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1.
Chinese Medical Journal ; (24): 1289-1298, 2021.
Article in English | WPRIM | ID: wpr-878153

ABSTRACT

BACKGROUND@#The significant morbidity and mortality resulted from the infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for urgent development of effective and safe vaccines. We report the immunogenicity and safety of an inactivated SARS-CoV-2 vaccine, KCONVAC, in healthy adults.@*METHODS@#Phase 1 and phase 2 randomized, double-blind, and placebo-controlled trials of KCONVAC were conducted in healthy Chinese adults aged 18 to 59 years. The participants in the phase 1 trial were randomized to receive two doses, one each on Days 0 and 14, of either KCONVAC (5 or 10 μg/dose) or placebo. The participants in the phase 2 trial were randomized to receive either KCONVAC (at 5 or 10 μg/dose) or placebo on Days 0 and 14 (0/14 regimen) or Days 0 and 28 (0/28 regimen). In the phase 1 trial, the primary safety endpoint was the proportion of participants experiencing adverse reactions/events within 28 days following the administration of each dose. In the phase 2 trial, the primary immunogenicity endpoints were neutralization antibody seroconversion and titer and anti-receptor-binding domain immunoglobulin G seroconversion at 28 days after the second dose.@*RESULTS@#In the phase 1 trial, 60 participants were enrolled and received at least one dose of 5-μg vaccine (n = 24), 10-μg vaccine (n = 24), or placebo (n = 12). In the phase 2 trial, 500 participants were enrolled and received at least one dose of 5-μg vaccine (n = 100 for 0/14 or 0/28 regimens), 10-μg vaccine (n = 100 for each regimen), or placebo (n = 50 for each regimen). In the phase 1 trial, 13 (54%), 11 (46%), and seven (7/12) participants reported at least one adverse event (AE) after receiving 5-, 10-μg vaccine, or placebo, respectively. In the phase 2 trial, 16 (16%), 19 (19%), and nine (18%) 0/14-regimen participants reported at least one AE after receiving 5-, 10-μg vaccine, or placebo, respectively. Similar AE incidences were observed in the three 0/28-regimen treatment groups. No AEs with an intensity of grade 3+ were reported, expect for one vaccine-unrelated serious AE (foot fracture) reported in the phase 1 trial. KCONVAC induced significant antibody responses; 0/28 regimen showed a higher immune responses than that did 0/14 regimen after receiving two vaccine doses.@*CONCLUSIONS@#Both doses of KCONVAC are well tolerated and able to induce robust immune responses in healthy adults. These results support testing 5-μg vaccine in the 0/28 regimen in an upcoming phase 3 efficacy trial.@*TRIAL REGISTRATION@#http://www.chictr.org.cn/index.aspx (No. ChiCTR2000038804, http://www.chictr.org.cn/showproj.aspx?proj=62350; No. ChiCTR2000039462, http://www.chictr.org.cn/showproj.aspx?proj=63353).


Subject(s)
Adult , COVID-19 , COVID-19 Vaccines , Double-Blind Method , Humans , SARS-CoV-2 , Vaccines, Inactivated/adverse effects
2.
Chinese Medical Journal ; (24): 2048-2053, 2021.
Article in English | WPRIM | ID: wpr-887657

ABSTRACT

BACKGROUND@#With the ongoing worldwide coronavirus disease 2019 (COVID-19) pandemic, an increasing number of viral variants are being identified, which poses a challenge for nucleic acid-based diagnostic tests. Rapid tests, such as real-time reverse transcription-polymerase chain reaction (rRT-PCR), play an important role in monitoring COVID-19 infection and controlling its spread. However, the changes in the genotypes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may result in decreased sensitivity of the rRT-PCR assay and it is necessary to monitor the mutations in primers and probes of SARS-CoV-2 detection over time.@*METHODS@#We developed two rRT-PCR assays to detect the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS-CoV-2. We evaluated these assays together with our previously published assays targeting the ORF1ab and N genes for the detection and confirmation of SARS-CoV-2 and its variants of concern (VOCs). In addition, we also developed two rRT-PCR assays (S484K and S501Y) targeting the spike gene, which when combined with the open reading frames (ORF)1ab assay, respectively, to form duplex rRT-PCR assays, were able to detect SARS-CoV-2 VOCs (lineages B.1.351 and B.1.1.7).@*RESULTS@#Using a SARS-CoV-2 stock with predetermined genomic copies as a standard, the detection limit of both assays targeting RdRp and N was five copies/reaction. Furthermore, no cross-reactions with six others human CoVs (229E, OC43, NL63, HKU1, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus) were observed using these assays. In addition, the S484K and S501Y assays were combined with the ORF1ab assay, respectively.@*CONCLUSIONS@#Four rRT-PCR assays (RdRp, N, S484K, and S501Y) were used to detect SARS-CoV-2 variants, and these assays were shown to be effective in screening for multiple virus strains.


