1.The effect of mutation of hepatitis B virus core protein dimer interface domain related to nucleocapsid formation on HBV replication
Kai DENG ; Dong JIANG ; Jinchao HAN ; Xiaoben PAN ; Hao WANG ; Lai WEI
Chinese Journal of Microbiology and Immunology 2010;30(8):730-736
Objective To investigate the effect of hepatitis B virus core protein (HBc) dimer interfaces amino acids mutation on nucleocapsid assembly and HBV DNA replication. Methods Based on HBc three dimension structure, four HBc dimer interfaces domain mutation plasmids, pHBc14-18M,pHBc120-135M,pHBc23-39M and pHBc122-139M were constructed in pcDNA3.1 vector by PCR site-directed mutagenesis, there was a flag-tag at the C-terminal of all mutants for easy detection. Wild type core protein plasmid 1-183flag was also constructed as a positive control. The 4 mutants were cotransfected HepG2 cells with pHBV1.2 core negative plasmid (pHBV1.2-core-) ,which contained 1.2 copies of HBV whole genome but the core protein would not express due to a stop codon. The capsid formation, HBV pregenome(pgRNA) and HBV DNA replication mediate were analyzed by native agarose gel electrophoresis and Western blot, Northern blot and Southern blot , respectively. The 4 mutants were also cotransfected HepG2 cells with HBV wild type plasmid pHBV1.2 and examined by Southern blot. Virions in the medium were determined by native agarose gel electrophoresis and Western blot. Results Cotransfecting HepG2 cells with pHBV1.2-core- plasmid, pHBc14-18M,pHBc120-135M and pHBc122-139M mutant groups formed nucleocapsid-like structure but pHBc23-39M could not, Northern and Southern blot displayed no signal in all mutants except 1-183flag conrol group. In pHBV1.2 cotransfection experiment, HBV DNA replication was blocked in pHBc14-18M, pHBc120-135M and pHBc122-139M mutant groups, sharply decreased in pHBc120-135M and pHBc122-139M groups, correspondingly virons production in medium were also inhibited. pHBc23-39M mutant exerted no influence on HBV replication. Conclusion pHBc23-39M mutant can neither form nucleocapsid-like structure nor interact with wild type HBc dimmer to interfere HBV replication.On the contrast, pHBc14-18M, pHBc120-135M and pHBc122-139M mutants can form nucleocapsid-like structure by themselves, but this structure does not support HBV DNA synthesis. Besides, they can effectively inhibit wild type HBV DNA replication by contacting with wild HBc dimmers resulting in nucleocapsid dysfunction.
2.Significance of serum hepcidin in assessment of liver inflammation activity among patients with chronic hepatitis B
Yinfei HU ; Tao HE ; Yunxia FEI ; Xiangbo ZHANG ; Jie WANG ; Ling GONG ; Xiaoben PAN ; Gongyin CHEN
Journal of Preventive Medicine 2022;34(3):240-243
Objective:
To investigate the value of serum hepcidin in assessment of liver inflammation activity among patients with chronic hepatitis B ( CHB ), so as to provide insights into the assessment of liver inflammation activity among CHB patients.
Methods:
A total of 79 CHB patients who were admitted to the Affiliated Hospital of Hangzhou Normal University were selected as the experimental group, while 40 healthy volunteers were randomly sampled as controls. Subjects'demographic data, liver function tests and iron metabolism parameters were collected from medical records, and serum hepcidin was measured using enzyme-linked immunosorbent assay ( ELISA ). In addition, ultrasound-guided liver biopsy was performed in CHB patients, and mild and moderate-to-severe CHB were classified according to liver inflammation activity and degree of liver fibrosis. Serum hepcidin levels were compared between the experimental and control groups and between patients with mild and moderate-to-severe CHB. The value of serum hepcidin in assessment of liver inflammation activity was examined among CHB patients using the receiver operating characteristic ( ROC ) curve analysis.
Results:
Subjects in the experimental group included 54 men ( 68.35% ) and had a mean age of ( 39.06±10.67 ) years, while the controls included 24 men (60.00%) and had a mean age of ( 42.43±11.44 ) years. Lower hepcidin levels were measured in the experimental group than in the control group [( 11.70±5.64 ) vs. ( 17.82±3.63 ) μg/L; P<0.05 ]. There were 54 patients with mild CHB ( 68.35% ) and 25 cases with moderate-to-severe CHB ( 31.65% ), and lower hepcidin levels were detected in patients with moderate-to-severe CHB than in those with mild CHB [ ( 6.92±2.21 ) vs. ( 13.95±5.36 ) μg/L; P<0.05 ]. The area under the ROC curve, optimal cut-off, sensitivity and specificity of serum hepcidin were 0.903 ( P<0.05 ), 10.365 μg/L, 100.0% and 72.2% for assessment of moderate-to-severe CHB, respectively.
Conclusion
Serum hepcidin is feasible to evaluate the liver inflammatory activity among patients with CHB.
3.Study on norovirus GⅡ.4/Sydney 2012 variant in China
Huan MAI ; Yan GAO ; Xiaoben PAN ; Jinchao HAN ; Xu CONG ; Lai WEI
Chinese Journal of Epidemiology 2014;35(2):157-162
Objective To study the nucleotide and amino acid sequences of norovirus G Ⅱ.4/Sydney 2012 variants,in China.Methods Twenty-two stool specimens,confirmed as G Ⅱ.4/Sydney 2012-positive were collected from Beijing in the winter of 2012-2013.RT-PCR was performed to target the complete capsid gene.G Ⅱ.4/Sydney 2012 strains from other regions in China were searched and obtained from the GenBank.Nucleotide and amino acid sequences of G Ⅱ.4/Sydney 2012 strains were analyzed,using the CLUSTAL X (Version 1.83)and followed by phylogenetic analysis using Mega version 5.1.Results The complete major capsid nucleotide sequences of thirty-eight G Ⅱ.4/Sydney 2012 strains from seven regions in China were obtained.The VP1 nucleotide and amino acid sequences diversity were 0.1%-3.3% and 0-3.1%,respectively.Result from phylogenetic analysis demonstrated that the G Ⅱ.4/Sydney 2012 variant shared a common ancestor with both the dominant norovirus G Ⅱ.4 variants Apeldoom 2008 and the New Orleans 2009.G Ⅱ.4/Sydney 2012 variants appeared to have had two A/D/E site combinations at the amino acid level,TSRN-GTT-SNT and TSRN-STT-SNT.Conclusion G Ⅱ.4/Sydney 2012 variant had been circulating in many regions in China.There seemed a high nucleotide and amino acid identity among G Ⅱ.4/Sydney 2012 strains collected from China.G Ⅱ.4/Sydney 2012 variants showed different A/D/E site combination from other pandemic G Ⅱ.4 variants.