Objective: To determine the relationship between dengue virus load and clinical characteristics, so as to provide basis for dengue fever prevention and treatment. Methods : The dengue viral load and typing of 120 patients in Gongshu District of Hangzhou from June to November 2017 were detected by real-time fluorescent quantitative RT-PCR;the clinical indicators of these dengue patients were collected and their correlation with the viral load was analyzed. Results: The DNA detection of dengue virus in 120 patients showed that they were all typeⅡ. The median dengue virus load was 3.91×104 copies/mL. All the patients had fever, the average peak temperature was（38.96 ± 0.69）℃. There were 102（85.00%）cases with asthenia;116（96.67%）cases with white blood cell count（WBC）less than 4× 109/L;119（99.17%）cases with platelet count（PLT）less than 100×109/L;114（95.00%）cases with glutamic oxaloacetate transaminase（GOT）more than 40 U/L;81（67.50%）cases with glutamic pyruvate transaminase（GPT）more than 52 U/L;58（48.33%）cases with creatine kinase（CK）more than 210 U/L. There was no significant correlation of dengue virus load with length of hospitalization, peak temperature,duration of fever, WBC,PLT, GOT, GPT and CK（P>0.05）. There were 75（62.50%）severe patients, and their median viral load was 9.29×104copies/mL, which was higher than 5.33×103copies/mL in non-severe patients（P<0.05）. Conclusion The dengue virus load is not related with length of hospitalization,peak temperature,WBC,PLT,GOT,GPT and CK,but with the severity of the disease.

Objective:To investigate the characteristics of clinical bacteria and H3N2 influenza A virus coinfection and variations in the hemagglutinin (HA) cleavage site of H3N2 among influenza-like cases.Methods:A total of 12 250 samples were collected from influenza-like cases for real-time PCR detection of H3N2 influenza virus from January 2013 to December 2018. To analyze the characteristics of co-infection, some H3N2-positive samples were selected and analyzed by real-time PCR to detect Staphylococcus aureus, Streptococcus pneumoniae and haemophilus influenzae type B. HA genes of H3N2 isolates were amplified by RT-PCR and sequenced. A phylogenetic tree was constructed based on HA gene sequences. Amino acid variations in cleavage sites were analyzed. Results:H3N2 influenza viruses had been detected every year since 2013, causing 44.69% influenza-positive cases. There were 295 randomly selected H3N2-positive samples, of which 29.2% had clinical bacterial infection. The HA cleavage sites of 210 H3N2 isolates were sequenced and 68 had variations, including 63 carrying K342R (PEKQTR to PERQTR) single-amino acid site variation. The co-infection rate was 31.25% (45/144) in unmutated samples and 23.53% (16/68) in mutated samples (χ 2=1.34, P>0.05). The H3N2 influenza viruses circulating in Hangzhou mainly belonged to two evolutionary clusters of 3c.3a and 3c.2a, and the viruses with K342R mutation at the cleavage site belonged to the evolutionary cluster of 3c.3a. Conclusions:H3N2 influenza virus played an important role in the epidemic of influenza virus in Hangzhou. There were some bacterial co-infections in influenza-positive cases. Cleavage site variations showed regional epidemic characteristics, but had no significant correlation with bacterial co-infection.

Objective To investigate the clinical and epidemiological characteristics of Saffold virus (SAFV)infection in outpatient and hospitalized children with acute respiratory tract infections(ARI). Methods A total of 1060 clinical specimens were collected from children with ARI in the Affiliated Children's Hospital,Zhejiang University School of Medicine from March 2011 to February 2014, including 256 samples of throat swabs from outpatients,and 804 samples of trachea suctions from hospitalized patients. Real time PCR(RT-PCR)was performed to detect 5'UTR segment of SAFV.SPSS 17.0 software was used to analyze the test results and clinical data.Results The positive detection rates of SAFV in outpatients and hospitalized children with ARI were 2.3%(6/256)and 13.2%(106/804), respectively(χ2=24.147, P<0.01).Among the hospitalized children,the positive detection rates of SAFV in children <1 year,1-<3 years,3-<6 years and 6-12 years were 14.0%,11.2%,11.1% and 8.3%, respectively(χ2=1.845,P>0.05).The positive rates of SAFV in males and females were 12.7% and 17.7%(χ2=0.279,P>0.05).The detection rate of SAFV in autumn was highest(21.2%), followed by that in spring (14.6%),winter(9.5%)and summer(8.8%)(χ2=15.625, P<0.01).The co-infection rates with other respiratory pathogens of hospitalized and outpatients children were 76.4%(81/106)and 66.7% (4/6).Among the hospitalized patients, the rate of co-infection with respiratory syncytial virus was the highest(36.8%),followed by rhinovirus(27.4%), metapneumovirus(10.4%)and parainfluenza virus (10.4%).Among children with ARI,the fever rate of SAFV-positive cases was lower than that of SAFV-negative cases(χ2=4.069,P<0.05).Conclusions The detection rate of SAFV in hospitalized children with ARI is significantly higher than that in outpatients,and SAFV infection was dominated by co-infection. The prevalence of SAFV in the Hangzhou area presents a certain local epidemic pattern.