Subject(s)
COVID-19 , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2 , Sensitivity and Specificity
3.
Article in English | WPRIM | ID: wpr-773386

ABSTRACT

OBJECTIVE@#This study was conducted to investigate the viral and bacterial etiology and epidemiology of patients with acute febrile respiratory syndrome (AFRS) in Qinghai using a commercial routine multiplex-ligation-nucleic acid amplification test (NAT)-based assay.@*METHODS@#A total of 445 nasopharyngeal swabs specimens from patients with AFRS were analyzed using the RespiFinderSmart22kit (PathoFinder BV, Netherlands) and the LightCycler 480 real-time PCR system.@*RESULTS@#Among the 225 (225/445, 51%) positive specimens, 329 positive pathogens were detected, including 298 (90.58%) viruses and 31 (9%) bacteria. The most commonly detected pathogens were influenza virus (IFV; 37.39%; 123/329), adenovirus (AdV; 17.02%; 56/329), human coronaviruses (HCoVs; 10.94%; 36/329), rhinovirus/enterovirus (RV/EV; 10.03%; 33/329), parainfluenza viruses (PIVs; 8.51%; 28/329), and Mycoplasma pneumoniae (M. pneu; 8.51%; 28/329), respectively. Among the co-infected cases (17.53%; 78/445), IFV/AdV and IFV/M. pneu were the most common co-infections. Most of the respiratory viruses were detected in summer and fall.@*CONCLUSION@#In our study, IFV-A was the most common respiratory pathogen among 22 detected pathogens, followed by AdV, HCoV, RV/EV, PIV, and M. pneu. Bacteria appeared less frequently than viruses, and co-infection was the most common phenomenon among viral pathogens. Pathogens were distributed among different age groups and respiratory viruses were generally active in July, September, and November. Enhanced surveillance and early detection can be useful in the diagnosis, treatment, and prevention of AFRS, as well as for guiding the development of appropriate public health strategies.


Subject(s)
Adolescent , Adult , Age Factors , Aged , Child , Child, Preschool , China , Epidemiology , Female , Humans , Infant , Male , Middle Aged , Nasopharynx , Virology , Seasons , Sentinel Surveillance , Severe Acute Respiratory Syndrome , Epidemiology , Virology , Young Adult
4.
Article in English | WPRIM | ID: wpr-258831

ABSTRACT

The first imported Middle East respiratory syndrome (MERS) case in China was identified in May 2015. We determined the kinetics of antibody (IgG and IgM) and neutralizing antibodies against MERS-coronavirus (MERS-CoV) in this case before discharge. Moreover, no seroconversion was found among 53 close contacts by anti-MERS IgG antibody enzyme-linked immunosorbent assay (ELISA) of paired serum samples. These findings suggest that neither community nor nosocomial transmission of MERS-CoV occurred in China.


Subject(s)
Antibodies, Neutralizing , Blood , Antibodies, Viral , Blood , China , Epidemiology , Contact Tracing , Coronavirus Infections , Blood , Epidemiology , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G , Blood , Immunoglobulin M , Blood , Male , Republic of Korea , Epidemiology , Travel
5.
Chinese Journal of Virology ; (6): 554-560, 2014.
Article in Chinese | WPRIM | ID: wpr-280328

