Objective:To investigate the effect of virus inactivation on weak positive result of 2019 novel coronavirus(2019-nCoV) nucleic acid test.Methods:A retrospective study was conducted on the nasopharyngeal swabs of three patients with positive PCR nucleic acid test for 2019-nCoV at different concentrations in the Second affiliated Hospital of Zhejiang University Medical College from January to February 2020.The virus in nasopharyngeal swab specimens were inactivated by water bath at 56 ℃ for 30 min, dry bath at 56 ℃ for 60 min and dry bath at 60 ℃ for 30 min respectively. After treatment, these samples RNA were extracted and then detected by three new commercial quantitative real-time polymerase chain reaction reagent kits for 2019-nCoV.Cycle threshold (Ct) value was used to evaluate the effect of virus inactivation on nucleic acid detection of 2019-nCoV.Results:There was no significant difference between the groups before and after inactivation. Ct values of ORF1ab gene before inactivation were 23.28±0.28, 25.25±0.25, 28.93±0.44, 32.06±0.47, 35.20±0.38, 32.89±0.38, 36.24±0.23, 33.30±0.46, and those after inactivation were, group 1:23.60±0.20, 27.29±0.30, 31.83±0.51, 37.41±0.46, group 2: 24.25±0.34, 27.18±0.42, 31.84±0.61, 34.99±1.01, 34.89±0.45,group 3: 23.37±0.17, 26.89±0.52, 32.05±0.50.Ct value of N gene before inactivation were 24.38±0.09, 26.64±0.11, 30.35±0.12, 33.29±0.33, 36.93±0.11, 34.50±0.12, 35.63±0.12, those after inactivation were, group 1: 24.66±0.11, 28.52±0.14, 32.71±0.14, 37.00±0.13;group 2: 25.41±0.10, 28.79±0.15, 33.29±0.28; group 3: 23.37±0.11, 28.68±0.11, 33.54±0.13, 37.18±0.23(ORF1ab gene: t=-1.416; N gene: t=-1.379, P>0.05). There was no significant difference among the three inactivation groups, the specific Ct values are shown above(ORF1ab gene: t=-0.460; N gene: t=-0.132, P>0.05). However, the Ct values of the inactivated groups (1,2,3) and the non-inactivated group at different dilution times were different (10 ×:Ct value of ORF1ab was 25.25±0.25 in the non-inactivated group, and 27.29±0.30, 27.18±0.42 and 26.89±0.52 in the inactivated group1,2 and 3, t(ORF1ab)=-7.327, P<0.01.Ct value of N gene in the non-inactivated group was26.64±0.11, those in inactivated group 1, 2 and 3 were 28.52±0.14, 28.79±0.15 and 28.68±0.11, respectively, t (N)=-19.340, P<0.01. 100 ×:Ct value of ORF1ab was 28.93±0.44 in the non-inactivated group, and 31.83±0.51,31.84±0.61 and 32.05±0.50 in the inactivated group1,2 and 3, t (ORF1ab)=-9.462, P<0.01. Ct value of N gene in the non-inactivated group was 30.35±0.12, those in the inactivated group 1, 2 and 3 were 32.71±0.14, 33.29±0.28 and 33.54±0.13, respectively, t (N)=-18.583, P<0.01. The positive detection rate of the non-inactivated group (7/11, 8/11, 5/11) was significantly different from that of the inactivated group (inactivated group 1:4/11, 4/11, 3/11, inactivated group 2:3/11, 3/11, 3/11, and inactivated group 3:3/11, 3/11, 2/11) ( Z=-2.670, P<0.01). There were no significant difference among the inactivated groups(inactivated group 1:4/11, 4/11, 3/11, inactivated group 2:3/11, 3/11, 3/11, inactivated group 3:3/11, 3/11, 2/11) ( Z=4.413, P>0.05) and among the three reagents(reagent 1:7/11, 4/11, 3/11, 3/11, reagent 2:8/11, 4/11, 3/11, 3/11, reagent 3:5/11, 3/11, 3/11, 2/11)(χ 2=1.199, P>0.05). Conclusion:The virus inactivation can degrade the nucleic acid of the 2019-nCoV, resulting in the decrease of the Ct value and the false negative results of the low-concentration specimens.

