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Objective@#To establish a method for the simultaneous identification of Zika, Chikungunya and Mayaro viruses.@*Methods@#The complete genome sequences of Zika, Chikungunya and Mayaro virus were retrieved from Global Shared Database for comparative analysis, estimate its conservative region and determine the target gene location, specific primers and probes were designed, then a triplex real-time RT-PCR assay was developed. The specificity, sensitivity and repeatability of the assay were assessed by viral nucleic acid of Zika virus, Chikungunya virus a, in vitro transcriptional RNA of Mayaro virus, normal human serum and related virus simulation sample.@*Results@#The result showed that the established method could detect Zika virus, Chikungunya virus, as well as simulated Mayaro virus samples, the limit of detection (LOD) of Zika and Chikungunya virus was 16.22 Copy/PCR and 12.02 Copy/PCR, respectively, the LOD for simulated Mayaro virus RNA was 2.82 Copy/PCR, no significant difference was detected between the triplex and monoplex assays. No cross reaction was found in the detection of dengue virus, Hantavirus, severe fever with thrombocytopenia syndrome (SFTS) virus, yellow fever virus and influenza virus, and 100 healthy adults blood samples, the specificity of the method was 100%. The repeatability result showed that the standard deviation of all three detections were blow 0.5 and the coefficient of variation was less than 2% by selecting viral nucleic acids or transcribed RNA with high, medium and low concentration gradients.@*Conclusions@#A triplex real-time RT-PCR assay for detection of Zika, Chikungunya and Mayaro virus has been established with an acceptable specificity, sensitivity and repeatability.
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Objective@#To obtain the optimum of lentiviral library packaging based on CRISPR/cas9 (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9).@*Methods@#Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence antibody (IFA) and enzyme linked immunosorbent assay (ELISA) were used to detect the lentivirus titers in condition of different ratio of packaging plasmids, different addition of lipofectamine 3000 reagent and different time points post-transfection. Then, high-throughput sequencing was performed to evaluate the representation and distribution of single guide (sg)RNAs in the library.@*Results@#The lentivirus titer was the highest when the molar ratio of psPAX2∶pMD2.0G∶Lentivirus library was 2∶1∶1, and the optimum addition of Lipofectamine 3000 reagent was 10 μl, while the result of ELISA were correspondent to that of RT-PCR. The IFA result showed that the lentivirus titer was the highest at 60 h post-transfecion. The coverage of sgRNAs in the lentivirus library packaged with the optimum we obtained was 99.3%, and the read counts of sgRNAs was observed in a normal distribution.@*Conclusions@#The optimal lentivirus library packaging was obtained, and this can provide basis for CRISPR/cas9-based screening.
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Objective To determine the safety and effectiveness of double balloon catheter for labor induction in women with previous cesarean section. Methods This was a retrospective case-control study. Data from Shenzhen maternal and child healthcare hospital and Shenzhen Longgang District Maternal and Child Healthcare Hospital between 2015. 01. 01 to 2018. 12. 31 were used. A total of 156 term pregnant women, with previous one low segment cesarean section (CS) and balloon catheter for labour induction were included as case group. A total of 156 term pregnant women with previous one low segment CS and sponta-neous onset of labor were included as control group. The vaginal delivery rate and maternal and infant out-comes were compared between the two groups. Results There was no significant difference in CS rate, va-ginal assisted rate and vaginal spontaneous rate between the two groups ( 20. 51％ vs 15. 38％, 8. 97％ vs 5. 77％, 70. 51％ vs 78. 85％ respectively, P >0. 05 ) . And there were no significant difference in the rates of intrauterine infection, uterine rupture, postpartum hemorrhage, blood transfusion, maternal and in-fant mortality and neonatal transfer between the two groups. Conclusions Our study indicates that induc-tion of labor with double balloon catheter is effective and safe in term pregnant women with previous one low segment CS. Women with previous CS and indications for labour induction should be informed about vaginal birth success rates and the alternative of elective repeat CS needs to be discussed.