Objective:To understand the viral genomic characteristics of a 2019-novel coronavirus (2019-nCoV) strain in the first COVID-19 patient found in Hangzhou, China.Methods:Viral RNA was extracted in throat swab and sputum sample of the patient and was performed real-time reverse transcription PCR detection and obtained viral genome by high-throughput sequencing method. Phylogenetic analysis was conducted using 29 2019-nCoV genomes and 30 β-coronavirus genomes deposited in NCBI GenBank. Fifteen genomes from Wuhan were grouped by mutation sites and others were identified by Wuhan's or specific mutation sites.Results:A 29 833 bp length genome of the first 2019-nCoV strain in Hangzhou was obtained, covering full length of the coding regions of coronavirus. Phylogenetic analysis showed that the genome was closest to the genome of a bat SARS-like coronavirus strain RaTG13 with an identity of 96.11% (28 666/29 826). Among the genes between two genomes, E genes were highly conserved (99.56%), while S genes had lowest identity (92.87%). The genome sequence similarities among 29 strains from China (Hangzhou, Wuhan, and Shenzhen), Japan, USA, and Finland, were all more than 99.9%; however, some single nucleotide polymorphisms were identified in some strains.Conclusion:The genome of Hangzhou 2019-nCoV strain was very close to the genomes of strains from other cities in China and overseas collected at early epidemic phase. The 2019-nCoV genome sequencing method used in this paper provides an useful tool for monitoring variation of viral genes.

Objective:To understand the viral genomic characteristics of a 2019-novel coronavirus (2019-nCoV) strain in the first COVID-19 patient found in Hangzhou, China.Methods:Viral RNA was extracted in throat swab and sputum sample of the patient and was performed real-time reverse transcription PCR detection and obtained viral genome by high-throughput sequencing method. Phylogenetic analysis was conducted using 29 2019-nCoV genomes and 30 β-coronavirus genomes deposited in NCBI GenBank. Fifteen genomes from Wuhan were grouped by mutation sites and others were identified by Wuhan's or specific mutation sites.Results:A 29 833 bp length genome of the first 2019-nCoV strain in Hangzhou was obtained, covering full length of the coding regions of coronavirus. Phylogenetic analysis showed that the genome was closest to the genome of a bat SARS-like coronavirus strain RaTG13 with an identity of 96.11% (28 666/29 826). Among the genes between two genomes, E genes were highly conserved (99.56%), while S genes had lowest identity (92.87%). The genome sequence similarities among 29 strains from China (Hangzhou, Wuhan, and Shenzhen), Japan, USA, and Finland, were all more than 99.9%; however, some single nucleotide polymorphisms were identified in some strains.Conclusion:The genome of Hangzhou 2019-nCoV strain was very close to the genomes of strains from other cities in China and overseas collected at early epidemic phase. The 2019-nCoV genome sequencing method used in this paper provides an useful tool for monitoring variation of viral genes.

