Objective:To establish a nucleic acid detection method for human parainfluenza virus (HPIV) based on reverse transcription recombinase-aid amplification (RT-RAA) combined with CRISPR/Cas12a.Methods:The type-specific primer pairs of RT-RAA and CRISPR RNA (crRNA) targeted on conserved sequence of nucleocapsid protein (N) gene of HPIV were designed. Fluorescence intensities from cleavage of fluorophore labeled probes mediated by Cas12a were measured for screening of crRNA and concentration optimization of crRNA, Cas12a as well as the probe. With the RNA transcribed in vitro and clinical specimen, the lower limit of detection and specificity of RT-RAA combined CRISPR/Cas12a detection were evaluated. Results:The crRNA specific to each type of HPIV 1-4, with strongest cleavage activity, were screened out. With the optimal concentration of crRNA, Cas12a as well as the probe, the lower limit of detection could reach 10 copies of target gene per reaction on fluorescence intensity measurement. No cross-reaction was found in clinical samples of eight other respiratory viruses detected by this method .Conclusions:The established HPIV1-4 fluorescence CRISPR nucleic acid detection method is rapid, specific, and does not require professional nucleic acid detection equipment.

Objective:To improve the efficiency and success rate of human adenovirus 3 (HAdV-3) whole genome sequencing, a high-throughput sequencing method for the whole genome of HAdV-3 based on multiplex PCR was established.Methods:Multiplex PCR primers suitable for the whole genome amplification of HAdV-3 were designed. The whole genome sequence of HAdV-3 was amplified by multiplex PCR, and the specificity of the amplification product was preliminarily verified by agarose gel electrophoresis. High-throughput sequencing of the multiplex PCR products was performed using Illumina second-generation sequencing. After obtaining the sequence, software such as CLC and IGV were used to analyze the effective data amount, average sequencing depth, and whole genome coverage obtained by high-throughput sequencing, then the sequencing quality was evaluated. Based on the whole genome sequencing result, a phylogenetic tree was constructed to confirm the virus type and analyze homology of the sequences, and then the accuracy of this method was evaluated.Results:A total of 70 (35 pairs) multiplex PCR amplification primers for the whole genome of HAdV-3 were designed, with amplicon size of approximately 1 200 bp. And the expected whole genome coverage could reach 99.8% (with a total genome length of approximately 35 240 bp). Agarose gel electrophoresis analysis showed that the size of the multiplex PCR products was consistent with expectations, and the amplification specificity was high. The high-throughput sequencing result showed that the whole genome sequences obtained by this method were complete and intact, and the genome coverage was consistent with expectations. Sequence quality analysis showed that the high-throughput sequencing method based on multiplex PCR could obtain more effective data and greater sequencing depth, resulting in more uniform whole genome coverage. Phylogenetic analysis showed that the evolutionary typing result of viral DNA sequenced after multiplex PCR amplification were consistent with those of viral DNA sequenced directly and had high homology, indicating that the multiplex PCR method had high amplification fidelity and the results obtained in combination with high-throughput sequencing were accurate.Conclusions:A high-throughput sequencing method for the whole genome of HAdV-3 based on multiplex PCR was established in this study successfully. This method could improve the efficiency and success rate of HAdV-3 whole genome sequencing, aiming to provide better technical support and reference for HAdV-3 pathogen surveillance and epidemic source-tracing based on whole genome sequencing.

