Clinical research progresss of antiviral drugs for the novel coronavirus pneumonia / 中华实验和临床病毒学杂志

The novel coronavirus (2019-nCoV or SARS-CoV-2) is a highly contagious and deadly virus that has infected more than 50 000 people and killed more than 1 000 people in 25 countries around the world. People who infected by the novel coronavirus may suffer from fever and cough, some may gradually appear breathing difficulties and other serious manifestations, some severe patients may have acute respiratory distress syndrome and septic shock leading to death. However, there are no definite and effective antiviral drugs for the novel coronavirus pneumonia all around the world. Therefore, this article aims to provide new idea for the effective treatment of the novel coronavirus pneumonia by summarizing the basic research and clinical progress of antiviral drugs at home and abroad.

Allele-31 C>T regulates binding activity to IL-1βgene promoter of nuclear transcription factor C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection / 中华临床感染病杂志

Objective To investigate the effects of allele-31 C>T on the binding activity to IL-1βpromoter of the nuclear transcription factor C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection.Methods The electrophoretic mobility shift assay ( EMSA) was performed to explore whether the nuclear transcription factor C/EBPβand PU.1 could bind to -31 region in IL-1βpromoter.The C/EBPβ-and PU.1-expressing vectors were constructed and co-transfected into HeLa cells with IL-1βpromoter luciferase vector.The expression of C/EBPβand PU.1 was confirmed using Western blotting assay, and the promoter activity was determined using Dual-Glo Luciferase system under various transfection conditions. Lentivirus-mediated RNA interference was used to explore the effects of C/EBPβand PU.1 on IL-1βexpression.GraphPad Prism 5.0 was used for data analysis.Results EMSA results showed that both C/EBPβand PU.1 could bind to -31 region in IL-1βpromoter.Both C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection could increase IL-1βpromoter activity, especially for the -31 T allele (t=22.33 and 7.98,PT can induce IL-1βpromoter activity and gene transcription through regulation of binding activity to C/EBPβand PU.1 induced by Mycobacterium tuberculosis infection.

Variation and significance of helper T cells and regulatory T cells in the peripheral blood before and after antiretroviral treatment of human immunodeficiency virus-1 infected patients / 中华传染病杂志

Objective To investigate the changes and clinical significance of helper T cells (Th)22,Th17,Th1 and regulatory T cells (Treg) in the peripheral blood before and after antiretroviral treatment (ART) of human immunodeficiency virus (HIV)-1 infected patients.Methods Forty HIV-infected patients were recruited into this study,and 30 healthy subjects were recruited as controls.Peripheral blood of the patients was collected at baseline and after 3 months of ART treatment.The frequencies of Th22,Th17,Th1 and Treg were detected by flow cytometry.Tests for homogeneity of variance and paired t test for comparison was adopted.Spearman rank test was used for correlation analysis.Results The frequencies of peripheral Th22,Th17,Th1 and Treg from HIV-infected patients before treatment were significantly decreased compared to the healthy controls ([0.59± 0.47] ％ vs [1.65 ± 0.56] ％ [t =8.544,P＜0.01],[4.46±1.84]％ vs [6.98±1.86]％[t=5.619,P＜0.01],and [16.75±6.72]％ vs [22.77±6.87]％; [t=5.311,P＜0.01].The frequencies of peripheral Th22 and Th17 after 3 months of treatment were significantly higher than those at baseline ([1.60± 1.10] ％ [t=5.268,P＜0.01] and [6.33±2.64]％ [t=3.663,P＜0.01] and no difference from those of healthy controls (t=1.783 and 1.143,respectively; both P＞0.05).However,the Th1 frequency showed no difference compared to the baseline.The frequency of Treg in the HIV infected patients was significantly increased compared with the healthy controls ([10.76±3.76]％ vs [7.01±1.88]％,t=5.003,P＜0.01).However,it gradually decreased along with the ART treatment ([9.22±2.56]％ after 2 months; [8.57± 2.36]％ after 3 months),which was still higher than that of healthy controls (t=2.984,P=0.004).The ratios of Th22/Treg,Th17/Treg and Th1/Treg of the HIV-infected patients were significantly decreased in comparison with the healthy controls (0.05±0.03 vs 0.25±0.10,t=11.69,P＜0.01; 0.46 ± 0.27 vs 1.07±0.42,t=7.728,P＜0.01; 1.56±0.89 vs 3.37± 1.02,t=7.052,P＜0.01),and those were significantly increased after 3 months of treatment (0.17±0.10[t=6.852,P＜0.01],0.81±0.46[t=4.253,P＜0.01] and 2.31±1.27[t=3.030,P＜0.01]).Correlation analysis showed that the ratio of Th17/Treg of the HIV-infected patients was positively correlated with the peripheral CD4+ T cell count (r=0.312 5,P=0.049 6),and negatively correlated with HIV RNA viral load (r=-0.474 7,P=0.002 0).The ratio of Th1/Treg of the HIV-infected patients was positively correlated with the peripheral CD4+ T cell count (r=0.333 5,P=0.035 5).Conclusions Th22,Th17,Th1 and Treg cells in the peripheral blood of HIV-infected patients are closely related to CD4+ T cell count.ART can partially recover immune imbalance,and help to rebuild immune function of HIV-infected patients.