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Objective To establish a suitable model for predicting the success of trial of labor after cesarean section (TOLAC) during the pregnancy at term.Methods Data for all deliveries at term with one cesarean delivery history in Shenzhen Maternity and Child healthcare hospital during 1 January 2012 to 31 December 2014 were reviewed.Variables associated with VBAC were identified and used to build a model to predict the outcome of TOLAC with multivariable logistic regression.Godness of fit and accuracy of the model were evaluated by ROC.Results A total of 531 women met inclusion criteria and underwent TOLAC.Of the women who underwent trial of labor, 448 (84.4％) had a successful VBAC, 83 failed, and 2 (0.38％) had uterine rupture.Multivariable logistic regression analysis showed that previous cesarean section (CS) time interval, neonatal birth weight (BW) and premature rupture of membranes (PROM) were independent factors affecting TOLAC outcome, and their Odds Ratios were 2.79, 1.002 and 0.244, respectively.The Logistic regression model was expressed as follows:P =1/[1 + exp (2.4 × neonatal BW + 1.03 × previous CS time interval-1.41 × PROM-10.24)].The Hosmer-lemeshow test showed that the model fitted well (x2 =123.45, P =0.996), and the prediction accuracy of the model was 86.77％.The model performed well with an AUC of 0.794 (P =0.000).Conclusions A predictive model, which contains three variables (previous CS time interval, neonatal BW and PROM), has been developed and its prediction efficiency and accuracy are satisfactory.The larger birth weight, the longer time interval from previous CS, and the absence of PROM are more likely to be failed in TOLAC.
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Objective@#To establish a multiplexed immunoassay for detection of IgG antibodies against viral hemorrhagic fever epidemic in Africa.@*Methods@#The recombinant antigens of hemorrhagic fever viruses (HFVs) epidemic in Africa were expressed and purified, and then coupled with the fluorescent microspheres. The coupling effects were evaluated by monoplexed detection of rabbit immune sera. Blood specimens were collected from people from Africa with fever, and multiplexed detection of IgG antibodies to HFVs was performed. Comparison of multiplexed assay and ELISA was performed by paired χ2 test using SPSS software.@*Results@#Both the purity and concentration of HFVs recombinant antigen met the standards for coupling and detection, and the antigens were successfully coupled with fluorescent microspheres. Seventy-eight sera samples of people from Africa with fever were multiplex detected. Among these, one was tested positive for LASV-specific IgG, one was tested positive for LUJV-specific IgG, 4 were tested positive for DENV-specific IgG and 6 tested positive for YFV-specific IgG. There was no statistically significant difference compared with ELISA, and the two method were highly correlated.@*Conclusions@#A multiplexed luminex-based immunoassay for detection of IgG antibodies to viral hemorrhagic fever epidemic in Africa was established, which laid the foundation for the differential diagnosis.
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Objective@#To identify whether the three imported yellow fever cases in China in March 2016 were infections by wild type strain of yellow fever virus in Angola in 2016, vaccine-associated disease or co-infection of both.@*Methods@#Sequences of three yellow fever virus strains were obtained by high-throughput sequencing with IonTorrent PGM platform from blood or urine samples of three yellow fever cases, and their genomic characteristics were analyzed. Then the regions with relatively great difference between the wild type strain and 17D vaccine strain were identified, and then served as the reference sequences when mapping the reads obtained by high-throughput sequencing.@*Results@#Partial yellow fever virus genomes were obtained from three samples of yellow fever patients, among them a full length coding region sequence was gained in sample 2. Comparing the genome sequences, the three newly obtained strains of yellow fever virus were highly similar to strain CNYF01R / 2016 which was isolated from the first imported yellow fever case to China in 2016 and strain Angola 71 from Angola in 1971, and they all belonged to Angola genotype of yellow fever virus. In this study, we found five regions in yellow fever virus genomes with great diversity between the vaccine strain and the wild type strain. In these five regions, a number of short reads obtained by high-throughput sequencing of the three samples were mapped to the sequence of wild type virus, while no short reads matched the vaccine strain.@*Conclusions@#There were no viral nucleic acid of 17D vaccine strain in the blood or urine samples of these three cases of yellow fever. They are all infected by wild type strains of Angola in 2016.
