Objective:To accurately ascertain the whole genome sequencing and the composition of open reading frames (ORFs) of non-replicating Tiantan strain of vaccinia virus (NTV) using next-generation long-read sequencing technology.Methods:NTV, obtained from our laboratory stock, was amplified and purified on chicken embryo fibroblast cells(CEFs), and the full-length genomic nucleic acid of NTV was extracted. The PacBio HiFi sequencing platform was utilized for de novo assembly to obtain the complete genomic sequence of NTV. Using a homology annotation strategy, we identified its ORF composition and compared it with known non-replicating vaccinia virus strains. Results:The total length of NTV′s genome was 171 729 bp, with a GC content of 33%. Its unique inverted terminal repeat (ITR) region comprised hairpin structures, two tandem repeat regions, and three non-repeat regions. NTV contained 166 ORFs, with major differences observed in the ITR and its surrounding regions when compared to MVA-BN and NYVAC. These three strains shared a common set of 138 ORFs. NTV encoded six unique ORFs related to virus evasion of host antiviral response.Conclusions:This study accurately determines the whole genome sequencing and ORFs composition of NTV, and reveals its similarities and differences with other replication-deficient vaccinia virus strains, which pave a way for the development and application of the next generation of monkeypox vaccines and novel viral vectors.

Objective:To develop a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a-based nucleic acid assay for monkeypox virus with high specificity and sensitivity.Methods:RAA primers and CRISPR RNA (crRNA) were designed based on the known conserved regions of the monkeypox virus gene and synthesized, and specific crRNAs were screened using fluorescence detection. The sensitivity and specificity of the detection system were evaluated.Results:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was developed with a sensitivity of 2.5 copies/reaction and high specificity without cross-reactivity with ectromelia virus and vaccinia virus.Conclusions:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was established, which would provide a powerful tool for efficient, rapid and specific detection of monkeypox virus.

Objective:Based on targeted amplicon technology combined with high-throughput sequencing technology and bioinformatic analysis technology, to understand the characteristics of the whole genome of the monkeypox virus and its variation, and to construct a method for the analysis of monkeypox virus variation and molecular traceability of the case in Qinghai province, and to provide technical support for the prevention and control of monkeypox epidemic in the future.Methods:The extracted viral DNA was used as a template, and the genome of monkeypox virus was specifically amplified by Ion AmpliSeq Monkeypox Panel with the number of amplicons 1 609 and the length of 125 bp-275 bp, and the sequencing library was constructed by Ion AmpliSeq Library Kit Plus, and sequenced by Ion Torrent GeneStudio S5. The sequencing library was constructed by Ion AmpliSeq Library Kit Plus, and the monkeypox virus genome was sequenced using Ion Torrent GeneStudio S5 sequencer. Monkeypox virus was analyzed for genomic profiling and mutation site analysis using the online analysis tool Nextclade. The genomic sequence of the case virus in this study was compared with some sequences in the GIASID monkeypox virus database and a phylogenetic tree was constructed to analyze the potential origin of the case virus.Results:The Ct values of monkeypox virus genes in the rash swab and oropharyngeal swab samples were 32.13 and 36.91, respectively. The rash swab sample had a reads number match of 99.99% and a genome coverage of 99.45% after whole-genome sequencing of monkeypox virus, and the sequences belonged to the IIb (West African branch) B. 1.3 type. The analysis of nucleotide mutation sites and phylogenetic tree showed that the sequences were in the same branch with four monkeypox virus genome sequences recently submitted by China and Japan in the GISAID monkeypox virus database, and had the closest evolutionary relationship with the sequence EPI_ISL_18059184 (sampled on 2023-07-03) submitted by Yunnan, China, which shared 82 single-nucleotide mutation sites, among which the sequence from Yunnan was only present in all of the shared 82 single-nucleotide mutation sites. The sequence in this study has 2 additional nucleotide mutation sites on top of the shared 82 single nucleotide mutation sites. The sequence submitted by Japan, EPI_ISL_17692269 (sampled on 2023-04-28), is more closely related in evolution, sharing 78 single nucleotide mutation sites, with 7 single nucleotide mutation site differences, and the Japanese sequence shares 78 single nucleotide mutation sites. The Japanese sequence shared 78 mutation sites with one additional nucleotide mutation site (G57786A), while the present sequence had six additional nucleotide mutation sites (G13563A, C21062T, G101241A, C142797T, G152866A, T169721A).Conclusions:The whole genome sequence of monkeypox virus of 197 084 bp was successfully obtained from a sample with low viral load, and the average. We constructed a method for sequencing and analyzing the whole genome of monkeypox virus.