Study on the NKG2D receptor expression on the surface of natural killer cells in the patients with hepatitis B / 中华传染病杂志

Objective To investigate difference of NKG2D receptor expression level on the surface of natural killer (NK) cells in the patients with hepatitis B and its clinical significance.Methods This was a four-arm study with different types of subjects,including patients with chronic hepatitis B (CHB,n =22),HBV carriers (HBVC,n=10),patients with acute hepatitis B (AHB,n=18) and healthy donors (HD,n=18).NKG2D protein and mRNA levels on the surface NK cells in the peripheral blood were examined by reverse transcription-polymerase chain reaction assay.The relationship between NKG 2D expression and serum hepatitis B virus (HBV) DNA level was analyzed.The data were compared by analysis of variance and linear regression.Results NKG2D mRNA expression levels in groups of HBVC, HD, AHB and CHB were 0.96±0.17, 1.03±0.12,1.53±0.30 and 1.51 ± 0.35,respectively; the differences among groups were statistically significant (q=7.586,7.485,7.920 and 7.880,respectively; all P＜0.01).NKG2D protein expression levels in groups of AHB,HD,CHB and HBVC were 0.87±0.14,0.89±0.17,0.67±0.09 and 0.59±0.13,respectively; the differences among groups were statistically significant (q=6.92,7.67,7.53and 8.16,respectively; all P＜0.01).The NKG2D mRNA expression levels on NK cells were negatively correlated with serum HBV DNA viral loads in patients with CHB,AHB or HBVC (r=-0.75,-0.66 and-0.69,respectively; all P＜0.01).The NKG2D protein levels on NK cells from patients with AHB and CHB were negatively correlated with serum HBV DNA levels (r=-0.47 and -0.45,respectively; both P＜0.05).Conclusion NKG2D mediated NK cytotoxicity may play a role in viral clearance in hepatitis B.

Expression and identification of recombinant Clostridium difficile toxin B using Bacillus megaterium system / 中华传染病杂志

Objective To express and purify recombinant and biologically active Clostridium difficile toxin B (rTcdB). Methods The genes of TcdB were amplified by polymerase chain reaction (PCR) using chromosomal DNA from a toxigenic strain, and cloned into a shuttle vector pHis1522.The sequences of TcdB genes in the vector were verified by DNA sequencing. The construction was transformed into Bacillus megaterium protoplasts and the protein expression was driven by a xylose promoter. The purified protein was tested for biological activity. Results rTcdB was successfully purified from bacterial crude extracts. Approximately 5-10 mg of highly purified recombinant toxin was obtained from one liter of bacterial culture. The expressed rTcdB had molecular mass similar to the native toxin, and its biological activity was proved to be similar to its native counterpart after an extensive examination. Conclusion rTcdB with biological activities is successfully expressed in Bacillus megaterium.

