Objective To observe the changes of CD4+CD25+ regulatory T cells in the spleen of mice infected with T.gondii. Methods Twenty-eight female C57BL/6 mice were randomly divided into four groups. Three groups of mice were inoculated intraperitoneally with 104 tachyzoites in 200 ?l sterile PBS. At 2, 4 and 6 days post-infection, the spleens were removed. The expression level of Foxp3 mRNA in splenic CD4+ T cells was quantitated by real-time PCR. The percentage of CD4+CD25+ regulatory T cells in CD4+ T cells was determined by flow cytometry, and the absolute numbers of splenic CD4+CD25+ regulatory T cells and CD4+ T cells were assessed. The fourth group was injected intraperitoneally with 200 ?l sterile PBS as control. Results The relative mRNA level of Foxp3 in splenic CD4+ T cells at day 4 (1.89?0.23) and day 6 (1.79?0.24) post-infection was significantly higher than control (1.00?0.12)(P

To prepare the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 for use as RNA standard and control in RNA virus detection, the genes coding the coat protein and maturase of E.coli bacteriophage MS2 were amplified by PCR and then cloned into prokaryotic expression vector pET32a to construct the intermediate vector pET32a-MS2. In addition, the gene sequences coding hemagglutinin (HA), neuraminidase(NA) and M protein of the H5N1 virus were also cloned separately to the down-stream of plasmid pET32a-MS2, thus constructing the prokaryotic expression vectors pET32a-NS2-HA, pET32a-MS2-NA and pET32a-MS2-M. These recombinant plasmids were then transformed separately to E.coli BL21(DE3) with induction by IPTG. to express the virus-like particles. The virus-like particles observed under electron microscopy were identified by RT-PCR ,while their stability was confirmed by real-time RT-PCR. In this way, the virus-like particles were successively constructed and identified through PCR amplification, enzymolysis identification and sequencing analysis. These virus-like particles observed under electron microscopy appeared to be circular in shape with a diameter of about 50 nm. Their stability was proved to be rather good. From these observations, it is apparent that these virus-like particles can be used as RNA standard and quality control in the detection of avian influenza virus H5N1.

Objective To explore the effect of culture supernatant of Toxoplasma gondii on CD4+CD25+ T cells in mice in vitro. MethodsCD4+CD25+ T cells were separated from spleens of C57BL/6 mice and incubated in the culture superntant of Toxoplasma gondii. The apoptosis percentage of the CD4+CD25+ T cells were detected by FACScan, and the suppression of the CD4+CD25+ T regulatory cells were examined by 3H-TdR incorporation. ResultsCompared with the CD4+CD25+ T cells incubated with RPMI-1640, (36.90?0.36)% CD4+CD25+ T cells took apoptosis after incubated with the culture supernatant of Toxoplasma gondii for 10 hours,and the Annexin-v positive rate increased by (13.60?2.15)%. Compared with RPMI-1640, the culture supernatant of Toxoplasma gondii incubating the CD4+CD25+ T regulatory cells for 5,10 hours reduced their suppressive potential on the proliferation of the CD4+CD25- T cells significantly. ConclusionsSome composition of the culture supernatant of Toxoplasma gondii might cause apoptosis of CD4+CD25+ T cells and as a result, could reduce their suppression on CD4+CD25- T cells.

Objective To prepare and study the immunogenicity of hepatitis B virus surface anti-gen (HBsAg)-tetanus toxoid (TT) conjugate vaccine. Methods Tr was activated by cyangen bromide and reacted with adipic acid dihydrazide, then HBsAg-TT conjugate was prepared by carbediimide. Conjugate, HBsAg or hepatitis B vaccine was injected subcutaneously into mice. Anti-HBsAg and HBsAg-specific T cell response elicited by these immunogens were assayed. Results New HBsAg-TT conjugate elicited higher levels of anti-HBsAg and HBsAg positive conversion rates after the immunization than did HBsAg alone or hepatitis B vaccine. Conjugate induced mesdy antibodies of the IgG2a subclass, while HBsAg alone or hepa-titis B vaccine mainly elicited anti-HBsAg in the IgG1 subclass. The number of IFN-γand IL-2 secreting T cells induced by conjugate was also significantly higher than that did by HBsAg or hepatitis B vaccine. Con-clusion This study indicated new HBsAg-TT conjugate can induce both stronger humoral and TH1 type of cellular immune response.

Objective:To establish a rapid, sensitive and specific detection method for important imported viruses based on the recombinase aided amplification (RAA) method and clustered regularly interspaced short palindromic repeats-associated protein 13a (CRISPR-Cas13a) system.Methods:In this study, we selected Japanese encephalitis virus (JEV), Yellow fever virus (YEV), West Nile virus (WNV), Middle East respiratory syndrome coronavirus (MERS-CoV)、Ebola virus (EBOV), Dengue virus (DENV), Rift Valley fever virus (RVFV), Zika virus (ZIKV) as subjects, and designed specific RAA primers and CRISPR RNA(crRNA). The sensitivity and specificity of the method were evaluated. We detected suspected clinical samples of dengue fever and compared with the fluorescent reverse transcriptase-polymerase chain reaction (RT-PCR) technology. Clinical simulation samples of the remaining seven viruses were also detected.Results:The RAA method combined with CRISPR-Cas13a can detect eight pathogens within 40-52 min at 39 ℃. The sensitivity was 1-10 copies/μl. There was no cross-reaction among eight viruses and all clinical samples could be detected by this method.Conclusions:The established RAA combined with CRISPR-Cas13a detection method can sensitively, specifically and quickly detect eight imported infectious disease pathogens.