Objective:To preliminarily observe the clinical efficacy of microwave hyperthermia combined with intensity-modulated radiotherapy (IMRT) and chemotherapy for patients with locally advanced gastric cancer.Methods:Forty patients who could not been operated or refused operation were enrolled in this clinical trial, who were confirmed as locally advanced proximal or distal gastric cancer by gastroscopy pathology and imaging. Radiotherapy was delivered by IMRT technology for 5 times per week with a total dose of 46 to 56 Gy (median dose of 50 Gy) in 25 to 28 fractions. Synchronous hyperthermia was given at 42 to 44℃ twice a week, 45 min/time. S-1 or capecitabine-based synchronous chemotherapy was performed, d1-14/3 weeks. The symptom remission rate, adverse reactions, objective remission rate (complete and partial remission) and survival were observed.Results:A total of 40 patients, aged between 56 and 83 years (median age of 71 years), were enrolled in this study. The male-to-female ratio was 7: 1. Among them, 38 cases (95%) showed symptom remission. The most common adverse reactions were grade 1-2 gastrointestinal reactions and leukopenia. The objective remission rate was 87.5%, the 2-year progression-free survival and overall survival rates were 68.6% and 70.5%, respectively.Conclusion:Preliminary findings demonstrate that microwave hyperthermia combined with chemoradiotherapy achieve satisfactory outcomes and yield tolerable toxicity in patients with locally advanced gastric cancer.

Objective:To establish and evaluate a rapid nucleic acid detection method for SARS-CoV-2 based on COYOTE ? Flash20 real-time fluorescent quantitative PCR instrument. Methods:A rapid reaction system was constructed by using specific primer and probe sets targeting ORF1ab and N gene of SARS-CoV-2, and the sensitivity and specificity of the system were verified. At the same time, 108 clinical samples of COVID-19 were used to evaluate the application of this method.Results:The detection method did not require nucleic acid extraction, and the manual operation time was only one minute. After the sample was sent to the system, the test could be completed in 30 minutes. The detection limit of this method was 4×10 2 copies/ml. It had no cross-reactivity with other human coronaviruses (including HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV and MERS-CoV) and other respiratory viruses. The evaluation of clinical sample application showed that the total coincidence rate with the conventional RT-qPCR which required nucleic acid extraction was 98.15%. Conclusions:Through the application evaluation of the rapid fluorescent quantitative PCR method of SARS-CoV-2, it was found that the method was simple, fast, specific and sensitive, and it was suitable for real-time and rapid detection needs in varieties of situations.

Influenza poses a serious threat to global public health and causes serious economic los-ses. Vaccination is the most effective measure to prevent influenza. However, the mismatch between the vac-cine strain and the epidemic strain, which results from antigenic drift and antigen conversion of influenza vi-rus, often invalidates the conventional vaccine stockpiles. mRNA vaccine is a relatively safe platform com-posed of nucleic acid. Improvements in genetic engineering technology and novel delivery systems have great-ly promoted the research and development of mRNA vaccines against influenza. mRNA vaccines are promis-ing candidates for the rapid and efficient prevention of seasonal influenza epidemics and influenza pandemics.

Objective To evaluate the expression profile of succinate dehydrogenase (SDH)B and SDHC in pheochromocytoma (PCC) and paraganglioma(PGL) (collectively abbreviated as PPGL), and their value in the early diagnosis of malignancy. Methods SDHB and SDHC immunohistochemistry were performed on 140 tumor specimens from 126 PPGL patients (PCC n＝62, PGL n＝61, PCC+PGL n＝3). Results (1) Germline mutation status of 67 patients were determined, of which, identifying 37(55.2%) patients with germline mutation: 2 (3.0%) SDHA, 18 ( 26. 9%) SDHB, 2 ( 3. 0%) SDHC, 5 ( 7. 5%) SDHD, 2 ( 3. 0%) VHL, 7 ( 10. 4%) RET, and 1(1.5%) NF1; and 30 (44.8%) individuals without known mutation. (2) Among 30 PPGLs from 27 patients with SDH-related (SDHx) mutations, 96.7%(29/30) stained negative for SDHB, 76.7%(23/30) stained negative for SDHC, while only 28.6%(14/49) and 18.4%(9/49) stained negative for SDHB and SDHC respectively in the 49 PPGLs without SDHx mutation (P<0.05). (3) The sensitivity of the SDH immunostaining in detecting the presence of germline SDHx mutation was 96.7%for SDHB and 76.7%for SDHC, while the specificity was 71.4%for SDHB and 81.6% for SDHC. ( 4 ) Among PPGLs without SDHB expression, 22. 9% were malignant. This percentage is significantly higher than that in PPGLs with preserved SDHB expression (3.8%, P<0.05). Conclusion SDHB and SDHC immunohistochemistry may serve as post-surgical screening tools to predict the presence of germline SDHx mutation in PPGLs. Negative SDHB expression calls for intense follow-up to rule out malignancy.