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 analyze the prevalence of influenza A (H3N2) virus in Hangzhou be-tween 2012 and 2017 and to investigate the genetic variations in hemagglutinin ( HA) and neuraminidase ( NA) . -ethods Throat swab samples were collected for viral isolation from 12185 patients with suspected influenza in Hangzhou area from January 2012 to December 2017. Influenza virus subtypes were identified by real-time RT-PCR. HA and NA genes of some isolated Influenza A (H3N2) viruses were amplified with spe-cific primers and then analyzed by sequencing and phylogenetic analysis. Results Influenza A (H3N2) virus was the predominant subtype circulating in Hangzhou during 2012 to 2017. It caused high morbidity in elderly people (Z=81. 039, P<0. 05). Most of the isolated influenza A (H3N2) viruses belonged to the phylogenetic clades of 3C. 3a and 3C. 2a. These viruses shared a homology of 96. 7％-100％ in nucleotide sequences of both HA and NA genes, but possessed several HA and NA mutations in antigenic sites. Con-clusions Influenza A (H3N2) virus was an important pathogen causing influenza epidemics in Hangzhou during 2012 to 2017. HA and NA genes showed many mutations in antigenic sites. No drug resistant virus was reported.

BACKGROUND:The establishment of a safe, reliable and easily repeatable mouse model of nonalcoholic fatty liver disease is the prerequisite for the study of the diagnosis and treatment of the disease.
OBJECTIVE:To establish a C57BL/6 mouse model of nonalcoholic fatty liver disease and observe changes of biochemical indicators, which can provide a theoretical basis for its pathogenesis and drug treatment.
METHODS:Sixty healthy male C57BL/6 mice were randomly divided into a control group of 30 cases (normal diet), and a model group of 30 cases (high fat diet). Models of nonalcoholic fatty liver were established. At 8 weeks, body mass, liver index, and homogenate superoxide dismutase activity in the liver were detected. Changes in serum alanine aminotransferase, aspartate aminotransferase, triglyceride glycerol, cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol were observed. Pathological examination was performed.
RESULTS AND CONCLUSION:(1) Pathological sections showed that large droplets and smal lipid droplets in the mouse liver and spread the whole liver. Swel ing of the liver cel s, visible cytoplasmic vacuoles and obviously inflammatory changes in liver cel s were observed in the model group. (2) Body weight and liver index were significantly higher in the model group than in the control group (P<0.05). Superoxide dismutase activity was significantly reduced in the liver (P<0.05). (3) Triglycerides, cholesterol, and low-density lipoprotein cholesterol levels were significantly higher, but high-density lipoprotein cholesterol levels were significantly lower in the model group than in the control group (P<0.05). (4) Nonalcoholic fatty liver mouse model is ideal for high-fat diet-induced animal model. The method is simple, repetitive, and can provide a stable animal model for the study on the mechanism of nonalcoholic fatty liver disease and drug treatment.

Objective To investigate the levels of blood pressure, blood lipid, blood glucose, body mass index (BMI) as well as the epidemiological characteristics of dyslipidemia, hyperglycemia, hypertension and metabolic syndrome of Hangzhou citizens. Methods A total of 28 990 citizens in Hangzhou city who underwent health checkup were recruited in this study, including 10 179 males and 18 811 females. The average age of subjects was 65.05 years. Subjects were asked to complete questionnaires regarding personal characteristics. The physical examination emphasized measurement of height, waist and blood pressure. Blood samples were collected and subjected to serum glucose, TC, HDL-C, LDL-C and TG measurements. The values of the examinations was described as xˉ± s . The ratios were compared with chi-square test. The trend analysis was conducted by linear correlation test. Results The prevalence of hypertension and metabolic syndrome was 17.1% and 11.2% respectively. And the prevalence of overweight/obesity and hyperglycemia was 36.3%,8.1%,16.4%respectively. It was indicated that the men had higher prevalence of hyperglycemia, hypertension, metabolic syndrome and overweight compared with women. However, as to the dyslipidemia, men and women were totally different. Women were more prone to suffer from hypercholesterolemia and elevation of low-density lipoprotein cholesterol. Men were apt to suffer from hypertriglyceridemia and reduction of high-density lipoprotein cholesterol. Divided the subjects by age into three groups, it was suggested that the rates of dyslipidemia, hyperglycemia, hypertension and overweight/obesity increased along with the increment of age in women. Although the rates of metabolic disorders were higher in the group of men, the trend of increase with age was not as significant as in women. It could be seen in men that dyslipidemia and overweight/obesity were reduced with the increase of age. Conclusion The metabolic disorders in Hangzhou citizens showed their own characteristics. It is suggested that multiple strategies targeting at different sexes and age-groups should be formulated to prevent and control the occurrence of metabolic diseases.