Establishment and application of neutralization assay based on H5N1 avian influenza pseudotyped virus in vitro / 中华临床感染病杂志

Objective To establish neutralization assay based on H5N1 avian influenza pseudotyped virus in vitro and to evaluate neutralizing titer of convalescent serum from 2 patients with H5N1 avian influenza.Methods pHR-Luc,pCMV△8.2 and CMV/R-SH or CMV/R-TH were cotransfected into 293T cell by co-precipitation with calcium phosphate.Pseudotyped virus supernatant was harvested 72 h posttranofection and identified the expression of HA and P24 by Western blot,and then we analyzed infective activity of 200 μL supernatant of pseudotyped virus.293T cell integrated HA was prepared and anti-HA antibodies in convalescent serum were measured with FACS assay.Neutralizing titers of convalescent serums against Shenzhen and Thailand pseudotyped virus were determined based on calculating IC50 with neutralizing assay.Results Pseudotyped virus involved P24 and HA,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Pseudotyped virus possessed better infective activity,and RLA value was about 2 × 104 with 200 μL supernatant.Both convalescent serums contained anti-HA antibodies and had cross-reactivity against different virus clades with FACS assay.Both convalescent serums had neutralizingactivity and could cross-neutralize different virus clades.However,both serums'neutralizing titers against Shenzhen virus were higher than Thailand.Conclusion We successfully constructed infectious pseudotyped virus which integrated HA of Shenzhen or Thailand virus,and it could be used for evaluation of serum neutralizing activity fast,efficiently and safely with broadly application prospect.

The effect of Th17 cells on A(H1N1) influenza virus clearance / 中华传染病杂志

Objective To investigate the phenotype, frequency of Th17 cells and the association between Th17 cells and viral clearance in patients with H1N1 influenza A. Methods Three groups including 70 confirmed patients with H1N1 influenza A, 30 patients with seasonal influenza as well as 68 healthy subjects as controls were enrolled in this study. The percentages of Th1, Th2, Treg and Th17 lymphocytes in the peripheral blood were determined by intracellular staining and flow cytometry. The levels of interferon-γ (IFN-γ), transforming growth factor-beta (TGF-β),interleukin-6 (IL-6) in plasma and supernatant of the peripheral blood mononuclear cell (PBMC)culture were quantified by enzyme-linked immunosorbent assay (ELISA). Viral load in nasopharyngeal swabs was detected by real time quantitative reverse transcription-polymerase chain reaction (RTPCR). Data were analyzed by one way ANOVA and liner correlation analysis. Results The percentage of Th17 cells in H1N1 influenza A patients was (2. 740±0. 210)%, which the percentage of was significantly decreased compared to healthy subjects (3. 443 ±0. 154)% and seasonal influenza patients (3. 443±0. 277) % (F=4. 242, P＜0. 05); while the percentage of Thl, Th2 and Treg cells were not significantly different among these groups. Moreover, the TGF-β level in plasma of H1N1 influenza A patients was (10±8) ng/mL, which was significantly lower than healthy subjects (43 ±32 ) ng/mL and seasonal influenza patient ( 18 ± 10) ng/mL ( F= 17.72, P＜0.01 ). The TGF-β level in the supernatant of PBMC culture of H1N1 influenza A patients was (782 ± 736) pg/mL, which was significantly lower than healthy subjects (1462±315) pg/mL and seasonal influenza patients (1481 ±348) pg/mL (F=5. 730, P＜0.01). Additionally, the viral clearance period was inversely correlated with the percentage of Th17 cells (r=-0.38, P=0.02). Conclusions The proportion of Th17 cells in patients with H1N1 influenza A is significantly decreased, which is closely correlated with the level of TGF-β. This decrease may results in the delayed viral clearance.