ABSTRACT

To compare different adjuvant formulation and explore the impact of Calcineurin B subunit(CnB) as adjuvant with a novel HBV protein particle (HBSS1) vaccine in mice, female C57BL/6 mice were immunized HBSS1 with Al(OH)3 only, or a normal dose (5 μg) CnB only, or (CnB+ Al(OH)3) mixture as the adjuvant. All immunized groups were primed twice at 4-week intervals; followed by boosting with recombinant adenoviral based HBV vaccine(rAdSS1) at 10-week intervals. We detected the antigen specific humoral response in mice, including total IgG antibody and IgG subtyping. Then, we characterized the specific cell-mediated immune (CMI) response by detection of γ-interferon secreting splenocytes after stimulaton with S or PreS1 peptide pools. No enhancement of immunity was found among the mice with 5 μg of CnB alone or combined with Al(OH), adjuvanted vaccine,which could not induce higher level of anti-PreS1 and anti-S antibodies and CMI than that of HBSS1 alone or Al(OH)3 adjuvanted vaccines. We concluded that CnB is not an effective adjuvant for a novel HBV subunit vaccine.


Subject(s)
Adjuvants, Immunologic , Pharmacology , Animals , Calcineurin , Pharmacology , Female , Hepatitis B Vaccines , Allergy and Immunology , Mice , Mice, Inbred C57BL , Protein Subunits
6.
Article in English | WPRIM | ID: wpr-270541

ABSTRACT

<p><b>OBJECTIVE</b>This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella, and Shigella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2).</p><p><b>METHODS</b>A two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated.</p><p><b>RESULTS</b>The detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mL for S. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL for Salmonella, 2.5×102 CFU/mL for Shigella, 2.1×102 CFU/mL for V. parahaemolyticus, and 1.2×102 CFU/mL for E. coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL.</p><p><b>CONCLUSION</b>A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).</p>


Subject(s)
Animals , Bacteria , Genetics , Food Microbiology , Methods , Milk , Microbiology , Multiplex Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity
7.
Chinese Journal of Virology ; (6): 325-332, 2014.
Article in Chinese | WPRIM | ID: wpr-356596

ABSTRACT

In addition to the structural genes of the coronavirus genome, S, E, M, and N, there are several additional genes called "group-specific or accessory genes". Their gene products are designated as "accessory proteins", as reports to date make it clear that these proteins are not essential for virus replication in vitro. Nevertheless, many of these genes are still maintained in the virus genome under selective pressure, suggesting that they might play a very important role in the survival of the virus in the natural environment of the infected host. This review will summarize the research progress in the functions of coronavirus accessory genes.


Subject(s)
Animals , Coronavirus , Genetics , Metabolism , Coronavirus Infections , Virology , Humans , Open Reading Frames , Viral Proteins , Metabolism
8.
Article in Chinese | WPRIM | ID: wpr-318091

ABSTRACT

<p><b>OBJECTIVE</b>To express HPV31 and 52 L2 fusion protein and detect its immunogenicity.</p><p><b>METHODS</b>According to the amino acid sequences of HPV31 and 52 L2 11-200AA published in the GenBank database, weartificially synthesized the HPV31 and 52 L2 fusion gene which was optimized according to Escherichia coli codon usage and encodes 11-200 amino acid of HPV31 and HPV52 L2, then cloned it into pET-9a vector. The HPV31 and 52 L2 fusion protein was expressed in Prokaryotic expression system and the mice were immunized with the fusion protein after purification. The immunogenicity was characterized in vaccinated mice.</p><p><b>RESULTS</b>HPV31 and 52 L2 fusion protein was highly expressed in E. coli, the amount of fusion protein is nearly 20% of the total bacterial protein. The purified fusion protein with aluminum adjuvant could induce specific high titer of IgG antibodies detected by ELISA, and also induce the neutralizing antibodies against pseudovirus of HPV31 and HPV52 and cross-neutralizing antibodies against pseudovirus of HPV45, 58, 16, 18.</p><p><b>CONCLUSION</b>HPV31 and 52 L2 fusion protein could induce neutralizing and cross-neutralizing antibodies against HPV pseudovirus. It provides laboratory basis for development of HPV L2 protein vaccine.</p>


Subject(s)
Animals , Antibodies, Viral , Blood , Escherichia coli , Genetics , Female , Mice , Mice, Inbred BALB C , Oncogene Proteins, Viral , Genetics , Allergy and Immunology , Papillomaviridae , Allergy and Immunology , Papillomavirus Vaccines , Allergy and Immunology , Recombinant Fusion Proteins , Allergy and Immunology
9.
Article in Chinese | WPRIM | ID: wpr-318082

