Objective To obtain the antigen and antibody of JC virus(JCV)VP2.Methods The JCV VP2 gene were amplified from a cerebrospinal fluid sample by polymerase chain reaction (PCR)and confirmed by sequencing.Then,the gene was cloned into plasmid pET32a(+)to construct recombinant prokaryotic expression vector pET-32a(+)-VP2.The recombinant plasmid was transformed into the competent E.coli BL21.Induced with isopropyl-β-D-1-1 thiogalactopyranoside (IPTG),E.coli BL21 were subsequently crushed by ultrasound.The gene expression in the supernatant was analyzed by Western blot.Thereafter,the expressed protein was purified by isoeleetric point method.The polyclonal antibody against JCV VP2 protein was obtained from the BALB/c mouse immunized with the purified protein.Results The VP2 fusion protein was expressed in the E.coli BL21.The recombinant fusion protein was expressed by IPTG induetion with relative molecular mass of 58.5×10~3.Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDSPAGE)analysis showed that the expression level was highter after 6-10 h of IPTG induction.The recombinant protein had good antigenicity which was confirmed by BALB/c mice immunized with the protein.Conclusions The successful expression and purification of VP2 fusion protein and the antibody will be valuable for the study on the biological function of VP2 and JCV epidemiologieal investigation.

Objective To investigate the capsular polysaccharide (CPS) serotypes and molecular characteristics of carbapenem resistant hypermucoviscous Klebsiella pneumoniae (CR-HMKP) and study the possible mechanism of carbapenem resistance. Methods A retrospective study was conducted on 18 nonduplicate CR-HMKP strains which were collected from the First Affiliated Hospital of Nanchang University from 2012 to 2016. The clinical data were retrieved from medical records. The capsular serotypes, resistance genes and virulence factors were detected by polymerase chain reaction and DNA sequencing. Antimicrobial susceptibility testing was determined on VITEK 2 compact system. The CR-HMKP strains were characterized molecularly by using PCR, multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). Modified carbapenem inactivation method was used to screen carbapenemase-producing strains. Plasmid conjugation transfer experiments were carried out to study transmission of carbapenem resistance. Results Eighteen (2.7％) CR-HMKP isolates were identified, which belonged to 4 serotypes, including wzi128-K1 (n=1), wzi206-K57 (n=1), wzi2-K2 (n=2), and wzi64-K14.64 (n=14). PCR and sequencing analysis identified blaNDM-1 gene in 2 CR-HMKP strains, blaKPC-2 gene in 17 strains, qnrS1 gene in 18 strains, blaCTX-M-3 gene in 3 strains, blaCTX-M-14 gene in 18 strains, blaTEM-1 gene in 16 strains, blaSHV-12 gene in 17 strains, and rmtB in 5 strains. All the 18 CR-HMKP strains carried virulence-associated genes, including rmpA (88.9％, 16/18), magA (5.6％, 1/18), iroN (83.3％, 15/18), aerobactin (27.8％, 5/18), rmpA2 (66.7％, 12/18) and mrkD (100％, 18/18). Three sequence types (STs) were identified by MLST, including ST11 (15 strains), ST86 (2 strains), and ST412 (1 strain). PFGE resulted in three major PFGE clusters, of which cluster A corresponds to ST1 isolates, and cluster B corresponds to ST86 isolates, and cluster C corresponds to ST412 isolates. All the blaKPC-2- positive strains belonged to ST11. Plasmid conjugation was successful in 5 (27.8％) of the 18 CR-HMKP isolates. Conclusions wzi64-K14.64 is the predominant capsule serotype of the CR-HMKP strains in this hospital. KPC-2 gene conjugationmay contribute to the emergence of CR-HMKP isolates. In addition, CRHMKP strain may be the highly prevalent ST11, and highly virulent CPS serotypes harboring K1/K2.