Analysis on clinical features, viral load and viral shedding period of patients with mild or severe H1N1 influenza A virus infection / 中华传染病杂志

Objective To analyze clinical and laboratory features, viral load and viral shedding period of patients with mild or severe H1N1 influenza A infection. Methods Seventy mild cases and 16 severe cases with concurrent pneumonia were included from Shcnzhen area for analysis.Nasopharyngeal-swab specimens of patients were collected and viral load was detected by real-time quantitative polymerase chain reaction (PCR) assay during their hospitalization. The viral load and viral shedding period were compared between patients over 14 years old and less than 14 years old, and between 70 mild cases without pneumonia and 16 severe cases with pneumonia. The statistic analysis was performed using t test and chi square test. Results The most common symptoms and signs of the patients were fever, cough and enlargement of tonsils. However, the severe cases suffered more frequently from cough, dyspnea and high fever compared with the mild cases (x2 = 10. 9 and 14.3, respectively, t=3.65; both P＜0.01 ). The levels of white blood cell (WBC) count and alanine arninotransferase (ALT) of severe patients were both significantly higher than those of mild patients(t= 3.2, 2.4,respectively; both P＜0.05). The chest radiology of the severe cases showed interstitial pneumonia,mostly with ground glass image. The viral load of patients under 14 years was significantly higher than those over 14 years [(4.86± 1.23) lg vs (4. 17±0.89) lg; t=2.3, P＜0.05], and the viral shedding period of patients under 14 years was significantly longer than those over 14 years [(5.33±0. 49) d vs(3. 63±0.28) d; t=3.4, P＜0.01]. The severe patients also displayed significantly higher viral load and prolonged viral shedding period than the mild patients [(6. 36±1. 44) lg vs (4. 35±0.99) lg, t=6.1,P＜0.01; (5.75±1.77) d vs (4. 24±1. 96) d, t=3.2, P＜0.01]. Conclusion Age anddisease severity of patients with H1N1 influenza A infection are significantly associated with viral load and viral shedding period.

Construction of pseudotype retrovirus which integrates hemagglutinin of H5N1 avian influenza virus isolated from human in Shenzhen / 中华微生物学和免疫学杂志

Objective To construct pseudotype retrovirus which integrates hemagglutinin(HA)of H5N1 avian influenza virus(AIV)isolated from human in Shenzhen.Methods AIV HA gene was amplified bv RT-PCR,then it was ligated with pGEM-T vector,and identified by restriction enzyme digestion and sequenced.HA gene was cloned into CMV/R vector at the site of Sal Ⅰ and BamH Ⅰ.pHR-Luc,pCMV&8.2 and CMV-HA were co-transfected into 293T cell by co-precipitation with calcium phosphate.The pseudotype virus supernatant was harvested 72 h post-transfection and ultracentrifugation,and the HA and P24 expression on the surface of pseudotype virus was analyzed by western blot.Meanwhile.the infection activity of HIV-HA pseudotype virus was identified in different kinds of cell lines,including MDCK,HeLa,CHO and 293T.Results A/Shenzhen/406H/06 belonged to subclade2.3 with open reading frame(ORF)of HA gene encoded 567 amino acides,whose accession number was EF137706 in GenBank.HA gene was cloned into CMV/R successfully.After co-transfection of above vectors,it revealed that HA protein could integrate pseudotype virus by western blot,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Finally.HIV-HA pseudotype virus could infect 4 kinds of cell lines,which indicated its property of infectivity and catholicity.Conclusion The pseudotype retrnvirns wassuccessfully constructed,which can integrate HA protein of A/Shenzhen/406H/06 and had property of infectivity.It call be used in the further research,including selection of neutralizing antibodies and epitope analysis.

Development of a serological ELISA kit for detection of EV71 infection associated with hand-foot and mouth disease and its clinical application / 中华检验医学杂志