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the infection of human parvovirus B19, human bocavirus (HBoV) and human parvovirus 4 (PARV4) in blood samples among patients with liver disease in Nanjing by molecular detection.</p><p><b>METHODS</b>Nested PCR assays were designed and validated to detect B19, HBoV and PARV4, respectively. The assays were used to screen three parvoviruses in blood samples from 95 patients with different liver disease in Nanjing. The parvovirus infection was analyzed statistically.</p><p><b>RESULTS</b>The detection limits were 10 copies of genomic DNA equivalents per reaction for each assays and the good specificity were observed. The frequency of B19 and HBoV were 2/95 (2.1%) and 9/95 (9.5%) in blood samples respectively. No PARV4 was detected. HBoV was detected in 3/5 patients with drug-induced hepatitis.</p><p><b>CONCLUSION</b>Both B19 and HBoV infection were detected in blood from patients with liver disease.</p>


Subject(s)
Adolescent , Adult , Aged , Child , Coinfection , Virology , Female , Human bocavirus , Humans , Liver Diseases , Virology , Male , Middle Aged , Parvovirus , Parvovirus B19, Human , Polymerase Chain Reaction , Viremia , Virology
10.
Chinese Journal of Virology ; (6): 65-70, 2013.
Article in Chinese | WPRIM | ID: wpr-339973

ABSTRACT

Coronaviruses are a large family of viruses which include viruses that cause the common cold and severe acute respiratory syndrome (SARS) in humans and other diseases in animals. There are considerable genetic diversities within coronaviruses due to their wide rang hosts and their special gene replication and transcription mechanisms. During this process, gene recombinations often occur, resulting in novel subtype or coronavirus emerge constantly. Of note are SARS-like-CoVs and novel HCoV-EMC identified in 2012. This minireview summarized major advances of recently identified coronaviruses, focusing on the genome structures and interspecies jumping mechanism of coronavirus.


Subject(s)
Animals , Coronavirus , Classification , Genetics , Coronavirus Infections , Humans , Phylogeny
11.
Chinese Journal of Virology ; (6): 106-111, 2013.
Article in Chinese | WPRIM | ID: wpr-339967

ABSTRACT

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.


Subject(s)
Animals , Coronavirus Infections , Metabolism , Virology , Coronavirus NL63, Human , Chemistry , Genetics , Metabolism , Escherichia coli , Genetics , Metabolism , Gene Expression , Humans , Mice , Mice, Inbred BALB C , Protein Engineering , Protein Structure, Tertiary , Receptors, Virus , Metabolism , Viral Envelope Proteins , Chemistry , Genetics , Metabolism
12.
Chinese Journal of Virology ; (6): 137-142, 2013.
Article in Chinese | WPRIM | ID: wpr-339962

ABSTRACT

Resequencing Pathogen Microarray (RPM) is a new pathogen detection and identification technology based on DNA microarray. In order to apply RPM in the detection of unexplained infection and as a result, to improve the emergency response capacity, a new RPM-based respiratory pathogens detection assay was developed to simultaneously detect 19 common respiratory viruses, 9 influenza A viruses (Flu A),11 human rhinoviruses(HRV), 28 enteroviruses and 18 rare respiratory viruses. The specificity of multiplex system was examined by confirmed positive specimens for 16 common respiratory virus. The sensi-tivity was evaluated by serial ten-fold dilutions of plasmids or in vitro-transcribed RNA. RPM could detect and differentiate 16 virus types/subtypes at 10 - 1 000 copies/reaction level. Nucleic acids of 8 throat swabs with unexplained respiratory tract infections were pooled and detected by the new assay. The RPM result was verified by common PCR followed by sequencing as well as PLEX-ID (Abbott). Except for a false-positive of PIV1, no difference among the three assays was found. These results indicate the assay based on the new RPM is a highly sensitive, high throughput test for the detection of respiratory virus infections, which is significant for the management of emergent and epidemic infectious disease.