Objective:To investigate the expression levels of interferon-α receptor (IFNAR), interferon-stimulated gene factor 3(ISGF3), double-stranded RNA-activated protein kinase(PKR) and ribonuclease L (RNase L) in patients with chronic hepatitis B (CHB) treated with interferon.Methods:From July 2014 to June 2017, 41 treatment naive CHB patients were enrolled in the Department of Infectious Diseases, First Affiliated Hospital of Nanchang University. Eighteen patients were treated with polyethylene glycol interferon α-2b, and 23 patients were treated with conventional interferon. The mRNA and protein expression levels of IFNAR1, IFNAR2, ISGF3, PKR and RNase L in peripheral blood mononuclear cells (PBMC) were detected by reverse transcription polymerase chain reaction and Western blot, respectively. The differences of these molecular expression levels in PBMC between the effective and ineffective groups were compared. The data were analyzed by t test. Results:After 24 weeks of treatment, 25 cases were effective, while 16 cases were ineffective. At four weeks of treatment, the mRNA expression levels of IFNAR1, IFNAR2 and PKR in PBMC of the effective group were 0.748±0.129, 1.169±0.125 and 1.047±0.091, respectively, which were all higher than those in the ineffective group (0.591±0.021, 0.689±0.059 and 0.791±0.033, respectively). The differences were statistically significant ( t=-4.304, 16.482 and -5.346, respectively, all P<0.01). The mRNA expressions of ISGF3 and RNase L in PBMC of the effective group were 0.739±0.159 and 0.780±0.140, respectively, while those in the ineffective group were 0.690±0.035 and 0.733±0.122, respectively, which were not significantly different ( t=-0.160 and -1.443, respectively, both P>0.05). The mRNA expression levels of IFNAR1, IFNAR2, ISGF3, PKR and RNase L at baseline, week eight, 12 and 24 of treatment in the effective group were all higher than those in the ineffective group (all P<0.01). The protein expression levels of IFNAR1, IFNAR2, ISGF3, PKR and RNase L in the effective group were all higher than those in the ineffective group (all P<0.01). Conclusion:After interferon treatment, the mRNA and protein expression levels of IFNAR1, IFNAR2, ISGF3, PKR and RNase L in PBMC of CHB patients are all increased, especially IFNAR2 and PKR levels increase in the early stage of treatment (four weeks).

Objective To investigate the antimicrobial resistant mechanisms of high level carbapenem resistant Klebsiella pneumoniae infection of burn patients .Methods A retrospective study was conducted on totally 18 non-repetitive high level CR-KP which were isolated from burn patients hospitalized between July 2014 and June 2015.MIC of antibiotics were determined by using the GN 13 cards and agar dilution method.The specific PCR and DNA sequence analysis were performed to confirm the β-lactamase type.Plasmid conjugation transfer experiments and southem hybridization were applied to study the mode of carbapenem resistance transmission .Outer membrane proteins ( Omps) were isolated and examined by PCR and ( sodium dodecyl sulfate polyacrylamide gel electrophores ) SDS-PAGE.Pulsed-field gel electrophoresis( PFGE ) and Multilocus sequence typing ( MLST ) was used to determine the genotypes . Results Susceptibility of antimicrobial agents indicated that all these strains with multiple drug resistance . The resistance rate to piperacillin/tazobactam, ceftriaxone, levofloxacin, ciprofloxacin, gentamicin, tobramycin, cefotetan, ceftazidime, cefepime, aztreonam, imipenem, and meropenem was 100％ (18/18).Moreover, the resistance rate of CR-KP isolates to amikacin was 72.2％ ( 13/18 ) , compound sulfamethoxazole was 61.1％(11/18), tigecycline was 0％(0/18).Conjugation study with Escherictda coli J53 resulted in the transfer of significant reduced carbapenem susceptibility from donors (MICs increased at least 8-fold).By PCR, eighteen strains of Klebsiella pneumoniae carried NDM-1 gene, 5 strains carried KPC-2 gene.The blaNDM-1 was transferable by plasmids.Southern blot hybridization indicated that the blaNDM-1 gene was located on plasmid in size of 46 kb.The plasmid belonged to incompatibility group IncX 3.Seven types of CR-KP were detected by PFGE.In addition, MLST assigned them to sequence type ( ST)11, ST395, ST17, ST37, ST263, ST14 and ST76 types.SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that there was absence of outer membrane proteins OmpK 36 in ST11, ST395, ST37 strains.However, the other STs strains expressed lower quantities of OmpK 36.Conclusions High level carbapenem resistance in K.pneumoniae causing infection in burn patients is attributable to production of plasmid-mediated metallo· β-laetamase NDM-1 combined with porin OmpK36 deficiency or low expression .The K.pneumoniae with NDM-1 and KPC-2 carbapenemase were detected .

