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The pre-S/S gene of hepatitis B virus (HBV) can encode for the production of large, medium and small surface protein. Different protein expression levels and their composition ratios have certain influences on the diagnosis, treatment and outcome of HBV infection. It is of great significance to clarify the functions of large, medium and small surface protein as serum markers and to explore their value in the diagnosis and treatment of HBV infection. In this paper, the expression status, detection methods and clinical significance of the three HBV proteins were reviewed.
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【Objective】 To analyze the characteristics of gene mutation in S region of blood donors with occult hepatitis B virus infection (OBI) in Huzhou area. 【Methods】 A total of 60 107 blood samples in Huzhou between October 2018 and June 2020 were collected by our blood station. Among them, 52 samples were NAT, yield and their epidemiological characteristics were analyzed. Twenty-seven OBI out of the 52 NAT yield samples were included in experimental group. Other eight HBV-infected individuals with positive HBsAg, core antibody (anti-HBc) and HBV-DNA were selected as positive control. Liver function and 5 serological markers of HBV were compared between the two groups, and HBV genotypes and amino acid mutation in S region in the two groups were analyzed. 【Results】 The number of NAT-yield samples were different by gender, age, and educational background (P0.05). Surface antigen (HBsAg) in the experimental group was significantly lower than that in the control group, while surface antibody (anti-HBs) and e antibody (anti-HBe) were significantly higher than those in the control group (P<0.05). Twenty sequences in S region were obtained from the experimental group, including 4 in S region and 16 in preSS region; 16 cases with type C and 4 cases with type B. 【Conclusion】 The follow-up of NAT-yield blood donors in Huzhou area should be conducted. Compared with HBV infected individuals with positive HBsAg, anti-HBc and HBV-DNA, those with OBI have a higher gene mutation rate in S region.
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Objective@#To analyze the characteristic mutations of epitopes in HBV Pre-S/S region in HIV/HBV co-infected patients’ peripheral blood to provide basic data for studying the pathogenesis of HIV/HBV co-infection.@*Methods@#The chronic hepatitis B infected patients admitted to the Infectious Disease Center of the Eighth People′s Hospital of Guangzhou from January 2009 to December 2011 were enrolled into HIV/HBV co-infected group and HBV mono-infected group according to the result of HIV antibody detection respectively before treatment. HBV DNA in serum was extracted and Pre-S/S region of HBV DNA was amplified by nested-PCR. After sequencing of the obtained PCR products (direct sequencing), ContigExpress software was used for sequence splicing and BioEdit software was used for sequence alignment. With reference to the standard sequence of the matched genotype HBV, mutants of HBV Pre-S/S region in HIV/HBV co-infected group and HBV mono-infected group were analyzed respectively. Statistical analysis was performed by chi-square test with SPSS19.0 statistical analysis software.@*Results@#HBV Pre-S/S fragments were successfully amplified from 150 patients, including 90 cases of HIV/HBV co-infected group and 60 cases of HBV mono-infected group, with matched gender, age, genotype, HBeAg status, alanine aminotransferase (ALT), aspartate aminotransferase (AST). The result of analyzing mutants of HBV Pre-S/S region indicated that the incidence of mutation in all epitopes for cytotoxic T cells (CTL cells) was higher in the HIV/HBV co-infected group, and Pre-S2 aa1-15 epitope was significantly higher (χ2=6.964, P=0.008). The incidence of deletions in PreS2 aa1-15 epitope in HIV/HBV co-infected group (11.1%) was higher than HBV mono-infected group (3.3%) (χ2=2.959, P=0.085). In the B cell epitopes, the incidence of mutations in Pre-S2 aa1-26 in the HIV/HBV co-infected group was significantly higher than HBV mono-infected group (χ2=6.924, P=0.010), and there was no statistical significance between two groups in other B cell epitopes. No differences in helper T cell (Th cell) epitopes were found between the two groups.@*Conclusions@#Co-infection with HIV increased the CTL cell epitopes’ mutations in the HBV Pre-S/S region, especially the 5′ end epitope mutations in Pre-S2 region, which indicated that HBV mutation is related to the host immune status, and showed guiding information for further study on the pathogenesis of HIV/HBV co-infection
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Hepatitis B virus (HBV) infection could lead to different clinical presentations and disease progresses, including acute and chronic hepatitis B, liver cirrhosis, hepatocellular carcinoma, and occult HBV infection, in which the interaction between virus and host plays an important role. Because HBV reverse transcriptase is lack of correction function, HBV is prone to generate mutations under the pressure of host immune response and antiviral drug treatment. Some mutations in the HBV S gene-encoding region can significantly attenuate antibody immune response against HBV and therefore affect clinical presentations and disease progression. Such mutations are termed immune escape-related mutations. In this paper, we mainly review the structure and functional characteristics of HBV S gene, the causes and forms of the immune escape-related mutation, its influence on clinical presentations and antiviral treatment response, as well as clinical detection methods.
