Analysis of genotype IV distribution of hepatitis E virus infections in Wuhan by sequencing the open reading frame 3 gene / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To determine the distribution of genotype IV among hepatitis E virus (HEV) infections in Wuhan by sequencing the open reading frame (ORF) 3 gene of HEV clinical isolates.</p><p><b>METHODS</b>Serum samples were collected from 103 individuals who tested positive for the anti-HEV IgM antibody, and total HEV RNA was extracted for targeted gene sequencing analysis. Reverse transcription-nested polymerase chain reaction (PCR) was used to amplify two fragments of the ORF3 gene (5020 to 5392 nt and 5347 to 5956 nt, EF570133). The two PCR products were sequenced and the sequences were stitched with the ContigExpress program and used to determine the HEV genotype.</p><p><b>RESULTS</b>Both ORF3 gene fragments were amplified in 18 out of the 103 anti-HEV IgM-positive serum samples. These 18 HEV isolates shared 92.5% to 99.4% identity with each other at the nucleotide level. Nucleotide sequence homology analysis of the HEV genotypes I, II, III, and IV indicated the highest homology was with genotype IV; specifically, homology with genotype I was 83.5% to 86.7%, with genotype II was 83.2% to 85.2%, with genotype III was 84.6% to 87.2%, and with genotype IV was 92.0% to 96.5%.</p><p><b>CONCLUSION</b>Targeted sequencing of the HEV ORF3 gene facilitated genotyping of clinical isolates. Using this method, it was determined that nearly 20% of HEV clinical isolates from Wuhan belong to genotype IV.</p>

The effect of endotoxemia on gluconeogenesis in livers and kidneys during acute hepatic failure / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To explore the effects of endotoxemia on gluconeogenesis in livers and kidneys during acute hepatic failure.</p><p><b>METHOD</b>Twenty-four healthy male SD rats were randomly divided into four groups (6 rats in each group) and all of them were injected intraperitoneally with solutions: group I with normal saline, group II with 400 mg/kg of D-galactosamine (D-GaLN), group III with 400 mg/kg of D-GaLN plus 50 microg/kg lipopolysaccharide(LPS), and group IV with 400 mg/kg of D-GaLN plus 500 microg/kg LPS. At 6 hours after the administration of different solutions intraperitoneally, blood samples were collected to examine blood urea nitrogen (BUN) and serum creatinine. Realtime PCR was used to study the expression of phosphoenolpyruvate carboxykinase (PEPCK) in the livers and kidneys.</p><p><b>RESULTS</b>No endotoxemia developed in group I or group II but it was evident in group III and group IV. The level of endotoxemia in group IV was higher than in group III (8.05+/-0.43, 3.50+/-2.25, P<0.05). After 6 hours of administration of LPS in group IV, hypoglycemia appeared, and blood glucose was normal in the other three groups. BUN and serum creatinine were all normal in the four groups, except that blood urea nitrogen was elevated in group IV. The mRNA of PEPCK in livers decreased gradually in all the four groups (2.54+/-1.32 vs 1.87+/-0.15 vs 0.91+/-0.13 vs 0.44+/-0.42, P<0.05). In the kidneys there was no change in the expression of PEPCK in group I and group II (0.75+/-0.03 and 0.77+/-0.04, P>0.05), but it increased in group III (0.75+/-0.03 vs 1.63+/-0.86, P<0.05), and decreased in group IV (0.75+/-0.03 vs 0.13+/-0.07, P<0.05).</p><p><b>CONCLUSION</b>During acute hepatic failure severe endotoxemia would damage the function of gluconeogenesis in livers and kidneys by inhibiting transcription of PEPCK and this can induce hypoglycemia.</p>

Efficacy and safety of reduced osmolarity oral rehydration salts in treatment of dehydration in children with acute diarrhea--a multicenter, randomized, double blind clinical trial / 中华儿科杂志