Objective To investigate the role of Th17/Treg balance in immune mechanism in severe exacerbation of hepatitis B.Methods The clinical data of 41 patients with chronic hepatitis B were collected,and according to the conditions during hospitalization,these patients were divided into exacerbation group (19 patients) and improvement group (22 patients).On admission,at weeks 1 and 2 of treatment,and at the end of treatment,flow cytometry was used to measure the frequencies of Th 17 and Treg cells in peripheral blood,and enzyme-linked immunosorbent assay was used to determine the serum levels of interleukin-17 (IL-17),interleukin-10 (IL-10),and transforming growth factor-β (TGF-β).The dynamic changes in the frequencies of Th17 and Treg cells were compared between the two groups,and the correlation between clinical indices for hepatitis and cytokines was analyzed.The t-test was used for comparison between groups,a one-way analysis of variance was used for comparison within one group across different time points,and Pearson correlation analysis was performed.Results With disease progression,the exacerbation group showed an increase in the frequency of Th 17 cells and a relatively low frequency of Treg cells;compared with the improvement group,the exacerbation group had a higher frequency of Th 17 cells and a lower frequency of Treg cells.Th1 7/Treg ratio gradually increased with exacerbation and decreased with improvement in conditions;in the exacerbation group and the improvement group,Th17/Treg ratio was positively correlated with total bilirubin and negatively correlated with prothrombin activity.In the exacerbation group and the improvement group,Th 17 cells were positively correlated with IL-17,and Treg cells were positively correlated with IL-10 and TGF-β.Conclusion Th1 7 and Treg cells play important roles in severe exacerbation of hepatitis B,and Th1 7/Treg ratio may be used as an immunobiological marker for the judgment of severity during severe exacerbation of hepatitis B.

Objective: To determine the clinical efficacy of platelet-rich plasma (PRP) injection combined with warm acupuncture and moxibustion for treating patients with knee osteoarthritis and cold dampness obstruction syndrome. Methods: One hundred and twenty-eight patients with knee osteoarthritis and cold dampness obstruction syndrome who visited the Rehabilitation Department and Orthopedics Department of the Second Affiliated Hospital of Anhui University of Chinese Medicine from January 2023 to March 2024 and who met the inclusion and exclusion criteria were randomly divided into an experimental (n=64) and control group (n=64) using the random number table method. The experimental group was treated with PRP injection combined with warm acupuncture and moxibustion, whereas the control group was treated with normal saline injection combined with warm acupuncture and moxibustion treatment. PRP and normal saline injections were administered once every two weeks, a total of four times. Patients were treated with warm acupuncture and moxibustion once a day, six times a week, for four consecutive weeks. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Traditional Chinese Medicine (TCM) syndrome, visual analog scale (VAS), and Lysholm scores were determined before treatment, at week 4 and week 8 of treatment, and week 16 of follow-up. Serum interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), tumor necrosis factor-α (TNF-α), osteoprotegerin (OPG), bone gla protein(BGP), and cartilage oligomeric matrix protein (COMP) levels were compared between the two groups before and after 8 weeks of treatment. The clinical efficacy and safety indicators between the two groups were also compared. Results: There was no statistical difference in baseline data such as gender, age, disease duration, and body mass index between the two groups of patients. Compared with before treatment, both groups showed decreased WOMAC total and subscale, TCM syndrome total score and symptom scores, and VAS scores, and an increase in Lysholm scores at 4, 8, and 16 weeks after treatment. After treatment, serum IL-6, MMP-3, TNF-α, and COMP levels decreased, whereas serum OPG and BGP levels increased (P<0.05). Compared with the control group, patients in the experimental group showed decreased WOMAC total and subscale, TCM syndrome total score and symptom scores, and VAS scores, and an increase in Lysholm score at 4, 8, and 16 weeks after treatment. Compared with the control group, patients in the experimental group showed decreased serum IL-6, MMP-3, TNF-α, and COMP levels and an increase in serum OPG and BPG levels after treatment (P<0.05). The total effective rate of the experimental group was 91.94%, higher than that of the control group (81.97%; P<0.05). Conclusion PRP injection combined with warm acupuncture and moxibustion can improve various TCM symptoms, improve knee joint function and bone metabolism, and reduce inflammation in patients with knee osteoarthritis and cold dampness obstruction syndrome.