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Objective To analyze the sequence of Pre-S gene in asymptomatic chronic HBV carriers (ASCs) with low-level HBsAg.Methods The serum samples were collected from 654 ASCs in the First Affiliated Hospital of Zhejiang University School of Medicine , Hangzhou Sixth People’s Hospital and the 903th Hospital of PLA.According to the level of HBsAg , ASCs were divided into low-level HBsAg group (≤10 IU／mL ) and high-level HBsAg group (＞10 IU／mL ).The pre-S／S gene amplification and sequencing were performed in 138 ASCs with low-level HBsAg and 100 age-matched ASCs with high-level HBsAg.A phylogenetic tree was constructed to determine the genotype , based on the successful sequencing results of Pre-S gene in the high level HBsAg group , the Pre-S gene reference sequences of the main ASCs genotypes in Eastern China were established.The sequence of Pre-S gene in low-level HBsAg group was analyzed and compared with the reference sequences.SPSS 12.01 statistical software was used to analyze the data.Results Sixty-three cases of Pre-S／S were successfully sequenced in 138 ASCs of low-level HBsAg group, including 52 cases of B genotype and 11 cases of C genotype.Among the 100 cases of high-level HBsAg group, 94 cases of Pre-S／S were successfully sequenced , including 48 cases of B genotype and 46 cases of C genotype.The sequence analysis indicated that in the B genotype , 81 amino acid mutation sites were found in the Pre-S protein of the low-level HBsAg group, including 4 significant mutations: F56I／V, T76A／N／P in the Pre-S1 region, P15L／S／T and Y21T／F／H／N in the Pre-S2 region; while 47 amino acid mutation sites were found in Pre-S protein of high-level HBsAg group, including 3 significant mutations :L34F, V49A and P59S／L in Pre-S1 region.The total number of amino acid mutation sites in the low-level HBsAg group of B genotype was higher than that of the high-level HBsAg group (χ2 ＝14.008, P＜0.05). In the C genotype, 19 amino acid mutation sites were found in the Pre-S protein of the low-level HBsAg group, including 3 significant mutations : W66V／G and A79V in the Pre-S1 region,V32A in the Pre-S2 region; while 39 amino acid mutation sites were found in Pre-S protein of the high-level HBsAg group, including 2 significant mutations: A79V in Pre-S1 region and T49I in Pre-S2 region.The total number of amino acid mutation sites of Pre-S protein in the C genotype was significantly different between the two groups (χ2 ＝7.571, P＜0.05).Conclusion Significant mutations in Pre-S gene may be associated with the persistent expression of low-level HBsAg in ASCs.
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Objective To reveal the characteristics of S gene sequence of hepatitis B surface antigen (HBsAg) in hepatitis B virus (HBV)-infected patients with low HBsAg level.Methods From February 2016 to December 2017, 1 308 serum samples of inactive HBsAg carriers were collected from the 903rd Hospital of PLA and Hangzhou Jianggan District People′s Hospital.The cases were divided into high-level group and low-level group according to the level of serum HBsAg (10 IU／mL) expression.The HBV S gene was sequenced in patients with low-level HBsAg expression.In addition, in patients with high-level HBsAg, 100 patients were randomly selected (stratified sampling) for HBV S gene sequencing based on the matching of age and serological pattern (hepatitis B e antigen [HBeAg] negative) of low-level HBsAg group.A comparative analysis was conducted between HBV S gene sequences from inactive HBsAg carrier in low HBsAg expression group and the HBV reference S gene sequences from inactive HBsAg carrier in high HBsAg expression group .The results of normal distribution data were expressed as Mean ±SD, and analyzed using t-test.The results of non-normal distribution data were expressed by M(QR), and analyzed using Mann-Whitney U test.Chi-square test or Fisher exact test was used to compare continuous variables and classification variables between the two groups .Results There were 276 serum samples from the low level group and 1 032 serum samples from the high level group , including 257 HBsAg／HBeAg／anti-HBc-positive cases, 753 HBsAg／anti-HBe／anti-HBc-positive cases, and 22 HBsAg／anti-HBc-positive cases.Successful HBV S gene sequencing was performed on 126 out of 276 patients in the low-level HBsAg group.According to the age inthe low-level HBsAg group, 100 samples with negative HBeAg in the high-level HBsAg group were randomly selected , among which 94 patients were genotyped and hemotyped.The results showed that there were statistically significant differences in HBV serological markers , HBV DNA level and HBV genotype distribution between the high level group (94 cases) and the low level group (126 cases) (all P＜0.05).The ASC-R-B and ASC-R-C genotypes reported in