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Objective@#To establish a method for detection of chikungunya virus(CHIKV) antigen.@*Methods@#CHIKV virus like particle(VLP), that contains all structural proteins, was prepared by baculovirus expression system. Mice and rabbits were immunized with the VLP to develop antibodies against CHIKV. A double antibody sandwich ELISA was established for detection of CHIKV antigens. The concentrations of the antibodies used and the reaction conditions were optimized. The detection limit and repeatability of the ELISA was evaluated.@*Results@#The sensitivity and specificity was estimated by 10 mimicking CHIKV sera, 90 health person sera, 40 other virus infected sera. It was show that the specificity of DAS-ELISA was 100%, the detection limit was 10 TCID50, the coefficients of variation (CV) within plate was <5%, the CV of different plates was <10%.@*Conclusions@#The double antibody sandwich ELISA established in this study can be used to detect the CHIKV antigen in acute phase serum of patient and provide a method for detection of CHIKV.
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Objective To investigate the expression of human papillomavirus E2 mRNA in cervical cytologi-cal specimens as well as the role of that in cervical carcinogenesis and its clinical significance in screening and evalat-ing prognosis of cervical lesions.Methods The expression of E2 mRNA in 582 cases of cervical cytological speci-mens with high risk human papillomavirus infection and cytological diagnosis of NILM,ASCUS and LSIL,was detected by RT -PCR.Thereafter,all cases were divided into the four groups by colposcopy and histopathological confirmation, including 414 cases of cervicitis,95 cases of CINⅠ,51 cases of CINⅡ,20 cases of CINⅢ and 2 cases of invasive cervical carcinoma as well.Results The expression of HPV -E2 mRNA decreased dramatically corresponding with pathological upgrading from groups of cervicitis to invasive cervical carcinoma (χ2 =132.72,P <0.05).The sensitiv-ity,specificity,positive predictive value and negative predictive value of HPV -E2 mRNA for screening potential cervical lesions in group of NILM,ASCUS and LSIL were 77.2%,96.8%,75.6%,96.0% and 81.0%,91.7%, 79.1%,92.6% as well as 95.9%,93.4%,94.7%,95.0% respectively.Conclusion Deletion of HPV -E2 induced by genetic disruption played an important role in the early stage of malignant transformation of cervical epithe-lial cells.Therefore,detection of the levels of HPV -E2 mRNA expression might be clinically valuable for the screen-ing and evaluating of prognosis in cervical lesions.
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To prepare monoclonal antibodies (mAbs) against structural proteins of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), BALB/c mice were immunized using purified inactivated SFTSV virions as the antigens. Subsequently, hybridoma cell lines that secreted monoclonal antibodies against nucleoprotein (NP) and glycoproteins (GP) were obtained using a hybridoma technique. The antigen specificities of prepared mAbs were examined by indirect immunofluorescence and immunoprecipitation assays. Functional analyses were then performed,including the detection of IFA antibody titers,the levels of neutralizing activity and antibody affinities. After cell fusion and cloning,13 hybridoma cell lines secreted mAbs specifically against SFTSV-GP and 7 hybridoma cell lines secreted mAbs specifically against SFTSV-NP. Immunofluorescence and immunoprecipitation assays showed that the mAbs had high levels of antigen specificity. Among the 13 anti-SFTSV-GP mAbs,6 recognized Gn,whereas the others reacted with Gc. IFA titers of most anti-SFTSV-GP mAbs were between 1,280 and 20,480, and four anti-SFTSV-Gn mAbs showed neutralizing activity. Seven of the obtained anti-SFTSV-NP mAbs reacted specifically with NP,of which the IFA titers ranged from 5,120 to 20,480 with no observed neutralizing activity. Furthermore, two anti-SFTSV-GP mAbs, 1C8 and 1G8, showed high levels of affinity via a non-competitive ELISA. Our study lays the foundation for the development of further diagnostic assays and basic research into SFTSV.
