ABSTRACT
ABSTRACT Objective This study verified the replication efficiency of the Rocio virus in a primary culture of mouse neural cells. Methods Mixed primary cultures (neurons/glia) obtained from the brains of newborn isogenic BALB/c mice were inoculated with Rocio virus on the 7 th day of culture, and the development of cytopathogenic effects was monitored. The infection was confirmed via immunocytochemistry (anti-ROCV), while viral replication was quantified in infected primary cultures. The titration method used depended on the infection period. Results Rocio virus efficiently infected primary cultured neural cells, with the highest viral titer causing cytopathic changes was observed at 2 days post infection. The virus-infected primary culture survived for up to 7 days post infection, and viral load quantitation showed viral replication kinetics compatible with the cell death kinetics of cultures. Conclusion The findings of this study suggest that mouse neural cell primary cultures support Rocio virus replication and could be used as an alternative system for studying Flavivirus infection in the central nervous system.
ABSTRACT
The COVID-19 pandemic caused by the SARS-CoV-2 virus has generated globally more than 110.7 million infections and 2.4 million deaths. The severity of this infection can range from asymptomatic, mild to severe. To know the possible associations between the presence of the virus and histopathological alterations found in tissues of fatal cases of COVID-19, the presence of the virus in the lung tissue of a patient with a clinical history of SARS-CoV-2 infection was evaluated. Lung tissue was histologically processed for immunohistochemical detection of SARS- CoV-2. In the histopathological study, morphological changes associated with pneumonitis of viral origin were observed. Likewise, the location of the SARS-CoV-2 virus was observed mainly in the cytoplasm of the cells of the inflammatory infiltrate.
La pandemia de COVID-19 causada por el virus SARS-CoV-2 ha generado más de 110,7 millones de infecciones y 2,4 millones de muertes a nivel mundial. Esta infección puede ser asintomática y sus manifestaciones clínicas pueden variar entre leves y graves. Para conocer las posibles asociaciones entre la presencia del virus y las alteraciones histopatológicas encontradas en los tejidos de casos fatales de COVID-19, se evaluó la presencia del virus en el tejido pulmonar de un paciente con antecedentes clínicos de infección por SARS-CoV-2. La muestra se procesó para la detección inmunohistoquímica del virus. En el estudio histopatológico, se observaron cambios morfológicos asociados con neumonitis de origen viral. Asimismo, el virus se localizó principalmente en el citoplasma de las células del infiltrado inflamatorio.
Subject(s)
COVID-19 , Lung , Immunohistochemistry , Antigens, ViralABSTRACT
Objective@#To investigate the expression of serum Neurensin 2 (NRSN2) in patients with nasopharyngeal carcinoma (NPC), and to analyze its relationships with epstein-barr virus (EBV) antibody and EBV-DNA.@*Methods@#120 patients with NPC admitted to our hospital from April 2016 to May 2018 were selected as the study group, and 56 healthy people in the physical examination center were selected as the control group. Enzyme linked immunosorbent assay (ELISA) was used to detect the expressions of serum viral capsid antigen-IgA (VCA-IgA), nuclear related tumor antigen-IgA (EBNA-IgA) and early antigen-IgG (EA-IgG). The expression of serum NRSN2 and EBV-DNA were detected by fluorescence quantitative polymerase chain reaction (qPCR). Receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of serum NRSN2 level for NPC. Spearman was used to analyze the relationship between serum NRSN2 and EBV antibody with EBV-DNA in nasopharyngeal carcinoma, multivariate logistic regression analysis was used to analyze the risk factors of NPC.@*Results@#The positive rates of serum VCA-IgA, EBNA-IgA and EA-IgG in the study group were significantly higher than those in control group (P<0.05); the level of serum NRSN2 and the positive expression rate of EBV-DNA in the study group was significantly higher than that in the control group (P<0.05); ROC curves showed that the area under curve (AUC) of serum NRSN2 level in diagnosing NPC disease was 0.759, with sensitivity 63.33%, specificity 80.36%; there were significant positive correlations between serum NRSN2 with VCA-IgA, EBNA-IgA, EA-IgG and EBV-DNA in NPC patients (P<0.05); multivariate regression analysis showed that VCA-IgA, EBNA-IgA, EA-IgG, EBV-DNA and NRSN2 expressions were supportive predictive value for diagnostic NPC (P<0.05).@*Conclusions@#Up-regulation of serum NRSN2 levels in NPC patients is positively correlated with serum VCA-IgA, EBNA-IgA, EA-IgG and EBV-DNA levels, which may provide a reference for the prediction, diagnosis and treatment of NPC.
