ABSTRACT
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.
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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 specicity 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.
ABSTRACT
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% (P0. 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% (P0. 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.