Objective To explore the potential mechanism of"hot flashes"in mice with type 2 diabetes with yin deficiency syndrome.Methods Ninety-nine 8-week-old db/db mice with fasting blood glucose≥16.7 mmol·L-1 were divided into yin deficiency syndrome group,type 2 diabetes group,type 2 diabetes with yin deficiency syndrome group,type 2 diabetes with Liuwei Dihuang Decoction low,medium,high-dose group,type 2 diabetes with yin-deficiency syndrome and Liuwei Dihuang Decoction low,middle,high-dose groups,21 littermates of wild control male m/m mice were control group.The model was established by the method of"heat engenders yin deficiency",and the performance of yin deficiency syndrome in mice was evaluated after 4 weeks.Afterwards,the corresponding doses of Liuwei Dihuang Decoction group were given by gavage for 4 weeks.During the experiment,the rectal temperature,the temperature of the precordial area,and the temperature of the limbs and paws were measured once a week at 8:30,15:00,and 20:00.Western blot was used to observe the expression of biological rhythm-related proteins in the hypothalamus and liver.Results The experimental study showed that the yin deficiency syndrome model of type 2 diabetes could be established by using the method of overheating and injuring the yin.The rhythm changes of Liuwei Dihuang Decoction showed that the high-dose group could effectively restore the body temperature rhythm.The expression of rhythm factors Clock and Cry2 increased in both the hypothalamus and liver;in the hypothalamus,the central rhythm regulation center,the expressions of Per2 and Cry2 increased significantly;in the peripheral target organ liver,the expressions of Bmal1,Per3 and Cry1 increased more significantly.Conclusion The abnormal expression of biological rhythm protein in the liver and hypothalamus may be one of the important mechanisms leading to the characteristics of"hot flashes"in type 2 diabetes with yin deficiency.

Objective:To discuss the role of ataxia telangiectasia and Rad3-related kinase (ATR) /check point kinase 1 (CHK1) signaling pathway in the occurrence and development of epithelial ovarian cancer.Methods:Human epithelial ovarian cancer cells OVCAR3 cells were cultured in vitro, siRNA and VE-822 were used to interfere with ATR in OVCAR3 cells, the effectiveness of interference with ATR expression was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, the cell proliferation inhibition was detect by CCK-8 method, cell cycle and apoptosis were detected by flow cytometry (FCM) , the mRNA expressions of Caspase-3, B-cell lymphoma-2 (Bcl-2) , Bcl-2-associated X protein (Bax) were detected by qRT-PCR, Western blot was used to detect the expression of ATR/CHK1 pathway related proteins.Results:After siRNA-ATR transfection and VE-822 intervention, compared with those in NC group and siRNA-sc group, the expression level of ATR mRNA [ (1.55±0.12) , (1.51±0.13) , (0.71±0.11) , (0.73±0.12) , (0.49±0.09) ] in OVCAR3 cells in siRNA-ATR group, VE-822 group and siRNA-ATR + VE-822 group was significantly lower ( P<0.05) , the inhibition rate of cell proliferation [ (0.00±0.00) %, (0.00±0.00) %, (32.84±1.08) %, (30.75±1.44) %, (43.90±1.57) %], ratio of G0/G1 phase [ (40.08±2.57) , (36.35±3.44) , (53.28±4.34) , (56.37±5.03) , (70.63±3.81) ], apoptosis rate [ (4.28±0.67) %, (5.35±0.94) %, (23.63±1.13) %, (24.57±1.20) %, (35.86±1.09) %], expression of Caspase-3 and Bax mRNA were significantly higher ( P<0.05) , and the cell proportions in S phase [ (32.93±3.02) , (35.35±2.82) , (25.79±3.61) , (23.74±3.54) , (18.04±2.37) ] and G2/M phase [ (26.99±2.84) , (28.30±2.72) , (20.93±3.01) , (19.98±2.87) , (11.33±2.11) ], Bcl-2 mRNA, ATR, p-CHK1/CHK1, cell division cycle protein 25C (CDC25C) and cyclin B1 protein expression were significantly lower ( P<0.05) . Compared with those in siRNA-ATR group, the expression of ATR mRNA in siRNA-ATR + VE-822 group was further decreased ( P<0.05) , the inhibition rate of cell proliferation, apoptosis, expression of Caspase-3 and Bax mRNA were further increased ( P<0.05) , and the expression of Bcl-2 mRNA, ATR, p-CHK1/CHK1, CDC25C and cyclin B1 protein was decreased continuously ( P<0.05) . Conclusion:ATR/CHK1 signaling pathway is activated during the proliferation of epithelial ovarian cancer OVCAR3 cells. Inhibition of ATR/CHK1 signaling pathway can inhibit the proliferation of epithelial ovarian cancer cells and induce G1/S cell cycle arrest and apoptosis.