OBJECTIVE To investigate the effect of Jiawei Tianwang Buxin Dan(JWBXD)on insomnia in rats exposed to simulated high-altitude conditions.METHODS ① Thirty SD rats were randomly divided into the normal control,model,model+Jiawei Tianwang Buxin Dan(JWBXD,9.6 mg·kg-1),model+Tianwang Buxin Dan(TWBXD,9.6 mg·kg-1),and model+diazepam(DZP,3 mg·kg-1)groups.Rats,except for the normal control group,were subjected to a low-pressure,low-oxygen animal experimental chamber simulating a 5000 m altitude.Respective drugs were ig administrated once daily at 9:00 for seven days,and signal acquisition and sleep analysis were conducted by a wireless physiological sig-nal telemetry system.②Forty rats were randomly divided into five groups as described in ①.Through-out the experiment,the general condition and body mass of the rats were observed daily.Drug adminis-tration lasted for seven days,and grip strength was tested one hour after the final administration.ELISA was used to measure the levels of corticotropin-releasing hormone(CRH),adrenocorticotropic hor-mone(ACTH),corticosterone(CORT),and melatonin(MLT)in serum.Western blotting was performed to measure the expression levels of core clock proteins period circadian regulator 2(Per2),circadian locomotor output cycles(Clock),cryptochrome 2(Cry2),brain-muscle arnt-like protein 1(Bmal1),nuclear receptor subfamily 1,group D member 1(NR1D1),glycogen synthase kinase-3β(GSK-3β),as well as acetylserotonin O-methyltransferase(ASMT)in the hypothalamus and pineal gland,respectively.RESULTS ① Compared with the normal control group,the model group exhibited a decrease in total sleep time(P<0.01),an increase in wakefulness(P<0.01),a significant reduction in slow wave sleep(SWS)(P<0.05)and the mean bouts duration(P<0.05).Compared with the model group,both DZP and JWBXD(P<0.01)prolonged sleep time and suppressed wakefulness(P<0.01)in the hypoxic envi-ronment.DZP and JWBXD prolonged SWS(P<0.05,P<0.01),while TWBXD had no significant effect.JWBXD improved the mean bouts duration of SWS in the model rats(P<0.01),whereas no such improvement was observed in model+DZP and model+TWBXD groups.② Compared with the normal control group,the model group showed a significant decrease in forelimb grip strength(P<0.01),increased levels of serum ACTH(P<0.05),CRH,and CORT(P<0.01),and decreased MLT levels(P<0.05).The expression levels of Per2,Cry2,GSK-3β,and NR1D1 in the hypothalamus were downregu-lated(P<0.05,P<0.01),while Bmal1 and Clock were upregulated(P<0.05,P<0.01).ASMT expression in the pineal gland was decreased(P<0.05).Compared with the model group,JWBXD and TWBXD enhanced forelimb grip strength(P<0.01),reduced serum CORT and ACTH levels(P<0.05),decreased CRH levels(P<0.01),and restored MLT levels(P<0.01).JWBXD upregulated the expression levels of Per2,Cry2,GSK-3β and NR1D1 in the hypothalamus(P<0.05,P<0.01),but downregulated Bmal1 and Clock expression(P<0.05,P<0.01).TWBXD downregulated Bmal1 expression in the hypothalamus(P<0.01)and increased NR1D1 expression(P<0.05).DZP significantly enhanced the expression levels of Per2,Cry2 and NR1D1 in the hypothalamus(P<0.01).JWBXD,TWBXD and DZP improved ASMT expression in the pineal gland(P<0.05).CONCLUSION JWBXD can improve sleep structure and prolong the duration of SWS in rats exposed to simulated high-altitude conditions.The mechanisms may involve the regulation of core clock protein expressions in the hypothalamus,promotion of mela-tonin secretion,and inhibition of HPA axis hyperactivity.

Tianwang buxin dan （TWBXD），as a classic prescription for treating insomnia ，in forms of tablet ，drop pill ，patch and others ，has been widely used in clinical practice . Its compound prescription and single ingredient can play a calming and tranquilizing role by regulating neurotransmitters ，inhibiting oxidative stress reaction and the expression of inflammatory factors . Based on the theory of traditional Chinese medicine and combined with modern pharmacological research ，this paper reviews single ingredient and active components of TWBXD ，calming and tranquilizing role of related couplet medicinals . The results showed that the single ingredients such as Rehmannia glutinosa ，the seeds of Zizyphus jujube ，the seeds of Platycladus orientalis ，Angelica sinensis，Schisandra chinensis ，Poria cocos and their effective components as well as related couplet medicinals of Z. jujube could improve the insomnia symptoms in different degrees . Except that the mechanism of the seeds of P. orientalis has not been studied ， other ingredients mainly act on nervous -endocrine-immune system . They can play the sedative and hypnotic effects by regulating neurotransmitters，inflammatory factors and oxidation .