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: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.

CRISPR-Associated Protein 9 ; Gene Editing ; CRISPR-Cas Systems/genetics*