Objective In order to assess the value of Echinoccocus granulosus cyclophilin A (EgypA) in immune diagnosis,this novel gene was cloned and expressed.Methods By screening the EST library,the coding region of EgCypA was identified,and the PCR primers were designed based on this sequence.Bioinformatic tools were used to deduce the amino acid sequence of EgCypA and analyzed its biological characteristics.EgCypA was amplified from E.granulosus cDNA library by using PCR.Then,it was cloned into the prokaryotic expression vector pET28a and transformed into E.coli BL21.The recombinant protein was expressed in E.coli BL21 after IPTG induction.The immunogenicity of EgCypA was evaluated by Western blot using the Echinoccocus granulosus and other parasites infected animals' sera.Results Results of SDS-PAGE electrophoresis show that the recombinant BL21 expressed 18 400-25 000 protein,which was identical with the molecular weight calculated by bioinformatic analysis.Western blot shows that the recombinant protein only reacted with its immune serum and E.granulosus cystic fluid immune serum,and EgCypA immune serum could react with the excretion and secretion antigen from E.granulosus protoscolexes,and no cross-reaction between EgCypA and sera from other parasites infected animals.Conclusions Cloning and expression of EgCypA are successful.EgCypA has good immunologic activity and could be a candidate molecular for immune diagnosis of echinococcosis in early stage.

phological changes in rat liver tissues. TLR4 and NF-κB expression in the liver tissues were measured by im-munohistochemistry . The results showed that compared with the normal group , the serum levels of transaminase ( ALT , AST ) and endotoxin of the model group were higher ( P < 0 . 01 ); and the degree of liver pathology injury was significantly increased; the TLR4 and NF-кB expression were increased (P < 0.01). Compared with the model group, the serum levels of transaminase (ALT, AST) and endotoxin of the experimental group were lower (P < 0.01), the degree of liver pathology injury was significantly lighter; the TLR4 and NF-кB expression were significantly lower (P < 0.01). It was concluded that the WYJDHY granules has a good role in the prevention and treatment of liver injury of rat model of hepatic failure IETM through the downregulation of liver expression of TLR4 and NF-кB in rat liver tissues , reducing serum levels of endotoxin , which may be one of the mecha-nisms on hepatic failure treatment .

Objective To observe the histopathologic injury of small intestine and intestinal permeability in chronic renal failure (CRF) rats. Methods Twenty male Sprague-Dawley rats were randomly assigned to CRF group (n=10) and control group (n=10). 5/6 nephrectomy was used to establish CRF rats, while sham operation for control. Blood biochemistry was regularly monitored until CRF model was successfully established. The model rats were fed with lactulose (L) and mannitol (M) through intragastric administration. Urine was collected after 6 hours, and the concentration of lactulose and mannitol in urine was measured using high pressure liquid chromatograph with refractive index detector (HPLC-RID), and the ratio of urinary excretion of L/M was calculated to evaluate intestinal permeability. Small intestinal mucosa were stained by hematoxylin-eosin (HE) and observed with light microscope (villus height, thickness of muscle layer and villus count), histological damage score was used to evaluate intestinal injury. Results The L/M ratio of CRF group was higher than that of control group (1.75±0.11 vs 1.20±0.06, P<0.01). The small intestinal mucosal villus height and thickness of muscle layer in CRF group were higher (P<0.01), and the number of villi was lower compared to control group (P<0.01). The score of histopathologic intestine damage of CRF group was higher than that of control group (1.00±0.71 vs 0, P<0.01). Conclusion The intestinal permeability of CRF rats is increased with varying degrees of intestinal damage.

Objective To observe the influence of sodium phosphocreatine on the hemodynamics,human heart fatty acid binding protein (hFABP),cardiac troponin Ⅰ (cTnI) during perioperative period.Methods Forty patients (ASA grade Ⅱ to Ⅲ ) with extensive myocardial ischemia diagnosed by preoperative electrocardiogram were divided into two groups:sodium phosphocreatine group (group A) and control group (group B) with 20 cases each by random digits table. Both groups underwent routine preoperative preparation and group A were given sodium phosphocreatine 2 g/d till anesthesia induction immediately in the operation day. Hemodynamic parameters including mean artery pressure (MAP),heart rate (HR),cardiac output (CO), stroke index (SI) and pulmonary vascular resistance (PVR) were recorded respectively at 3 d before treatment (T0), anesthesia induction immediately before ( T1 ), tracheal intubation immediately after (T2),tracheal extubation immediately after (T3). And blood hFABP and cTnl levels were detected. Results Compared with group B, there was no significant difference in HR of group A at To to T3 (P ＞ 0.05 ). MAP,CO, SI at T1 to T3 of group A were significantly higher than those of group B(P＜ 0.05 ) ;whereas PVR,blood hFABP and cTnI levels were lower than those of group B (P ＜ 0.05 ). The incidence of arrhythmogenesis of group A [20%(4/20)] was lower than that of group B [45%(9/20)](P＜0.05). Conclusion Sodium phosphocreatine has good protective effect on the myocardium in the patients with myocardial ischemia during perioperative period.