Subject(s)
Child , Child, Preschool , Female , Humans , Male , Oligonucleotide Array Sequence Analysis , Methods , Respiratory Tract Infections , Diagnosis , Virology , Sensitivity and Specificity , Viruses , Classification , Genetics
13.
Chinese Journal of Oncology ; (12): 810-815, 2012.
Article in Chinese | WPRIM | ID: wpr-307288

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system, and evaluate its immunogenicity and antitumor efficacy in vaccinated mice.</p><p><b>METHODS</b>The HPV16L2N120E7E6 fusion gene, its codons were optimized to increase the expression of the protein, was constructed by overlap extension PCR and inserted into prokaryotic expression vector pET9a. Then the fusion protein was expressed by inducing with IPTG in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and further detected by SDS-PAGE and Western-blot. Finally, the humoral and cellular immune responses were measured by ELISA and ELISPOT, respectively, in vaccinated mice with the purified HPV16L2N120E7E6 fusion protein, and the antitumor efficacy was assessed in mice using the TC-1 tumor challenge model.</p><p><b>RESULTS</b>The codon-optimized HPV16L2N120E7E6 fusion gene was highly expressed in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and the amount of fusion protein was nearly 48.6% of the total bacterial protein. The purified fusion protein could induce high titer of specific antibody against L2, E7 and E6 in vaccinated mice. When accompanied with the adjuvant CpG, the fusion protein was able to elicit strong and moderate cellular immune responses in vaccinated mice against peptide HPV16E7(49-57) and peptide pools of HPV16E6, respectively. Furthermore, the tumor therapeutic experiment showed that HPV16L2N120E7E6 + CpG could prevent the tumor formation in 80.0% (8/10) vaccinated mice.</p><p><b>CONCLUSIONS</b>The data of this study suggest that HPV16L2N120E7E6 fusion protein could be a promising candidate vaccine for treatment of chronic HPV16 infection and post-operative adjuvant therapy for cervical cancer.</p>


Subject(s)
Adjuvants, Immunologic , Pharmacology , Animals , Cancer Vaccines , Allergy and Immunology , Therapeutic Uses , Capsid Proteins , Genetics , Allergy and Immunology , Metabolism , Cell Line, Tumor , Cell Proliferation , Codon , Escherichia coli , Allergy and Immunology , Metabolism , Female , Humans , Immunization , Methods , Immunotherapy , Methods , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Oligodeoxyribonucleotides , Allergy and Immunology , Oncogene Proteins, Viral , Genetics , Allergy and Immunology , Metabolism , Papillomavirus E7 Proteins , Genetics , Allergy and Immunology , Metabolism , Papillomavirus Vaccines , Allergy and Immunology , Therapeutic Uses , Plasmids , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Metabolism , Repressor Proteins , Genetics , Allergy and Immunology , Metabolism
14.
Article in Chinese | WPRIM | ID: wpr-305076

ABSTRACT

<p><b>OBJECTIVE</b>To construct the recombinant virus-like particles containing HCV envelope glycoprotein E1E2 based on sindbis virus vector.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>


Subject(s)
Animals , Cells, Cultured , Cricetinae , Genetic Vectors , Hepacivirus , Genetics , Plasmids , Recombination, Genetic , Sindbis Virus , Genetics , Viral Envelope Proteins , Genetics
15.
Article in Chinese | WPRIM | ID: wpr-305026

ABSTRACT

<p><b>OBJECTIVE</b>To develop and optimize the molecular detection assays for recently identified human coronavirus (HCoV) infection.</p><p><b>METHODS</b>Based on the 208 base pair(bp) sequence of novel HCoV reported by HPA of UK, we designed and obtained several pairs of primer (F-1, R-1; F-2, R-2) and Taqman probes (TZ1,TZ2) for detection of novel HCoV. Two of probes were modified with LNA (LNA-TZ1, LNA-TZ2). Then, RT-PCR and various real time RT-PCR assays were developed and optimized in this study. We also compared our assays with the real time RT-PCR assays reported recently by Europe team based on upE or ORF1b target.</p><p><b>RESULTS</b>The RT-PCR or real time RT-PCR assays for novel HCoV were developed without cross-reactivity with other HCoV and several common respiratory viruses using clinical specimen panel. The analytical sensitivity of assays were less than 50-500 copies per reaction and the detection was improved when Taqman probe modified with LNA-tagged, compared to no LNA-tagged in real time RT-PCR assays. The upE and LNA-TZ1 based assays were better than others.</p><p><b>CONCLUSION</b>The molecular detection sensitivity and specificity of TaqMan-based real time PCR assay could be improved when probe tagged with LNA. The upE or LNA-TZ1 based real time RT-PCR assay was recommend for detection of novel HCoV. This study laid a foundation for improving the performance of novel HCoV detection.</p>


