levels based on the near-infrared POCT device was rapid and highly sensitive with wide scope and was thus suitable for rapid and early clinical diagnosis of cardiac impairment.</p>


Subject(s)
Antibodies , Biomarkers , Heart Diseases , Diagnosis , Humans , Immunoassay , Methods , Infrared Rays , Natriuretic Peptide, Brain , Blood , Peptide Fragments , Blood , Point-of-Care Testing , Reagent Strips , Sensitivity and Specificity
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.
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.

10.
Article in Chinese | WPRIM | ID: wpr-325576

ABSTRACT

<p><b>OBJECTIVE</b>To sequence and analyze the whole genome of Japanese encephalitis virus (JEV) isolated from mosquitoes in Liaoning province in 2008.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>


Subject(s)
China , Encephalitis Virus, Japanese , Classification , Genetics , Encephalitis, Japanese , Virology , Genome, Viral , Humans , Molecular Sequence Data , Phylogeny , Viral Proteins , Genetics
11.
Article in Chinese | WPRIM | ID: wpr-332414

ABSTRACT

<p><b>OBJECTIVE</b>To identify the virus isolated from a mosquito Culex modestus collected from Tongliao city of Inner Mongolia Autonomous Region.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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%.</p><p><b>CONCLUSION</b>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.</p>


Subject(s)
Animals , Cell Line , China , Coltivirus , Classification , Genetics , Allergy and Immunology , Culicidae , Virology , Insect Vectors , Virology , Mice , Molecular Sequence Data , Phylogeny , Reoviridae Infections , Allergy and Immunology , Virology
12.
Article in Chinese | WPRIM | ID: wpr-254136

ABSTRACT

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


Subject(s)
Animals , Animals, Newborn , Cell Line , China , Culicidae , Virology , Encephalitis Virus, Japanese , Classification , Genetics , Encephalitis, Japanese , Blood , Virology , Genotype , Insect Vectors , Virology , Mice , Phylogeny , RNA, Viral , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
13.
Article in Chinese | WPRIM | ID: wpr-254132

ABSTRACT

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


Subject(s)
Adolescent , Adult , Aged , Antibodies, Viral , Blood , Cerebrospinal Fluid , Child , Child, Preschool , China , Encephalitis Virus, Japanese , Allergy and Immunology , Encephalitis, Viral , Blood , Cerebrospinal Fluid , Virology , Enterovirus , Genetics , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin M , Blood , Cerebrospinal Fluid , Male , Meningitis, Viral , Blood , Cerebrospinal Fluid , Virology , Middle Aged , Mumps virus , Allergy and Immunology , RNA, Viral , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Simplexvirus , Allergy and Immunology , Young Adult
14.
Article in Chinese | WPRIM | ID: wpr-248809

ABSTRACT

<p><b>OBJECTIVE</b>To establish a convenient method for diagnosis of lethal avian influenza virus infection.</p><p><b>METHODS</b>Ninety-six-well microtiter plates were coated with 100 microl of H5N1 protein (0.1 microg/ml) diluted in coating buffer. These plates were used for enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Four of the 23 suspected borderline cases of lethal avian influenza virus infection were positive by the ELISA, and all the healthy controls (n=234) were negative. These results were confirmed by hemagglutination-inhibition (HI) and neutralization tests, and results were totally consistent (100 percent) with each other among the three methods.</p><p><b>CONCLUSION</b>The ELISA may be used in the screening for lethal avian influenza virus infection.</p>


Subject(s)
Antibodies, Viral , Blood , Enzyme-Linked Immunosorbent Assay , Methods , Humans , Influenza A Virus, H5N1 Subtype , Allergy and Immunology , Influenza, Human , Diagnosis , Allergy and Immunology
15.
Article in Chinese | WPRIM | ID: wpr-305547

ABSTRACT

<p><b>BACKGROUND</b>To develop a rapid and more sensitive TaqMan RT-PCR assay specific for Banna virus.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>


Subject(s)
Animals , Coltivirus , Genetics , Culicidae , Virology , Humans , RNA, Viral , Genetics , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Methods , Taq Polymerase , Metabolism
16.
Article in Chinese | WPRIM | ID: wpr-333012

ABSTRACT

<p><b>BACKGROUND</b>To study the molecular characteristics of YN92-4 strain isolated from mosquitoes in Yunnan Province and define its classification.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The YN92-4 strain belongs to Batai virus, this is the first report of molecular biological identification of Batai virus in China.</p>


Subject(s)
Amino Acid Sequence , Animals , Bunyamwera virus , Classification , Genetics , China , Culicidae , Virology , DNA, Complementary , Chemistry , Genetics , Molecular Sequence Data , Phylogeny , RNA, Viral , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Homology, Amino Acid
17.
Article in Chinese | WPRIM | ID: wpr-333008

ABSTRACT

<p><b>BACKGROUND</b>To develop a rapid, specific and sensitive method for detecting Sindbis virus (SINV) with SYBR GREEN I real time PCR.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>