Objective To develop an ELISA(Enzyme-Linked Immunosorbent Assay)diagnostic kit for early rapid detection of sarum anti-EV71 antibody and evaluate its clinical application value.Methods Recombinant protein VP1 of EV71 were prepared and purified as an immobilized antigen for establishment of an indirect ELISA for detection of serum anti-EV71 IgM and anti-EV71 IgG.Compared with RT-PCR.isolation of EV71 and micro-neutralizing assay.the clinical application value of anti-EV71 IgM and anti-EV71 ISG in the diagnosis of EV71 disease was evaluated.Results In comparison with RT-PCR.the sensitivity,specificity,positive predictive value and negative predictive value of anti-EV71 IgM antibody were 83%,85%,81%and 87%,respectively.The sensitivity,specificity,positive predictive value and negative predictive value of anti-EV71 IgG antibody were 72%,74%,68%and 77%.respectively.Compared with viral isolation assay.the sensitivity and specificity of anti-EV71 IgM antibody were 85%and 97%,respectively.The sensitivity and specificity of anti-EV71 IsG antibody were 75%and 77%,respectively.In addition.the titers of anti-EV71 IgG antibody were significantly correlated with the titers of neutralizing antibody to EV71 by linear regression analysis(r=0.72,P＜0.05).Finally,the serum titers of anti-IgG from patients with EV71 associated hand food and mouth disease at convalescent stage exhibited significantly higher than that of the same patients at acute stage(P＜0.01),but the titers of anti-IgM had no significant difference(P＞0.05).Conclusions With VP1 recombinant protein used as an immobilized antigen,an indirect ELISA diagnostic kit was successfully develooed for detection of serum anti-human EV71 IgM and anti-human EV71 IgG antibodies.

The role of CD4+CD25+Foxp3 regulatory T cells in patients with chronic hepatitis B / 中华传染病杂志

Objective To investigate the role of CD4+CD25+Foxp3 regulatory T cells in chronicity of hepatitis B and viral clearance of hepatitis B virus(HBV).Methods Nineteen patients with chronic active hepatitis B(CAH).21 HBV carriers(AsC)and 12 patients with resolved HBV infection and 1 5 healthy controls were enrolled.The frequency and phenotype of peripheral CD4+CD25+Foxp3+ T cells were detected by flow cytometry.CD4+CD25+T cells were sorted by magnetic-activated cell sorting(MACS)assay.Level of Foxp3 mRNA in CD4+CD25+T cells was examined by real time polymerase chain reaction(PCR)assay.The data were analyzed by one-way ANoVA or nonparametric statistics.Results Both frequencies of CD4+CD25+Foxp3+T cells and levels of Foxp3 mRNA in CD4+CD25+T ceils in patients with CAH or AsC were significantly higher than those in healthy controls Or resolved HBV infection(F=6.8,F=3.72,respectively;both P<0.05).Accumulation of Foxp3+T cells in liver tissue of CAH patients was higher than that of healthy controls,while that in AsC was lower than CAH.The frequency of CD4+CD25+Foxp3+T cells of hepatitis B e antigen(HBeAg)positive patients(including CAH and AsC)was significantly higher than that of HBeAg negative patients(t=2.3,P<0.05),and that of antFHBe negative patients were significantly higher than anti-HBe positive patients(t=2.4,P<0.05).Furthermore,the frequency of CD4+CD25+Foxp3 regulatory T cells was positively correlated with serum HBV DNA level of patients with chronic hepatitis B(r=0.56,P<0.01).Conclusion The findings have important implication in the understanding of the role of CD4'CD25'regulatory T cells in chronicity and viral clearance in HBV infection.

Chest X-ray imaging of patients with SARS / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the chest X-ray manifestations of SARS cases.</p><p><b>METHODS</b>A retrospective study was conducted among 52 clinically confirmed SARS patients from February 9 to May 10, 2003. Chest X-ray scanning was performed at a interval of 1 - 3 days according to the requirements. The manifestations and special features of SARS in X-ray were analyzed.</p><p><b>RESULTS</b>Small or large patchy shadows with intensive density in both lungs were observed in 31 cases, ground-glass like opacification in 16, small patchy shadows in one lung lobe or one lung segment in 18, nodular shadows in one lung segment in 1, and increased lung marking in lung interstitial tissues in 2. Rapidly changing consolidations revealed in chest X-ray images were found to be associated with SARS infections, and they were not affected by treatment with antibiotics.</p><p><b>CONCLUSION</b>Chest X-ray provides a sensitive and specific method for the diagnosis and treatment of SARS, and those present with symptoms and signs should undergo chest X-ray scanning every 1 - 3 days.</p>