<p><b>OBJECTIVE</b>To assess the efficacy and safety of reduced osmolarity oral rehydration salts (ROORS) in treatment of mild to moderate dehydration caused by acute diarrhea in children.</p><p><b>METHODS</b>A multicenter, randomized, double-blind, positive drug controlled clinical trial was conducted in 125 cases aged 1 to 17 years. These children with acute diarrhea and signs of dehydration were randomly assigned to receive either ROORS (trial group, n = 62) or oral rehydration salts II (ORS II) (control group, n = 63). The volume of intravenous infusion were recorded. The improvements of systemic symtoms and signs, diarrhea, dehydration and total scores were compared between the two groups. The adverse events and changes of electrolyte and other laboratory tests during treatment were also observed and analyzed.</p><p><b>RESULTS</b>The overall effective rates in trial group and control group were 96.8% and 96.8%, respectively. The recovery of systemic symptoms, dehydration signs and diarrhea occurred in 96%, 97% and 78% patients in trial groups, and 96%, 98% and 85% patients in control group. The scores of symptoms and signs in both groups decreased significantly after treatment. All the above parameters and the number of cases who needed intravenous infusion (41 vs. 39) were not statistically different between two groups. However, the average volume of intravenously infused fluids in trial group was (450.98 +/- 183.07) ml, 24.5% less than that in the control group (597.30 +/- 343.37) ml (P < 0.05). The mean serum Na(+) concentration elevated from (137.48 +/- 4.55) mmol/L to (139.52 +/- 3.25) mmol/L (P < 0.01) in control group after treatment, but the change was not statistically significant in trail group. Serum K(+), Cl(-), HCO(3)(-) and other laboratory result did not change significantly after treatment. The total scores in both groups decreased obviously after treatment, but no significant difference was demonstrated between two groups (P > 0.05). A case in trial group had mild abdominal distention and recovered spontaneously.</p><p><b>CONCLUSION</b>ROORS was shown to be effective and safe in the treatment of mild and moderate dehydration induced by acute diarrhea. Compared to ORS II, ROORS could decrease the intravenous supplement of fluid and lower the risk of hypernatremia.</p>

Infection of tupaia hepatocytes with hepatitis C virus in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Tupaia belangeri (tree shrew) has a close phylogenetic relationship with primates and has been shown to be susceptible to a variety of human viruses. This study was conducted to investigate whether or not hepatitis C virus (HCV) could infect primary tupaia hepatocytes (PTHs) in vitro.</p><p><b>METHODS</b>Serum-derived HCV was cultivated with PTHs, and then positive and negative strand HCV RNA in PTHs, as well as the encapsidated HCV RNA in the culture medium were detected to evaluate the infection. Virus from the culture medium of the infected PTHs was passed to naïve PTHs, and the quasispecies of HCV were compared among the inoculum and PTHs after infection and passage.</p><p><b>RESULTS</b>Both positive and negative strand HCV RNA were detected in PTHs after infection. The negative strand RNA was detectable from day 5 to day 10 after infection, while the positive strand RNA was positive up to day 14. HCV RNA, which was RNase resistant, could be detected from the culture medium of the infected PTHs from day 3 to day 14. Production of infectious virons of PTH were demonstrated by passage HCV to naïve PTHs. Compared analysis of HCV quasispecies after infection and passage showed that PTHs were selectively infected with defined HCV quasispecies, and new quasispecies emerged in PTHs after passage.</p><p><b>CONCLUSION</b>The present study strongly indicates that PTHs could be infected by HCV and support HCV replication in vitro. Our results would be helpful for the establishment of a tupaia model of HCV infection.</p>

Distribution of hepatitis B virus genotypes in Hubei province and its clinical significance / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the distribution of hepatitis B virus genotype in Hubei province (China) and its clinical significance.</p><p><b>METHODS</b>Serum samples from 190 HBV DNA positive patients with chronic HBV infection,including 52 asymptomatic HBV carriers (ASC), 56 chronic hepatitis (CH), 32 fulminant hepatic failure (FHF), 22 liver cirrhosis (LC), and 28 hepatocellular carcinoma (HCC) patients were collected and tested for HBV genotypes by type-specific primers.</p><p><b>RESULTS</b>A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes of hepatitis B virus (HBV). Of the 190 patients, 140 (73.7%) were genotype B and 42 (22.1%) were genotype C. Genotype B was more prevalent in the FHF and HCC patients than in the ASC patients; the ALT value was significantly higher in genotype B than in genotype C patients. The rate of anti-HBe was significantly higher in genotype B than in genotype C except in the patients of the ASC group.</p><p><b>CONCLUSION</b>The system we used seems to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys. Genotype B, genotype C and BC combination exist in Hubei province, and genotype B is the major genotype in this area especially in FHF and HCC patients.</p>