Objective:To identify the viral etiologies and epidemic characteristics of acute respiratory infection (ARI) in Fuzhou.Methods:The information and respiratory tract specimens of ARI cases from three different types of hospitals in Fuzhou were collected in 2019. Real-time quantitative fluorescent PCR (Polymerase Chain Reaction, PCR) was uesd to detect adenovirus (AdV) and human bocavirus (HBoV), Real-time quantitative fluorescent RT-PCR (Reverse Transcription-PCR, RT-PCR) was used to detect influenza virus A/B (IFV-A/B), respiratory syncytial virus (RSV), parainfluenza virus 1-4 (PIV-1~4), human metapneumovirus (HmpV), coronavirus (CoV)-229E/OC43/HKU1/NL63 and rhinovirus (RV). Chi-square test and Fisher exact test were used to analyze the epidemiological characteristics of ARI cases. Results:Of the 726 ARI cases enrolled, 316 (43.53%) were positive for at least one viral pathogen, of which, 40 cases had dual infection and 9 had triple infection. The viral infection rates in different age groups were significantly different. The main viral pathogens in ARI in Fuzhou in 2019 were IFVs (12.40%), followed by Adv (10.61%), RV (8.82%), and RSV (5.51%). The AdV infection was more common in male ARI cases, and IFV-B, PIV, RSV, RV, and AdV were identified more commonly in children. In addition, the infection with IFVs, RSV, and RV had obvious seasonality.Conclusions:The main pathogens of ARI cases in Fuzhou in 2019 include IFVs, AdV, RV, RSV, etc. The infection of respective virus in ARI cases represent different age-distribution and seasonality.

Objective:To isolate and identify the yellow fever virus (YFV) from the specimens of the imported yellow fever (YF) cases in Fujian province in 2016.Methods:Sixteen positive YFV nucleic acid samples including serum, urine and saliva were inoculated into C6/36 cells, respectively. The isolated strain was identified by YFV real-time RT-PCR. The complete gene sequence of this strain was obtained by high-throughput next-generation sequencing, and the phylogenetic tree was drawn.Results:Only one strain was isolated from the serum of one case three days after onset, and identified as a YFV strain by real-time RT-PCR. BLAST analysis showed that the complete gene sequence of this strain was identical to the strain CNYF01R/2016(KX268355) isolated from the first YF imported case in China in 2016. The phylogenetic tree showed that this strain belonged to the same phylogenetic branch as the epidemic strain Angola71 in Angola in 1971, and was significantly different from the 17D vaccine strain (X03700), indicating that the YFV strain isolated in this study belonged to the wild YFV strain of Angola genotype.Conclusions:An Angola genotype YFV strain was successfully isolated from samples of imported YF cases in Fujian Province in 2016.

Objective: To study the etiological characteristics of an imported Chikungunya fever (CHIK) epidemic in Fujian province in 2018. Methods: Serum samples collected at different days after the onset of the two CHIK cases were detected by real-time RT-PCR and ELISA. Structural protein E1 gene was amplified by RT-PCR and sequenced for nucleotide characteristics analysis and phylogenetic tree analysis. Results: RNA of Chikungunya virus (CHIKV) was detected in the 4 serum samples collected on the first 5 days of the disease, and the earliest IgM antibodies were detected in specimens on the 5th day of the disease, however, IgG antibodies were only detected in specimen on 10th day. Compared with the S27-African prototype strain, 12 mutant points were found in the amino acids of E1 genes in this study. The E1 genes of the two CHIK cases were exactly the same, and they were closest to the evolutionary relationship with the strain isolated in the Philippines in 2014. Their genotype was Asian genotype. Conclusions This epidemic was confirmed to have been imported from the Philippines after the infection with the Asian genotype CHIKV, which suggests that Fujian province should strengthen the monitoring of persons entering from the CHIK epidemic area, so as to prevent imported cases from causing local outbreaks.