Subject(s)
Coronavirus , Classification , Genetics , DNA Primers , Genetics , Humans , Oligonucleotides , Genetics , RNA, Viral , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Methods , Sensitivity and Specificity
16.
Article in Chinese | WPRIM | ID: wpr-246187

ABSTRACT

<p><b>OBJECTIVE</b>To determine the antigen characteristics of different fragments of SARS-CoV N protein expressed in E. Coli and their application in the serological diagnosis.</p><p><b>METHODS</b>Based on preliminary analysis of 39 different segments of the N protein, We choosed six purified N protein for further antigenicity characterization in this study, including that PN360 (1 -360aa), PN301 (1-301aa), PN199 (30-228aa), PN185 (30-214aa), PN155b (60-214aa), and PN125 (90-214aa). We developed Western-Bolt and ELISA to detect antibody reactivity between truncated N fragments with sera from SARS-CoV-negative normal adults or SARS-CoV patient convalescent sera.</p><p><b>RESULTS</b>Western-Bolt results show that all the six fragments have reacted with the SARS patient convalescent sera, but the PN360 and PN301 showed obvious cross-reaction with sera from SARS-CoV-negative normal adults; sensitivity analysis using an ELISA coating with PN199, PN185, PN155b, PN125 as antigen showed that the PN185 and PN155b are better than PN125.</p><p><b>CONCLUSION</b>Truncated N protein PN185 and PN155b expressed in E. Coli are better antigen candidates used for detection of SARS-CoV specific antibody.</p>


Subject(s)
Antibodies, Viral , Blood , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Genetics , Humans , Nucleocapsid Proteins , Allergy and Immunology , Peptide Fragments , Allergy and Immunology , Recombinant Proteins , Allergy and Immunology , Serologic Tests , Severe Acute Respiratory Syndrome , Diagnosis
17.
Article in Chinese | WPRIM | ID: wpr-246170

ABSTRACT

<p><b>OBJECTIVE</b>To character HBV antigen expression in vitro and in vivo transduced by different transgenic plasmids carrying infectious genome of hepatitis B virus (HBV).</p><p><b>METHODS</b>We constructed four different lentiviral transfer plasmids (carrying 1.3 full-length genome of HBV, by replacing the EGFP express box in pCS-CG plasmid with HBV genome and with different structural element, named as pCS-HBV1.3 (pCS-HBV1.3 X, pCS-HBV1.3 P, pCS-HBV1.3 N and pCS-HBV1.3 K). We detected the expression of HBsAg and HBeAg by ELISA in different time after transfected Huh 7 cells or hydrodynamic injection into C57 BL/6 mice with transfer plasmids pCS-HBV, respectively.</p><p><b>RESULTS</b>We detected significant expression of HBsAg over 5 days after transfected Huh 7 cells (in vitro) or hydrodynamic injection into C57 BL/6 mice (in vivo) with transfer plasmids pCS-HBV1.3 X, pCS-HBV1.3 P and pCS-HBV1.3 K. The expression level and dynamics of HBsAg and HBeAg in the sera of mice is consistent with that of in the supernatant of Huh-7 cell. Furthermore, the expression of HBV antigens were modulated by the direction and position of HBV insert, also by some lentiviral vector cis-elements (cPPT and RRE).</p><p><b>CONCLUSION</b>The optimal lentiviral transfer plasmids (pCS-HBV1.3 X, pCS-HBV1.3 P and pCS-HBV1.3 K) could be further used for establishment and application of HBV transgenic cell or animal model.</p>


Subject(s)
Animals , Disease Models, Animal , Female , Genome, Viral , Hepatitis B Antigens , Genetics , Lentivirus , Genetics , Mice , Mice, Inbred C57BL , Plasmids , Transduction, Genetic , Methods
18.
Chinese Journal of Virology ; (6): 297-302, 2012.
Article in Chinese | WPRIM | ID: wpr-354732

ABSTRACT

Due to the progress of targeted recombination and reverse genetics technique, it is possible to express foreign genes using coronaviruses as a vector via its unique transcription mechanism. Two types of coronavirus-based expression vectors have been developed; one is helper-dependent expression system and the other a single genome-based expression system. By modification of the infectious cDNA of the genome of coronaviruse, it has led to an efficient (> 50 microg/10(6)cells) and stable (> 30 passages) expression of the foreign gene. Moreover,several other features of coronavirus make it attractive as a vector. Firstly, the virulent coronaviruse can be converted into nonvirulent viruse by deleting the nonessential, group-specific genes. Secondly, the species and tissue tropism of coronaviruses can be manipulated by modification of their attachment and fusion protein S. Thus, coronavirus-based vector is becoming a promising and useful tool for vaccine development and gene delivery.