The construction of transferrin receptor- mediated HSV-TK gene transfer system and its effect on human hepatocellular carcinoma cells in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct the localization system involving anti-TfR monoclonal antibody (McAb) and AFP promoters and assess its effect on human hepatoma cell lines.</p><p><b>METHODS</b>The conjugate of anti-TfR McAb and polylysine (PLL) was made by SPDP and purified by molecular screen chromatography. DNA blocking test determined that the ratio of one pEBAF/tk to six Ab-PLL was the most suitable to couple them. The pEBAF/tk recombinant plasmid bearing HSV-TK gene was coupled to Ab-PLL by noncovalent bond. The pEBAF/tk was transferred into human hepatoma cell line HepG2, SMMC7721 and pulmonary cancer cell line A549 by receptor-mediated gene delivery (Ab-PLL-DNA) and liposome procedure. The growth inhibitory rates of HepG2, SMMC7721 and A549 cells were measured by MTT assay.</p><p><b>RESULTS</b>The inhibitory rates of HepG2/tk in 100 mg/L and 1 mg/L of GCV were 60.5% and 24.3%, respectively. The inhibitory rate of GCV to SMMC7721 was 23.2% in 3 days. The pulmonary cancer cell A549, A549/tk (Ab) and A549 /tk (lipo) could not be inhibited by the addition of GCV.</p><p><b>CONCLUSION</b>The localization system employed in this paper has high specificity, effectiveness and safety for gene therapy. It would be a promising strategy for gene therapy.</p>

The association of HLA-DRB1 allele polymorphism with the genetic susceptibility to liver cirrhosis due to hepatitis B virus / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the association of human leucocyte antigen (HLA)-DRB1 allele with the genetic susceptibility to cirrhosis due to hepatitis B virus(HBV).</p><p><b>METHODS</b>One hundred and six patients with cirrhosis due to HBV in Hubei area were investigated for HLA-DRB1 gene by polymerase chain reaction-sequence specific primers technique. The results were compared with those from 108 normal healthy people.</p><p><b>RESULTS</b>The frequency of HLA-DRB1*1201/1202 allele was 20.28% in patient group, which was significantly higher than the frequency (6.01%) in control group, the relative risk (RR) being 4.9878 (P<0.01). The frequency of HLA-DRB1*1501/1502 allele was decreased in patient group (patient 6.6%, control 16.67%, RR=0.3043, P<0.05), while the frequencies of other HLA-DRB1 alleles were not significantly different(P>0.05).</p><p><b>CONCLUSION</b>HLA-DRB1*1201/1202 allele may be the susceptibility gene in patients with cirrhosis due to HBV in Hubei Han nationality; HLA-DRB1*1501/1502 allele is a resistant gene in patients with cirrhosis.</p>

Influence of SEN virus infection on their response to lamivudine in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the influence of SEN virus infection on their response to lamivudine in patients with chronic hepatitis B (CHB).</p><p><b>METHODS</b>SEN virus-D and -H DNA were detected in 45 CHB patients who received lamivudine 12 months with nested-PCR, and YMDD motif mutations in HBV DNA were investigated with gene chip.</p><p><b>RESULTS</b>The positive rate of SEN virus DNA was 11.1% (5/45), and there were four out of the five SEN virus DNA positive patients whose HBV DNA was positive, among them, two patients existed YMDD motif mutation. While ten out of the forty SEN virus DNA negative patients appeared HBV DNA positive. The response rate was significant lower in SEN virus-infected patients than that in uninfected patients (chi 2=3.97, P<0.05).</p><p><b>CONCLUSION</b>Coinfection with SEN virus in chronic hepatitis B patients may adversely affect the outcome of treatment with lamivudine</p>

The investigation of biofilm formation,alginate biosynthetic genes expression and sequence of Pseudomonas aeruginosa / 中华传染病杂志

Objective To investigate the biofilm formation,the alginate biosynthetic gene ex- pression and analyze the mucA gene sequence of mucoid Pseudomonas aeruginosa PA17 and nonmu- cold Pseudomonas aeruginosa PA01.Methods The modified plate culture method was used to estab lish the biofilm model in vitro.Semi-quantitative RT-PCR was used to determine the expression level of algD in planktonic condition and during the formation of biofilm.The mucA gene of PA17 and PA01 was amplified and the products were sequenced.Results PA17 biofilm was mature at 6th day, and PA01 biofilm was mature at 3rd day.The structures of the biofilms were both like pellicle.In planktonic condition,the algD expression of PAl7 was higher than PA01;in biofilm formation,the algD expression was maximal when the biofilm was mature.There was a 166～333 deletion mutation and 342A→G in mueA gene of PA17,the mucA gene of PA01 was the same with the sequence of Genbank.Conclusions The mucA gene mutation of PA17 was a new type,which maybe the reason for the little expression difference of algD between PA17 and PA01 during the biofilm formation than it in planctonic condition and the same structure of PA17 and PA01 biofilm.