Objective: To elucidate the epidemiological and etiological features of a local outbreak of dengue fever (DF) in Taijiang district in Fuzhou, Fujian province in 2017, and speculate possible viral source based on phylogenetic analysis. Methods: The clinical and demographic data of cases were collected through field investigation and the outbreak was characterized epidemiologically by descriptive method. The patient′s serum were collected and the adult mosquitoes were captured by anti-mosquito double-net method for the laboratorial test and viral isolation. The viral isolates were typed by real-time fluorescent RT-PCR and their full length of viral envelope (E) genes were amplified by RT-PCR and sequenced. The E gene sequences obtained in this study, together with the reference sequences, were used for the phylogenetic analysis. Results: A total of 13 cases of autochthonous DF were confirmed in the outbreak. All cases presented obvious clinical manifestations and clustered spatially and temporally. The Breteau Index (BI) of mosquito larva density was the highest in epidemic foci of Xingang street and was relatively low in surrounding areas. Four DENV-1 strains, three from patients and one from the captured adult Aedes albopictus, were isolated and identified by real-time RT-PCR. Full length E gene sequences of the four isolates were completely identical and phylogenetic analysis showed that the isolates were genetically closest to the strain (GenBank No. KT825033) from Vietnam in 2014, rather than the DENV-1 strains found in Fujian previously. Conclusions The DF outbreak occurred in Fuzhou in 2017 was caused by DENV-1 which was imported possibly from somewhere outside of Fujian province and subsequently led to local DF transmission in human via the mosquito Aedes albopictus.

ObjectiveTo investigate the effects of morphine preconditioning on myocardial ischemiareperfusion (I/R) injury and the expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2)in rats with chronic heart failure.MethodsForty-eight healthy male SD rats weighing 220-250 g were randomly divided into 6 groups ( n =8 each):control group (group C),sham operation group (group S),I/R group and preconditioning with low,median and high doses of morphine groups (groups MP1-3 ).Chronic heart failure was induced by iv edriamycin 2.0 mg/kg once a week for 6 weeks in groups S,I/R and MP1-3.Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured using ultrasound,and left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated at the end of 14th day after the end of adriamycin administration.Blood samples from the carotid artery were collected after ultrasonography for determination of the plasma brain natriuretic peptide (BNP) concentration.Myocardial I/R was induced by 30 min occlusion of left anterior descending branch of coronary artery followed by 120 min reperfusion at 2 day after ultrasonography in groups I/R and MP1-3.In groups MP1-3,iv morphine 0.015,0.030 and 0.050 mg/kg were repeated 3 times at 5 min interval at 30 min before ischemia respectively,while normal saline 5 ml/kg was given in group I/R.The animals were sacrificed at the end of reperfusion in groups S,I/R and MP1-3,and the hearts were removed to measure the area at risk (AAR),infarct size (IS),and IS/AAR ratio was calculated.The p-ERK1/2 expression in myocardium was assessed by Western blot.ResultsThe LVESD and plasma BNP concentration were significantly higher,while the LVEF and LVFS lower in the other 5 groups than in group C (P ＜0.01).No myocardial infarction was found in group S.The p-ERK1/2 expression was significantly lower in groups I/R and MP1 than in group S (P ＜ 0.05).IS and IS/AAR ratio were significantly lower,and p-ERK1/2expression was significantly higher in groups MP2.3 than in group I/R ( P ＜ 0.05).There were no significant differences in IS,IS/AAR ratio and p-ERK1/2 expression between groups MP1 and I/R (P ＞ 0.05).IS and IS/AAR ratio were decreased gradually,and the p-ERK1/2 expression was up-regulated gradually in groups MP1-3 ( P ＜0.05).ConclusionMorphine preconditioning can confer cardioprotection against myocardial I/R in a dose-dependent manner in rats with chronic heart failure.Up-regulation of p-ERK1/2 expression is involved in the underlying mechamism.