In vitro inactivation of SARS-CoV-2 by commonly used disinfection products and methods.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is currently a global pandemic, and there are limited laboratory studies targeting pathogen resistance. This study aimed to investigate the effect of selected disinfection products and methods on the inactivation of SARS-CoV-2 in the laboratory. We used quantitative suspension testing to evaluate the effectiveness of the disinfectant/method. Available chlorine of 250 mg/L, 500 mg/L, and 1000 mg/L required 20 min, 5 min, and 0.5 min to inactivate SARS-CoV-2, respectively. A 600-fold dilution of 17% concentration of di-N-decyl dimethyl ammonium bromide (283 mg/L) and the same concentration of di-N-decyl dimethyl ammonium chloride required only 0.5 min to inactivate the virus efficiently. At 30% concentration for 1 min and 40% and above for 0.5 min, ethanol could efficiently inactivate SARS-CoV-2. Heat takes approximately 30 min at 56 °C, 10 min above 70 °C, or 5 min above 90 °C to inactivate the virus. The chlorinated disinfectants, Di-N-decyl dimethyl ammonium bromide/chloride, ethanol, and heat could effectively inactivate SARS-CoV-2 in the laboratory test. The response of SARS-CoV-2 to disinfectants is very similar to that of SARS-CoV.

HTCC as a highly effective polymeric inhibitor of SARS-CoV-2 and MERS-CoV

The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the healthcare measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of previously developed by us HTCC compound (N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride), which may be used as potential inhibitor of currently circulating highly pathogenic coronaviruses - SARS-CoV-2 and MERS-CoV.

A data-driven drug repositioning framework discovered a potential therapeutic agent targeting COVID-19

The global spread of SARS-CoV-2 requires an urgent need to find effective therapeutics for the treatment of COVID-19. We developed a data-driven drug repositioning framework, which applies both machine learning and statistical analysis approaches to systematically integrate and mine large-scale knowledge graph, literature and transcriptome data to discover the potential drug candidates against SARS-CoV-2. The retrospective study using the past SARS-CoV and MERS-CoV data demonstrated that our machine learning based method can successfully predict effective drug candidates against a specific coronavirus. Our in silico screening followed by wet-lab validation indicated that a poly-ADP-ribose polymerase 1 (PARP1) inhibitor, CVL218, currently in Phase I clinical trial, may be repurposed to treat COVID-19. Our in vitro assays revealed that CVL218 can exhibit effective inhibitory activity against SARS-CoV-2 replication without obvious cytopathic effect. In addition, we showed that CVL218 is able to suppress the CpG-induced IL-6 production in peripheral blood mononuclear cells, suggesting that it may also have anti-inflammatory effect that is highly relevant to the prevention immunopathology induced by SARS-CoV-2 infection. Further pharmacokinetic and toxicokinetic evaluation in rats and monkeys showed a high concentration of CVL218 in lung and observed no apparent signs of toxicity, indicating the appealing potential of this drug for the treatment of the pneumonia caused by SARS-CoV-2 infection. Moreover, molecular docking simulation suggested that CVL218 may bind to the N-terminal domain of nucleocapsid (N) protein of SARS-CoV-2, providing a possible model to explain its antiviral action. We also proposed several possible mechanisms to explain the antiviral activities of PARP1 inhibitors against SARS-CoV-2, based on the data present in this study and previous evidences reported in the literature. In summary, the PARP1 inhibitor CVL218 discovered by our data-driven drug repositioning framework can serve as a potential therapeutic agent for the treatment of COVID-19.

Pyroptosis induced by different Enteroviruses infection in SH-SY5Y cell / 中华实验和临床病毒学杂志

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.

Clinical features and molecular characteristics of influenza A (H1N1) viral pneumonia in 17 elderly patients / 中华实验和临床病毒学杂志

Objective@#To analyze the clinical manifestations and results of etiological examinations of 17 elderly patients with influenza A (H1N1) viral pneumonia, and to understand the clinical features of pneumonia and molecular characteristics of influenza A (H1N1) virus infection in the elderly.@*Methods@#The elderly patients with pneumonia who were hospitalized in the Department of Respiratory Diseases of Nanjing First Hospital from January 2018 to March were enrolled. The cases were confirmed by nucleic acid examination for influenza virus and the clinical data were collected. After the amplification of the whole genome of influenza virus, the high throughput sequencing and bioinformatics analysis were performed.@*Results@#The mean age of the 17 enrolled patients was 73.8±10.8. All of them had at least 1 underlying disease, and 7 cases had co-infection. Respiratory symptoms and fever were the most prominent clinical manifestations. Lesions in both lungs were found in 76.5% of the patients. The result of high throughput sequencing showed that all the viruses were highly homologous to the vaccine strain, and the HA gene belonged to the 6B.1 subgroup. Furthermore, three variations of antigenic locus (H138Y, S74R and S164T in HA) and a drug-resistant variation (H275Y in NA) were detected in the circulating strains.@*Conclusions@#Elderly patients with influenza A (H1N1) virus pneumonia often have underlying diseases and are prone to have co-infection. The molecular characteristics of the virus and the variation of key amino acid loci should be closely monitored in order to provide evidence for epidemic prevention and clinical antiviral treatment.

Rapid detection of human adenovirus by recombinase polymerase amplification assay and lateral flow dipstick / 中华实验和临床病毒学杂志

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.

Preparation of the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 and its application / 中国人兽共患病学报

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.

Preparation and immunological properties of hepatitis B virus surface antigen-tetanus toxoid conjugate vaccine / 中华微生物学和免疫学杂志

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.

Effects of culture supernatant of Toxoplasma gondii on CD4~+CD25~+ T cells in mice in vitro / 中国血吸虫病防治杂志

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.

Changes of CD4~+CD25~+ T Cells in the Spleen of Mice Infected with Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

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