ABSTRACT
Objective To investigate the expression of serum Neurensin 2 (NRSN2) in patients with nasopharyngeal carcinoma (NPC),and to analyze its relationships with epstein-barr virus (EBV) antibody and EBV-DNA.Methods 120 patients with NPC admitted to our hospital from April 2016 to May 2018 were selected as the study group,and 56 healthy people in the physical examination center were selected as the control group.Enzyme linked immunosorbent assay (ELISA) was used to detect the expressions of serum viral capsid antigen-IgA (VCA-IgA),nuclear related tumor antigen-IgA (EBNA-IgA) and early antigen-IgG (EA-IgG).The expression of serum NRSN2 and EBV-DNA were detected by fluorescence quantitative polymerase chain reaction (qPCR).Receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of serum NRSN2 level for NPC.Spearman was used to analyze the relationship between serum NRSN2 and EBV antibody with EBV-DNA in nasopharyngeal carcinoma,multivariate logistic regression analysis was used to analyze the risk factors of NPC.Results The positive rates of serum VCA-IgA,EBNA-IgA and EA-IgG in the study group were significantly higher than those in control group (P ＜0.05);the level of serum NRSN2 and the positive expression rate of EBV-DNA in the study group was significantly higher than that in the control group (P ＜ 0.05);ROC curves showed that the area under curve (AUC) of serum NRSN2 level in diagnosing NPC disease was 0.759,with sensitivity 63.33％,specificity 80.36％;there were significant positive correlations between serum NRSN2 with VCA-IgA,EBNA-IgA,EA-IgG and EBV-DNA in NPC patients (P ＜ 0.05);multivariate regression analysis showed that VCA-IgA,EBNA-IgA,EA-IgG,EBV-DNA and NRSN2 expressions were supportive predictive value for diagnostic NPC (P ＜ 0.05).Conclusions Up-regulation of serum NRSN2 levels in NPC patients is positively correlated with serum VCA-IgA,EBNA-IgA,EA-IgG and EBV-DNA levels,which may provide a reference for the prediction,diagnosis and treatment of NPC.
ABSTRACT
Objective To compare and evaluate the performance and applications of three assays in preliminary screening of dengue infections.Methods This study was designed as a retrospective study.Clinical data were obtained from 2 137 borderline cases from September to October in 2014 and 2015.Markers of dengue infections of serum samples were detected by NS1 antigen captured ELISA, dengue NS1 detect rapid test and dengue IgM detect rapid test, respectively.Chi square test was used to compare the preliminary screening value of these 3 methods.Forty-eight diagnosed patients were also examined in the 1st-3rd day, 4th-5th day, 6th-7th days and 14th day after infection to access the sensitivities and specificities of three assays.Results Nine hundred and fifty-seven ( 57.2%) cases in 2014 and 48 ( 10.4%) cases in 2015 were diagnosed to be Dengue fever of 2 137 borderline cases.The overall sensitivity of NS1-ELISA was superior to NS1 and IgM rapid detect test (χ2 =40.865,P<0.001;χ2 =151.383,P<0.001).No significant differences were found in specificity between three assays(χ2 =0.661,P=0.416;χ2 =0.548,P=0.459; χ2 =2.397,P=0.122).The NS1 detecting assays were sensitive in 7 days after infection, but the sensitivity of IgM detecting assay increased over time.Conclusions NS1 detecting assays had good sensitivities and specificities, which can be used as an important method in preliminary screening of dengue infection.ELISA or rapid test can be selected according to epidemic situation.
ABSTRACT
The severe infectious diseases caused by virus are occurring with increasing frequency, which poses a rising global threat to human health and life.There are many kinds of viruses that cause a wide variety of viral diseases.The same kind of virus is able to cause different diseases, meanwhile a disease can be caused by different viruses.All these conditions make the relationship between viral pathogens and infection diseases complicated.At present, no effective laboratory detection methods for most of virus infectious diseases are developed.But the rapid development of molecular diagnostic technologies makes it possible for clinical laboratory to detect viral infection diseases rapidly and simultaneously.This review summarized the present and development perspectives of laboratory tests for the diagnosis of clinical virus infections, attempting to give some advices for laboratory diagnosis procedure of viral infections.