Objective To investigate the efficacy and safety of intravesical instillation of BCG vaccine in the prevention of early recurrence of middle and high risk non-muscle invasive bladder cancer.Methods From July 2015,patients with non-muscle invasive bladder cancer aged 18-75 years with informed consent were screened and underwent transurethral resection of bladder tumor (TURBT).Immediately intravesical instillation of epirubicin 50 mg was given postoperatively.After pathology was comfirmed,patients was enrolled in group 1 (BCG15) or group 2 (BCG 19) or the control group (epirubicin 18) randomly with SAS 9.3 software.Data of follow-up and Adverse event was collected and analyzed.Results By May 31,2019,531 patients were enrolled in the study.The drop-off rate was 20.1％.167 patients (143 males and 24 females)in group 1,172 patients (141 males and 31 females)in group2 and 84(75 males and 9 females) in the control group with follow-up data were analyzed.There were no significant differences in age,gender,BMI,ECOG score,risk stratification between the three groups (P =0.8641,P =0.2906,P =0.9384,P =0.6126).The median follow-up time makes no statistical difference between the groups (P =0.9251),12.0 (6.0,22.5) months,13.0 (6.0,22.3) months,and 13.0 (7.0,22.3) months.The median recurrence time of the three groups was 4.0 (3.0,6.0) months,4.5 (3.0,9.8) months,4.5 (3.0,8.8) months.There was no statistical difference between the three groups (P =0.2852).Risk stratification in the patients got no significant difference between the three groups (P ＞ 0.05).The 1-year recurrence-free survival rates were 80.0％ in the group 1 and 88.3％ in the group 2 and 73.7％ in the control group.The group 2 was superior to the group 1 and the control group (P =0.0281,P =0.0031).There was no significant difference between group 1 and control group (P =0.2951).There was no significant difference in the cumulative recurrence-free survival between the experimental group 1 and the experimental group 2,(95％ CI 0.80-2.43,P =0.2433).The cumulative recurrence-free survival in the group 1 and the group 2 was better than the control group (95 ％ CI 0.31-0.92,P =0.0266;95 ％ CI 0.20-0.65,P =0.0008).All the cases underwent instillation were analyzed for adverse events.The incidence of overall AE(adverse events) in group 1 was 68.5％ (152/222),the incidence of grade Ⅰ-Ⅱ AE was 53.2％ (118/222),the incidence of grade Ⅲ-Ⅳ AE was 15.3％ (32/222).The incidence of overall AE in the group 2 was 71.8％ (160/223),the incidence of grade Ⅰ-Ⅱ AE was 60.1％ (134/223),and the incidence of grade Ⅲ-Ⅳ AE was 11.7％ (26/223).The overall AE rate in the control group was 53.2％ (59/111),of which the incidence of grade Ⅰ-Ⅱ AE was 42.4％ (47/111),and the incidence of grade Ⅲ-Ⅳ AE was 10.8％ (12/111).There was no difference in the incidence of overall AE between the group 1 and the group 2 (P =0.4497).The incidence of AE in the two experimental groups was higher than that in the control group (P =0.0062,P =0.0008).There was no difference in the incidence of grade Ⅲ-Ⅳ AE between the three groups (P =0.3902).Conclusions BCG(19 instillation schedule) has a better effect on preventing recurrence after 1 year of bladder surgery,which is superior to epirubicin group.The long-term efficacy of BCG in preventing recurrence and the efficacy of different schedules need to be further followed up.The lower urinary tract symptoms,which are mainly urinary frequency,are one of the causes of case fallout and should be fouced in future.Compared with epirubicin,BCG perfusion does not increase the incidence of grade Ⅲ-Ⅳ adverse reactions,and is safe to use.

