Objective To evaluate the practicability of using CRISPR/Cas9 genome editing tech-nology for inhibition of hepatitis B virus ( HBV) replication. Methods Two sgRNA targeting sites were de-signed for the S region of HBV genome. The CRISPR/Cas9 expression plasmids specific for HBV were con-structed and then transfected into a cell line expressing HBV genome(HepG2-N10). The cytotoxicity of cells transfected with different expression plasmids were detected by MTT assay. The levels of hepatitis B surface antigen ( HBsAg ) were determined by using chemiluminescent immunoassay ( CLIA ) . The expression of HBV at mRNA level was analyzed by quantitative real-time PCR ( qRT-PCR) . The qPCR was performed for the detection of extracellular and intracellular HBV DNA. The next-generation sequencing ( NGS) Illumina MiSeq Platform was used to analyze HBV genome editing. Results No significant cytotoxic effects were de-tected in HepG2-N10 cells transfected with different expression plasmids. Compared with the cells carrying pCas-Guide-GFP-Scramble, the levels of HBsAg in the supernatants of transfected cell culture harboring pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were decreased by 24. 2% (P<0. 05) and 16. 9% (P>0. 05), respectively. The levels of HBsAg in cells transfected with pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were respectively decreased by 16. 4% (P>0. 05) and 32. 1% (P>0. 05) as compared with that of pCas-Guide-GFP-Scramble transfected group. The expression of HBV at mRNA level was inhibited as indica-ted by the results of qRT-PCR. Moreover, the levels of extracellular HBV DNA were respectively suppressed by 23% (P>0. 05) and 35% (P<0. 05), and the levels of intracellular HBV DNA were respectively sup-pressed by 7. 2% (P>0. 05) and 18% (P>0. 05). Different types of insertion/deletion mutation were de-tected in HBV genome by high-throughput sequencing. Conclusion HBV-specific CRISPR/Cas9 system could inhibit the expression of HBV gene and the replication of virus. Therefore, the CRISPR/Cas9 genome editing technology might be used as a potential tool for the treatment of persistent HBV infection.

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 evaluate the compatibility laws of effective-component compatibility of Bufei Yishen formula Ⅲ (ECC-BYFⅢ) in regulating mucus hypersecretion of chronic obstructive pulmonary disease (COPD).Methods:According to the efficacy of the original Chinese medicine, the components of ECC-BYFⅢ were divided into four categories: Buqi (Ginsenoside Rh1+Astragaloside), Bushen (Icariin), Huatan (Nobiletin), and Huoxue (Paeonol). The four categories were divided into 14 groups based on the method of mathematical permutation. ① The rats were divided into control group, model group, ECC-BYFⅢ, and different components compatibility groups according to the random number table, totaling 17 groups. COPD rat model in stable phase was established by cigarette smoke exposure combined with repeated bacterial infections. The corresponding drugs were given by gavage at the 9th week of modeling, and the samples were collected at the end of the 16th week. The levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of metalloproteinase 1 (TIMP-1) in serum and bronchoalveolar lavage fluid (BALF), and the levels of mucin (MUC) 5AC in lung tissue and BALF were detected by enzyme linked immunosorbent assay (ELISA). ② Human lung epithelial cells BEAS-2B were divided into blank group, model group, and different components compatibility groups. Hypoxia-induced mucus hypersecretion model of human lung epithelial cells BEAS-2B was established 4 hours after corresponding drug pretreatment. The mRNA expressions of MUC5AC, MUC5B, and MUC1 were detected by quantitative polymerase chain reaction (PCR). The mucus secretion indexes of rats and BEAS-2B cells were evaluated by Region (R) value comprehensive evaluation method.Results:① Compared with the control group, MMP-9 in serum and BALF from the model group were significantly increased, the level of TIMP-1 was significantly decreased, and MUC5AC in lung tissue and BALF were significantly increased. The results of R value comprehensive evaluation showed that except for the Buqi and Bushen groups, ECC-BYFⅢ and other components compatibility groups significantly corrected mucus hypersecretion in COPD rats, ECC-BYFⅢ, Bushen Quxie, Fuzheng Huatan, and Quxie groups were much better (R values were 2.15±0.42, 2.11±0.23, 2.16±0.23 and 2.16±0.55, respectively), compared with the model group (R value: 3.00±0.00), the differences were statistically significant (all P < 0.05). ② Compared with the blank group, the mRNA expressions of MUC5AC, MUC5B, and MUC1 increased in the model group. But different components compatibility groups had no significant effects on the mucus secretion of BEAS-2B cells. ③ The comprehensive evaluation results of R value about each in vivo and in vitro index showed that ECC-BYFⅢ, Huoxue, Quxie, Bushen Huoxue, Fuzheng Huatan, Buqi Quxie groups significantly corrected the mucus hypersecretion (R values were 2.30±0.43, 2.33±0.44, 2.12±0.68, 2.27±0.64, 2.24±0.27 and 2.29±0.47, respectively), compared with the model group (R value: 3.00±0.00), the difference was statistically significant (all P < 0.01). The order was: Quxie > Fuzheng Huatan > Bushen Huoxue > Buqi Quxie > ECC-BYFⅢ > Huoxue. Conclusions:Different components compatibility of ECC-BYFⅢ had different effects on COPD mucus secretion. The components containing Huatan (Nobiletin) or Huoxue (Paeonol) showed a better inhibitory effect on mucus secretion.

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.