ABSTRACT
Objective To establish an evaluation system about animals infected with hantavirus,an observation of the BALB/c mice infected with hantavirus was made.Methods BALB/c mice were infected with hantavirus by intramuscular injection with stock solution.The specific antigen from BALB/c mice tissues after 3 days was detected with enzyme-linked immunosorbent assay (ELISA) and viral RNA with real-time polymerase chain reaction (RT-PCR).Results Within a short term,the specific antigen and viral RNA were detected from the brain and liver at day 3 after infection,but not be detected from the heart,spleen,lung,and kidney samples.Conclusions The results provided ones with some information on animals infected with hantavirus.
ABSTRACT
Objective To construct a eukaryotic vector which contains avian H5N1 influenza virus hemagglutinin (HA) antigen and the cholera toxin B subunit (CTB) and to investigate its expression in COS7 cells,and the ability to induce specific immune responses in vivo in different periods.Methods After cloned by polymerase chain reaction (PCR),CTB and HA genes were digested with BamH Ⅰ and connected into CTB-HA gene with T4 ligase.The connected gene was referred to as CH.After double digestion,CH gene was inserted into a eukaryotic recombinant plasmid pCI-neo.The pCI-CH plasmid was then transfected into COS7 cells.Western blot was used to detect the expression of HA antigen.After New Zealand white rabbits were immunized,the titer of HA antigen-specific antibody in serum and its specificity with other strains such as H1N1,H9N1,H3N2 and influenza B virus were determined by indirect enzyme-linked immunosorbent assay.Results The pCI-CH vector (DNA vaccine) was successfully constructed,which could be efficiently expressed in COS7 cells and induce specific antibodies against pCI-CH in rabbits.Cross reactions indicated that DNA vaccine pCI-CH specific antisera could not only react with H5N1 strain (P/N＞2.1),but also H1N1,H9N1 and H3N2 strains,but did not cross react with influenza B virus.Conclusion The newly constructed avian H5N1 influenza virus nucleic acid vaccine has good immunogenicity.
ABSTRACT
Objective To compare the detection of flu A by nucleic acid amplification assav and rapid antigen assay in nasopharynx swabs and oropharynx swabs of flu-like patients.Methods A total of 170 flu-like patients were recruited in out-patient of Youan Hospital from September to October in 2009.Both nasopharynx swabs and oropharynx swabs were collected.Flu A virus was detected by both real-time reverse transcriptation polymerase chain reaction (RT-PCR) and rapid antigen assay.The data were analyzed by chi square test.Results For nasopharynx swabs,the positive rate of nucleic acid amplification assay was 74.1%(126/170),while that of rapid antigen assay was 65.9%(112/170)(X2=2.75,P>0.05).However,for oropharynx swabs,the positive rate of nucleic acid amplification assay was much higher than that of rapid antigen assay(62.9% vs 38.8%)(X2=19.78,P<0.01).Moreover,for nucleic acid amplification assay,the positive rate of nasopharynx swabs were higher than that of oropharynx swabs (X2=4. 90, P<0. 05). For rapid antigen assay, the positive rate of nasopharynx swabs was also higher than that of oropharynx swabs (X2=24.95, P<0.01). Based on the outcome of flu A detected with nasopharynx swabs by the nucleic acid amplification assay,the sensitivities of oropharynx swabs by nucleic acid amplification assay,oropharynx swabs by rapid antigen assay, nasopharynx swabs by rapid antigen assay were 81.7%,50.0% and 94.8%, respectively; the specifieities were 90.9%, 93.2% and 95.5%, respectively;the positive predictive values were 96. 3%, 95. 5% and 98.2%, respectively; the negative predictive values were 63.5 %, 39.4 % and 72.40%, respectively; Kappa coefficients were 0.64, 0.30 and 0.75,respectively; the total coincidences were 84.1%, 61.20% and 89.4%, respectively. Conclusions The detection of flu A with nasopharynx swabs is more sensitive than oropharynx swabs, and nucleic acid amplification assay is more sensitive than rapid antigen assay.