Objective:To investigate the 2 years’ efficacy of intravesical instillation of domestic BCG versus epirubicin in the prevention of recurrence of intermediate-risk or high-risk non-muscular invasive bladder cancer and predictive factors of BCG instillation.Methods:From July 2015 to June 2020, 18-75 years old patients with moderate to high-risk non muscle invasive bladder cancer (NMIBC) confirmed by pathological examination were involved. The ECOG score was 0-2. Exclusion criteria included ①immune deficiency or impairment (such as AIDS), using immunosuppressive drugs or radiotherapy, suspected allergic to BCG or epirubicin or excipients of the two drugs, fever or acute infectious diseases including active tuberculosis or receiving anti tuberculosis treatment, with severe chronic cardiovascular and cerebrovascular diseases or chronic kidney disease; ②combined with other urogenital system tumors or other organ tumors; ③combined with muscle invasive bladder urothelial carcinoma (≥T 2); ④undergoing chemotherapy, radiotherapy or immunotherapy within 4 weeks (immediate instillation after surgery not included); ⑤ pregnant or lactating women; ⑥ comfirmed or suspected bladder perforation; ⑦gross hematuria; ⑧cystitis with severe bladder irritation that may affect the evaluation; ⑨participat in other clinical trials within 3 months; ⑩alcohol or drug addiction; ?any risk factors that may increasing the risk of patients. Epirubicin 50 mg was irrigated immediately after the operation(TURBT or laser resection). The patients were randomly divided into BCG15 group, BCG19 group and epirubicin group by the ratio of 2∶2∶1, and the patients were maintained intravescical instillation for 1 year. The recurrence and adverse events of the three groups were compared. Univariate and multivariate analysis was performed to predict the risk factors of BCG irrigated therapy failure. Result:By June 15, 2020, the median follow-up duration was 22.1 months(12.1, 32.3), and there was no statistical difference between the groups ( P=0.9024). There were 274 patients enrolled in BCG19 group, 277 patients enrolled in BCG15 group and 130 patients enrolled in the epirubicin group. The drop-off rate was 16.6%(113 cases)and made no difference between groups( P=0.6222). There were no significant difference in age, gender, BMI, or ECOG score( P>0.05). During the follow-up, 116 cases was detected recurrence or progression. The recurrence rate of the three groups was 14.2% and 14.8% in BCG19 group and BCG15 group, and 27.7% in the epirubicin group. There was no difference in recurrence rate between BCG19 and BCG15 group( P=0.9464). The recurrence rate of BCG19 group was lower than that of the epirubicin group ( P=0.0017). The recurrence rate of BCG15 group was lower than that of the epirubicin group ( P=0.0020). There was no difference in the cumulative recurrence free survival rate between BCG19 and BCG15 group (95% CI0.57-1.46, P=0.7173). The cumulative recurrence free survival rate of BCG 19 group was better than that of the epirubicin group( HR=0.439, 95% CI0.26-0.74, P=0.0006), and the cumulative recurrence free survival rate of BCG15 group was better than that of the epirubicin group ( HR=0.448, 95% CI0.29-0.80, P=0.0021). The total incidence of adverse events in 19 BCG19, BCG15 and epirubicin group were 74.5%, 72.6% and 69.8% respectively. There was no difference in the incidence of adverse events between BCG19 and BCG15 group( P=0.6153). The incidence of adverse events in epirubicin group was lower than that of BCG19( P=0.0051) and BCG15( P=0.0167) groups.There was no significant difference in the incidence of serious adverse events (SAE) among the three groups ( P=0.5064). Log rank test univariate analysis and Cox risk regression model multivariate analysis showed that the history of bladder cancer recurrence( HR=6.397, 95% CI1.95-20.94, P=0.0001)was independent risk factor for BCG irrigation failure. Conclusions:The 2 years’ efficacy of intravesical instillation of domestic BCG is better than than of epirubicin with good tolerance and safety. There is no difference between BCG19 and BCG15 group. BCG doesn’t increase SAE compared with epirubicin. Recurrence status was an independent prognostic factor regarding recurrence-free survival.

COVID-19 is caused by a novel coronavirus-severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which has being spreading around the world, posing a serious threat to human health and lives. Neutralizing antibodies and small molecule inhibitors for virus replication cycle are the main antiviral treatment for novel coronavirus recommended in China. To further promote the rational use of antiviral therapy in clinical practice, the National Center for Infectious Diseases (Beijing Ditan Hospital Capital Medical University and the First Affiliated Hospital, Zhejiang University School of Medicine) invited experts in fields of infectious diseases, respiratory and intensive care to develop an Expert Consensus on Antiviral Therapy of COVID-19 based on the Diagnosis and Treatment Guideline for COVID-19 ( trial version 10) and experiences in the diagnosis and treatment of COVID-19 in China. The consensus is concise, practical and highly operable, hopefully it would improve the understanding of antiviral therapy for clinicians and provide suggestions for standardized medication in treatment of COVID-19.