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Animals , Female , Humans , Mice , Antibodies, Monoclonal , Allergy and Immunology , Antibodies, Viral , Allergy and Immunology , Antibody Specificity , Bunyaviridae Infections , Allergy and Immunology , Virology , Hybridomas , Allergy and Immunology , Mice, Inbred BALB C , Phlebovirus , Allergy and Immunology , Viral Structural Proteins , Allergy and ImmunologyABSTRACT
To understand the immunogenicity of purified inactivated severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), concentration by ultrafiltration as well as molecular-sieve chromatography (MSC) were used for purification of inactivated SFTSVs. Inactivated viruses in purified samples were analyzed and identified by western blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the glycoprotein (GP) and nucleoprotein (NP) antigen titers of which were detected using a double-sandwich enzyme-linked immunosorbent assay (ELISA). Purified inactivated SFTSVs were enriched and observed by electron microscopy, and the total protein concentration detected using the bicinchoninic acid assay. Purified inactivated SFTSVs were applied to New Zealand rabbits via two immunization programs to evaluate immunogenicity and to compare the immune effect. After SFTSVs were inactivated and concentrated by ultrafiltration, MSC revealed two typical elution peaks. The sample of one peak was identified as inactivated virions, in which GP and NP were detected by SDS-PAGE, western blotting and ELISA. Main corponent of the other peak was NP. After concentration by ultrafiltration, purified inactivated SFTSVs with purity >90% and total protein concentration of 1. 1 mg/mL were obtained, and the typical electron microscopy of bunyavirus was observed. In the sera of animals immunized with purified inactivated SFTSVs, SFTSV-specific IgG antibody and neutralizing antibody were detected at high titers. However, antibody titers were affected by the immunization program. Effect of immunization on days 0, 14 and 28 was significantly better than that on days 0, 7 and 28. Our work revealed that cultivation of SFTSVs contained intact virus particles and large amounts of free NP. Using MSC, purified inactivated SFTSVs of high purity could be obtained. Purified inactivated SFTSVs induced high titers of neutralizing antibody and virus-specific IgG antibody showing satisfactory immunogenicity, which provides important clues for further study on a vaccine for the inactivated virus.
Subject(s)
Animals , Humans , Rabbits , Antibodies, Neutralizing , Allergy and Immunology , Antibodies, Viral , Allergy and Immunology , Bunyaviridae Infections , Allergy and Immunology , Virology , Neutralization Tests , Phlebovirus , Classification , Genetics , Allergy and ImmunologyABSTRACT
To explore a new method for stable expression of virus-like particles (VLPs) of the severe fever with thrombocytopenia syndrome (SFTS) virus, an expression plasmid for the membrane glycoprotein (GP) and nucleocapsid protein (NP) of the SFTS virus was constructed by fusion of the two proteins via a serine residue, and a yellow fluorescence protein (YFP) gene was introduced into the plasmid as a reporter. CHO-K1 cells were transfected with this plasmid, and stable cell lines constructed using the limited dilution method. Cellular colonies were hand-picked based on YFP with the help of fluorescence microscopy and expanded without selection pressure. Stability of cell lines was evaluated by monitoring of fluctuation of the intensity of YFP for 40 passages. VLP production was characterized using an indirect fluorescence assay, immunoblotting, and electronic microscopy. We showed that GP and NP fusion proteins could be assembled into VLPs in vivo, and that VLPs had similar morphologies to virus particles. Selected cell lines were stable for YFP expression: no significant fluctuation was detected in 40 passages. These data demonstrated the effectiveness of this new method for expression of structural proteins of the SFTS virus and screening for stable cell lines. Our results could provide new concepts for the production of biopharmaceuticals.
Subject(s)
Animals , Cricetinae , Bunyaviridae Infections , Virology , CHO Cells , Cloning, Molecular , Methods , Cricetulus , Gene Expression , Phlebovirus , Genetics , Metabolism , Plasmids , Genetics , Metabolism , Viral Proteins , Genetics , Metabolism , Virion , Genetics , Metabolism , Virus AssemblyABSTRACT
This study performed phylogenic analysis on a dengue strain isolated from an outbreak of dengue fever in 2009 at Yiwu City of Zhejiang Province,China,and further to analyze the immunogenicity of E protein of this viral isolate.Firstly,the viral genome was amplified by RT-PCR and phylogenetic trees were constructed by MEGA 4 based on both nucleotide and amino acid sequences of E and NS1 proteins.The phylogenetic analysis showed that the similarity of Yiwu strain with the Guangzhou GZ1D3 strain and the India GWL-25 strain was over 99％.Secondly,the expression plasmid of E protein was constructed and transfected into 293T cells.The secreted E protein were then purified by sucrose density gradient centrifugation and used to inoculate BALB/c mice.The humoral immunity was evaluated by ELISA and neutralizing antibody analysis.Resuits showed that the E protein of Yiwu strain could induce dengue specific IgG antibodies and neutralizing antibodies.Therefore,the study found that the Yiwu strain was classified into the subtype Ⅲ of dengue virus type 3 (DENV-3),and the E protein of this strain had strong immunogenicity