OBJECTIVE To assess the prevalence and analyze the influence factors of laryngopharyngeal reflux disease(LPRD) in the Fuzhou region, in order to provide a theoretical basis for the development of prevention and control measures for LPRD. METHODS A questionnaire survey in residents in Fuzhou by a random cluster sampling was carried out. Individual information, reflux symptom index(RSI) of Belafsky and risk factors were included. Patients more than 13 scores of RSI were defined as LPRD. Data were statistically analyzed. RESULTS A total of 4100 residents were investigated, 4063 of them were available. The prevalence of LPRD was 5.00%. Often eating too much, often drinking strong tea, menolipsis, rhinitis, tonsillitis were closely related to LPRD. CONCLUSION The prevalence of LPRD in Fuzhou region were closely related to many factors.

Objective:To establish an HPLC method for the determination of paraquat in human serum.Methods:The analytical column was a Kromasil C18column (200mm×4.6mm,5μm).The mobile phase was a mixture of acetonitrile and water ( containing 0.03 mol·L-1sodium heptanesulfonate and 0.24 mol·L-1phosphoric acid) (3 :97,pH was adjusted to 2.0 by triethylamine),the detection wavelength was set at 258 nm,the column temperature was 25℃,the injection volume was 20 μl,and the flow rate was 0.8 ml·min-1.Results:The calibration curve of paraquat was linear within the range of 0.106-10.6 mg·L-1( r =0.999 3),and the lower limit of detection was 0.065 mg·L-1. The absolute recovery of paraquat at low,medium and high concentration was more than 89.4%,and the method recovery was more than 94.4%. The intra-day RSDs were 0.12%-1.74%,and the inter-day RSDs were 0.44%-2.89%.Conclusion:The method is simple,quick,accurate,sensitive and specific,and can be used for detecting paraquat con-centration in human serum.

Objective To investigate the feasibility of using recombinant infectious clones of hu-man coronavirus OC43 (HCoV-OC43) as a vector for the expression of exogenous genes and to analyze the insertion sites. Methods Based upon pBAC-OC43FL, a full-length cDNA infectious clone of HCoV-OC43, three recombinant expression plasmids ( pBAC-OC43-GFPΔNS2, pBAC-OC43-GFPΔNS12. 9 and pBAC-OC43-N-GFP) were respectively constructed by replacing NS2 and NS12. 9 genes with the reporter gene en-coding the green fluorescent protein ( GFP ) and inserting the reporter gene after the N gene by using the overlapping-PCR and in vitro ligation. Reverse genetics techniques were used for viral rescue. All of the res-cued virus strains were characterized by immunofluorescence assay ( IFA) and Western blot ( WB) assay af-ter transfecting BHK-21 cells with the recombinant viruses. Results Two recombinant viruses, OC43-GFPΔNS2 and OC43-GFPΔNS12. 9, could be successfully rescued by transfection the BHK-21 cells with pBAC-OC43-GFPΔNS2 and pBAC-OC43-GFPΔNS12. 9 plasmids. The expressed GFP was observed in BHK-21 cells transfected with pBAC-OC43-GFPΔNS2 or pBAC-OC43-GFPΔNS12. 9 plasmids, but not in the cells transfected with the pBAC-OC43-N-GFP plasmid. An efficient and stable expression of GFP was observed in the pBAC-OC43-GFPΔNS2 plasmid-transfected cells. The 10th generation of OC43-GFPΔNS2 virus was ob-tained after repeated freezing and thawing. The expression of GFP and N protein were detected in cells infec-ted with the OC43-GFPΔNS2 virus after 10 passages. Conclusion The NS2 gene of HCoV-OC43 could be used as a promising insertion site of the pBAC-OC43 FL infectious clone for the expression of exogenous genes. This study might provide a platform for further researches on the replication of HCoV-OC43 and the development of human coronavirus-based vectors.