With the continuous research on nanotechnology, nanosensors for the detection of various biomolecular have emerged one after another. As one of the main nanosensors, quantum dots have unique optical and particle properties, and quantum dot probes for pathogen detection have been widely used. The emerging fluorescent biomarkers, quantum dot probes, have unparalleled advantages over other fluorescent probes, such as strong fluorescence signal, good photostability, long fluorescence lifetime, and large Stokes shift. They can detect the target simply, quickly, sensitively and specifically. This article provides an overview of the basic properties of quantum dots and the application foundation of quantum dot probes as detection sensors. We focus on the types and preparation method of quantum dot probes, as well as their applications in pathogen detection in recent years. Finally, the application precautions of quantum dot probes as detection sensors were summarized, and the application prospects of quantum dot probes in the field of pathogen detection were also discussed.

Objective: To investigate the pyroptosis induced by different enteroviruses in human neuroblastoma cells SH-SY5Y and the differences among them. Methods: SH-SY5Y cells were infected with nine strains of enterovirus respectively, including enterovirus A71 (EV-A71), Coxsackievirus A (CA), Coxsackievirus B (CB), Echovirus (Echo). The cellular morphology of infected and control groups were observed and activity of Caspase-1 of infected and control groups were detected by flow cytometry at 48 h post infection. Results: The activity of Caspase-1 induced by EV-A71 was higher than control (P<0.001), and the activity of Caspase-1 induced by EV-A71 isolated from severe case was significantly higher than that induced by EV-A71 isolated from common case (P<0.001). The activity of Caspase-1 induced by CA was at a lower level. The activity of Caspase-1 induced by CB and Echo were both significantly higher than that induced by EV-A71 and control (P<0.001). Cytopathic effects (CPE) were found to be related with the activity of Caspase-1. Conclusions EV-A71, CB and Echo all could induce pyroptosis mediated by Caspase-1 in SH-SY5Y cells, but the ability of CA to induce pyroptosis was at a lower level, which may provide evidences for further study on mechanism of neuropathy caused by enterovirus.

Objective: To establish a rapid and sensitive isothermal amplification assay for the detection of human Adenovirus. Methods: Primers and probe used for recombinase polymerase amplification(RPA)were designed based on the conserved region of the adenoviruses hexon gene. After optimizing the reaction temperature and times, the products of RPA were detected by capillary electrophoresis and lateral flow dipstick(LFD). Sensitivity and specicity of the assay were evaluated. The diagnostic value of the RPA-LFD assay was verified using clinical samples which were simultaneously tested by real time PCR assay. Results: The analytical sensitivity of RPA-LFD assay was 2 copies DNA molecules per reaction and no cross reaction with other pathogens was observed. Compared with real-time PCR assay, the sensitivity, and specificity of the present assay were all 100%. Conclusions The RPA-LFD assay developed in this study has the characteristics of high specificity, sensitivity, rapid and no requirement of expensive equipment which provided a new tool for rapid detection of human adenovirus.

Objective:To establish a rapid method for detecting the activity of 2019-nCoV2.Methods:The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection system was screened after propidium monoazide (PMA) treatment and exposure to heat-inactivated 2019-nCoV samples, and the PMA pretreatment conditions were optimized to establish the 2019-nCoV PMA-RT-qPCR detection method . The established method was used to detect virus inactivated by different temperatures and chlorine-containing disinfectant, to evaluate its effect in detecting virus activity.Results:For the PMA-RT-qPCR assay, 200 μmol/L of PMA, 10 min of incubation time, 15 min of exposure time, and the CDC ORF1ab detection system were selected; there was no significant difference in the result of PMA-RT-qPCR and direct RT-qPCR for the active virus; the Ct values of PMA-RT-qPCR for virus inactivated by 95 ℃ and chlorine-containing disinfectant were higher than that of control groups at different dilutions; only partial dilutions of 70 ℃ and 56 ℃ heat-inactivated virus had higher Ct values than control groups. Conclusions:The established PMA-RT-qPCR for 2019-nCoV activity detection method has a good detection effect on the virus inactivated by 95 ℃ heat and chlorine disinfectant, and provides an auxiliary means for judging the infectivity of the virus in the sample.

Objective:The entire genome sequences of 25 novel coronaviruses in Jiangsu province were analyzed and their evolutionary characteristics were studied.Methods:High-throughput sequencing was used to sequence the throat swab samples from confirmed cases. Single nucleotide polymorphisms were analyzed using CLC Genomics Workbench 12.0 software. Evolution characteristics were analyzed by MEGA 5.1.Results:A total of 52 single-base substitution mutations were detected in 25 strains. Phylogenetic analysis showed 25 stains were clustered into two clades. Viruses in clade 1 contain 8 682 and 28 144 CT SNP links. While viruses in clade 2 contain mutations in those two bases, i. e., 8 682 (ORF1ab: C8 517T, synonymous mutation) and 28 144 (ORF8: T251C, L84S). Among clade 2, five stains subclustered into one group based on SNP links in 24 034 (S: C2 472T, synonymous mutation), 26 729 (M: T207C, synonymous mutation), and 28 077 (ORF8: G184C, V62 L). There were no significant differences in the distribution of different clades/subclusters in the population and the disease types.Conclusions:We have found some SNPs occurred in new coronaviruses. The effects of different SNPs on virus transmission and pathogenicity need to be further studied.