ABSTRACT
Objective To construct prokaryotic expression vector carrying jc virus(JCV)t-antigen gene,express and purify this fusion protein.Methods The JCV t-antigen gene from a cerebrospinal fluid sample was amplified using polymerase chain reaction(PCR)method.After sequencing.the gene was cloned into plasmid pET32a(+)to construct recombinant prokaryotic expression vector pET32a(+)-t.The t-antigen fusion protein was expressed by isopropy-~D-thiogalactoside(IPTG)induction and prepared in large scale,then purified by Ni+affinity column chromatography.The polyclonal antibody was obtained from the BAI.B/C mouse immunity by the purified protein.Results The relative molecular nlass of recombinant protein expressed by pET32a(+)-t was about 41 000.Sodium dodeeylsulfate-polyaerylamide gel electrophoresis(SDS-PAGE)showed that the fusion protein W&S highly expressed after 3.5～20.Oh of IPTG induction.The antigenicity of the purified protein Was well confirmed by Western blot.The anti-mousepolyclonal antibody was obtained successfully from immunized BALB/c mice.Conclusions The prokaryotic expression vector pET32a(+)-t is successful constructed and the fusion protein is expressed and purified.Furthermore,the antibody of JCV small envelop protein t is successfully prepared.This work provides vMuable information for further study on epidemiology and biological function of t antigen.
ABSTRACT
Objective To establish immortalized human hepatocyte lines for studies of bioartificial liver,hepatocyte transplantation,and drug metabolism in vitro.Methods Primary human hepatocytes were isolated by 4-step perfusion technique with collagenase and transfected with recombinant retrovirus containing Simian virus 40 large T antigen(SV40 LT).Subsequently,immortalized human hepatocytes were evaluated by analysis of gene expression and functional characteristics in vitro.Results Two immortalized human hepatocyte lines,HepLi2 and HepLi3,were obtained after primary human hepatocytes being infected by SV40 LT containing recombinant retrovirus for 3-4 weeks.The immortalized human hepatocytes showed classical appearance of hepatocyte observed by phase contrast microscope.The protein expression of SV40 LT in HepLi2 and HepLi3 cells were detected by Western blotting.The mRNA expressions of albumin(Alb),glutathione S-transferase(GST-p),human blood coagulation factor X(HBCF-X)and β-actin in HepLi2 and HepLi3 cells were detected by reverse transcription polymerase chain reaction(RT-PCR),and the mRNA expressions of cytochrome(CY)450 subtypes(CYP3A5,CYP2E1,CYP2C8-19 and CYP3A4)in HepLi2 and HepLi3 cells were also observed by RT-PCR.Levels of alanine transaminase (ALT),lactate dehydrogenase(LDH)and Alb were detected in the supernatant of immortalized human hepatoeyte culture.Conclusions The immortalized human hepatocyte lines have the biological characteristics of primary human hepatocytes and have the CYP450 functions of hepatocytes,which may be heIDful for the studies of bioartificial liver,heoatocvte transplantation and drug metabolism in vitro.
ABSTRACT
Objective To evaluate the feasibility and utility of three different assays on early diagnosis and monitoring of neonatal congenital cytomegalovirus infection. Methods Ninety-eight neonates whose mother was CMV-IgM positive during pregnancy were examined on the 14th days after birth for CMV antigen in blood and PCR-CMV-DNA in the saliva. Three different methods were applied including CMV antigenemia assay, PCR for CMV-DNA and ELISA for serum CMV-IgM. Neonates were followed up for six months. Results (1) Forty-eight of the 98 neonates were diagnosed as congenital CMV infection including 7 symptomatic infection and 41 asymptomatic. None of the 98 subjects was CMV-IgM positive. Among the 7 symptomatic cases, the positive rates of CMV antigen and PCR-CMV-DNA were 100%(7), 71.4%(5), and 70.7%(29/41), 46.3%(19/41) in the asymptomatic group, respectively. The sensitivity of CMV antigenemia assay and PCR was 75.0% and 54.2%, respectively. The CMV antigenemia index of the symptomatic cases was significantly higher than that of asymptomatic ones [(16-52)/50 000 vs (3-31)/50 000 white blood cells, P
ABSTRACT
Objective To investigate the immortalization of human melanocytes by transfection with SV40 T antigen ( SV40T ). Methods By using SofastTM,a gene transfection reagent, the reconstructed eukaryotic expression vector SV40T-pEGFP was stably transfected into cultured primary human melanocytes, then the positive cells were selected with G418. After the positive cells were expanded in culture, the expression of SV40T gene was detected by RT-PCR and PCR, and the protein expression of SV40T by Western blotting. Results The genome DNA and total RNA were isolated from the positive cell clones, and a 288 bp fragment, which was specific for the SV40T antigen gene, was amplified. The results of immunohis-tochemistry and Western blotting confirmed the expression of SV40T protein in transfected cells. Conclusion SV40T antigen gene can successfully induce the immortalization of human melanocytes.