Subject(s)
Animals , Coronavirus , Genetics , Physiology , Gene Expression , Gene Transfer Techniques , Genetic Vectors , Genetics , Physiology , Humans , Virus Replication
19.
Chinese Journal of Virology ; (6): 336-344, 2012.
Article in Chinese | WPRIM | ID: wpr-354726

ABSTRACT

This paper investigated the envelope protein E1/E2 quasispecies genetic characterization of 4 HCV positive sera (Genotype 1b: 274, 366, 383; Genotype 2a: 283) in China. Nucleotide acid was extracted and glycoprotein E1/E2 (191-764aa) coding genes were obtained by RT-PCR, positive clones were randomly selected for sequencing. The phylogenetic relationships and the homology of nucleotide and amino acid were analyzed based on E1/E2 coding genes, and some vital functional regions of E1/E2 were characterized. A total of 43 sequences (274: 10; 283: 12; 366: 13; 383: 8) were obtained showing high genetic heterogeneity in HVR1 and HVR2 regions, while sequences of the neutralizing epitopes, transmembrane domain I, II and N-terminal ectodomain were comparatively conservative. Single base (C) insertion mutation at nt1279 ( E1 region, aa313), resulting in a mutated E1 coding protein (beginning at aa 313) and interruption at N terminus (aa 398) of HVR1 region of E2, was dominant quasispecies sequence(11/12) found in serum 283 . This is the first report on E1/E2 quasispecies in Chinese HCV patients and this novel pattern of insertion mutation provides important information for further study on HCV pathogenesis and immune evasion.


Subject(s)
Amino Acid Sequence , Base Sequence , DNA Mutational Analysis , Hepacivirus , Genetics , Virulence , Hepatitis C , Virology , Humans , Molecular Sequence Data , Mutagenesis, Insertional , Viral Envelope Proteins , Chemistry , Genetics
20.
Chinese Journal of Virology ; (6): 34-43, 2011.
Article in Chinese | WPRIM | ID: wpr-286081

ABSTRACT

The purpose is to screen and identify the specific H-2d restricted T-cell epitopes. These epitopes are used to investigate the cellular immune response of BALB/c (H-2d) mice immunized with a HIV-1 vaccine which expresses six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C. A replicating DNA vaccine and a non-replicating recombinant vaccinia virus vector, both expressing the six antigens mentioned above, were used to immune BALB/c (H-2d) mice in a prime-boost regiment. The six peptide libraries of HIV B'/C corresponding respectively to the six complete antigens were pooled according to a designed matrix format and used to test for IFN-gamma production from splenocytes of immunized mice by an enzyme-linked immunospot (IFN-gamma ELISPOT) assay. The ELISPOT data indicated that two of seven Gag-specific T-cell epitope peptides were identified to be the novel epitopes. One of three Pol-specific T-cell epitope is unreported. One novel epitope was confirmed in two gp160-specific T-cell epitope peptides. One Nef-specific T-cell epitope was identified. Three Tat-specific T-cell epitope peptides were continuous sequences in Tat peptide library and all contained either complete or partial sequence reported. Rev-specific T-cell epitope was not be found. The specific T-cell epitopes (H-2d restricted) were identified by IFN-7 ELISPOT assay, which could be used to detect the cellular immune response of BALB/c mice immunized with the HIV-1 vaccine expressing six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C.


Subject(s)
Amino Acid Sequence , Animals , Enzyme-Linked Immunospot Assay , Methods , Epitopes, T-Lymphocyte , Chemistry , Genetics , Allergy and Immunology , Female , H-2 Antigens , Chemistry , Genetics , Allergy and Immunology , HIV Antigens , Chemistry , Genetics , Allergy and Immunology , HIV Infections , Allergy and Immunology , Virology , HIV-1 , Classification , Genetics , Allergy and Immunology , Histocompatibility Antigen H-2D , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptide Mapping , Methods
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