Subject(s)
Alphavirus Infections , Diagnosis , Virology , DNA, Complementary , Chemistry , Genetics , Organic Chemicals , Chemistry , RNA, Viral , Genetics , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Methods , Sensitivity and Specificity , Sindbis Virus , Genetics
18.
Article in Chinese | WPRIM | ID: wpr-281793

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the epidemic state of arboviruses in the downstream area of Lancang river in Yunnan province.</p><p><b>METHODS</b>Mosquitoes were collected from Lancang river downstream area (including Lancang county and Simao city) during summer in 1998 and stored in liquid nitrogen after classification. The mosquito pools were homogenized and centrifuged, then the supernatant was inoculated into C6/36 cells for virus isolation. New isolates were identified by neutralization test(NT), ELISA, immunofluorescence assay(IFA) and polyacrylamid gel electrophoresis(PAGE).</p><p><b>RESULTS</b>Totally 22 isolates of arbovirus were obtained from 233 mosquito pools by inoculation of C6/36 cells and positive rate of the isolation was 9.4%. Ten strains were resistant to both ether and 5 prime-IDU. So they were non-enveloped double-stranded (ds) RNA virus. Twelve segmented RNAs were shown by PAGE and PAGE profiles from the ten strains were 6-6 with minor variation. The isolates can be neutralized by immunized mouse ascites fluid of BJ95-75 strains of coltivirus by NT, and reacted with monoclonal antibody against BJ95-75 by ELISA. These viruses were identified as coltivirus. Nine isolates were sensitive to ether and resistant to 5 prime-IDU. So they were non-enveloped RNA viruses. PAGE showed 10 segmented RNA, and they were identified to be orbiviruses. Three isolations were sensitive to ether. One of them can be neutralized with JEV A2 strain antibody by NT and was positive to the homologous antibody by IFA. It was identified being strain of JE virus. One strain(YN92-4) can be reacted with anti-bunyavirus group specific immune ascites fluid by both IFA and ElISA, but reacted neither with anti-alpha virus group, nor with anti-flavivirus group JE virus ascites fluid. The virions are spherical and about 87 nm in diameter with surface projections by negative staining. Conclusion Twenty-two isolates have been obtained from wild caught-mosquitoes of Lancang river down-stream area in Yunnan province. Among them ten, nine, one and one were identified as coltivirus, orbivirus, JE virus and bunyavirus, respectively. One is under identification. This is the first report on bunyavirus isolated from mosquitoes in China.</p>


Subject(s)
Animals , Arboviruses , Classification , China , Coltivirus , Culicidae , Virology , Encephalitis Virus, Japanese , Insect Vectors , Virology , Orbivirus , Orthobunyavirus
19.
Article in Chinese | WPRIM | ID: wpr-281786

ABSTRACT

<p><b>OBJECTIVE</b>To classify the Chinese isolates of Coltiviruses.</p><p><b>METHODS</b>Three sets of primers were selected among them two were specific to the 9th and 12th segments of subgroup B2, and one was for the 12th segment of subgroup B1-All the Chinese isolates of Coltivirus selected in the experiment were classified according to the lengths of different amplicons of the reverse transcriptase-polymerase Chain reaction (RT-PCR). The homogenicity of the nucleic acids of the isolates BJ95-75 and YN-6 was also compared with other Coltivirus strains belonging to subgroup B2.</p><p><b>RESULTS</b>With the primers 12-854-S/12-B2-R, which were specific to the 12th segment of Coltivirus subgroup B2-850 bp amplicons were obtained from Beijing isolate BJ95-75 and all the Yunnan isolates such as YN-6, -67-1, -68-1, -69, -70-1, -70-2, -90, -92-2, -93 of Coltivirus 492 bp DNA fragments were also amplified from all of them with the segment 9th specific primers 9-JKT-S/9-JKT-R. However no positive results were obtained from Northeast isolates NE97-12, NE97-31 and control viruses YN-99(Orbivirus),YN-151-1(JEV) with the same two sets of primers. With 12-B1-S/12-B1R primers specific to the 12th segment of subgroup B1, no amplicons of right length were obtained from any of the Chinese isolates of Coltivirus and the control viruses. When compared the nucleic acid sequences of BJ95-75 and YN-6 with other Coltivirus strains such as Bannavirus, JKT6423, JKT6969, JKT7043, the amplicons from segment 12th of these two strains had more than 89.4% homology with the other strains, especially to the earlier Chinese isolate Bannavirus, the homolog was more then 98.9%. Nearly 96.5% and 99.2% of the nucleic acids of the amplicons from segment 9th of the two strains were being homologous to Bannavirus and about 84.0% to JKT6423, which had been classified into type B2a. But the maximal homogenicity was about 53% when compared with the other two coltivirus strains. JKT6969 and JKT7043 which had been classified into type B2b.</p><p><b>CONCLUSION</b>Genotyping the recent Chinese isolates of coltivirus for the first time in our country. Most of the Chinese isolates belong to subgroup B2, more exactly type B2a. The Northeast isolates NE97-12 and NE97-31 were not correctly grouped with the available primers.</p>


Subject(s)
Animals , Base Sequence , China , Coltivirus , Classification , Genetics , Culicidae , Virology , Genotype , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid
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