this study had high homology (99.6%-100.0%) with those reported in Shanghai , Chengdu, Wuhan, Yunnan and Beijing of China , and high homology (98.2%-99.6%) with those reported in Japan and Korea of NCBI genotype B and C reference sequences, but had low homology with patients far away from China (98.2% in Canada and 98.7% in Indonesia).In genotype B of the low level group , the amino acid mutation number of SHB protein was 71, and the hot spot mutation number was 19, both higher than those in the high level group (39 and 8, respectively). The difference was statistically significant (χ2 ＝12.303 and 4.766, respectively, both P＜0.05).Amino acid mutation sites in the low HBsAg group were mainly distributed on both sides of the major hydrophilic region (MHR) (amino acid residues 40 -49 and 198 -220).There were no significant differences in amino acid mutation number and hot spot mutation number between the two groups of C genotype (χ2 ＝0.383 and 0.409, respectively, both P＞0.05).For genotype B, 12 single point mutations and 4 dual co-mutations were found in low level group.Among them, one single point mutation (S210R) and 3 dual co-mutations (G44E／V+T45P／I, G44E／V+L49P／R and N40S+I208T) were not hot spot mutations , while 2 dual co-mutations and 2 single point mutations were found in high level group.The difference between two groups was statistical significant (χ2 ＝7.533,P ＝0.006).For genotype C, 5 single point mutations ( T5A, A45T, T47A／K, Q101R and I126S／T) were found in low level group and 1 single point mutation (N3S) in high level group.The difference in mutation frequency between two groups were statistical significant (χ2 ＝47.914,P＝0.000).Conclusions Significant mutations in multiple regions and at multiple sites ( including co-mutations) on both sides of the MHR may be one of the causes of low HBsAg expression level in this population .
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Objective To determine the reactivity of occult HBV infection (OBI)-related S-gene mutations to various anti-HBs antibodies (Abs) and HBsAg detection reagents.Methods Nine representative S-gene mutations M1-M9 (including 5 novel mutations M1-M5) from 1 OBI patient and 3 OBI blood donors were investigated.S-gene recombinant plasmids harboring 9 mutants or wild-type sequences constructed before were transfected into Chinese hamster ovary (CHO) cells,respectively.HBsAg expression levels and reactivity with various anti-HBs Abs and regular HBsAg detection reagents of all the mutants were analyzed.Results Compared to wild-type strain,intracellular HBsAg levels of the 9 mutants were obviously reduced upon Roche quantitative Elecsys assay.In contrast,analysis of the same samples using anti-His-tag Ab showed that the levels of HBsAg-Histag fusion protein were significantly reduced only in mutants M1,M6,and M7.The results of reactivity of mutant HBsAg against 6 anti-HBs Abs (S/CO values) showed that poor reactivity was observed for most mutants.Specifically,M1-,M4-,M7-,and M9-produced HBsAg with Ab4,and M9-produced HBsAg with Ab6 had the worst reactivity (S/CO＜1) compared to the wild-type.The results of reactivity of mutant HBsAg with 6 commercial HBsAg detection reagents showed that the reactivity for most mutants was significantly lower than that of the wild-type (P＜0.05).The miss rates of ELISA reagents D,E,and F were 11.1％,22.2％,and 55.6％,respectively.Conclusions Lower-affinity of studied mutant HBsAg with anti-HBs is one of the major causes of OBI presentation.The regular HBsAg detection reagents used currently in clinic show a significant deficiency in detection of the mutant HBsAg and thus need to be improved.
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Objective To study the prevalence of the occult hepatitis B virus infection (OBI) and the mutation of amino acid sequence in S gene of voluntary blood donors in AnHui/FuJian/Jiang Xi Province Blood centers.Methods Serologic testing for anti-HBc by ELISA was performed with HBsAg-HBV DNA+ samples from voluntary blood donors in three province blood centers.The S region of HBV of those samples was amplified and sequenced.The genotype and mutation of amino acid sequence were analyzed by MEGA6.Results 21 in 123046 blood donors from AnHui Province blood center were HBsAgHBV DNA+,the prevalence of OBI was 0.017％,and 76.2％ of these-OBI samples was positive in anti-HBc,S region was amplified by nest-PCR in 15 OBI samples,8 of them were B genotype,the others were C genotype.39 samples of 51 OBI blood donors from FuJian Province blood center were anti-HBc positive,16 samples of those OBI donors were amplified S region,14 were B genotype,the others were C genotype.There are 30 OBI blood donors from JiangXi Province blood center,24 of them were anti-HBc positive,S region was amplified in 4 samples,1 was B genotype,the others were C genotype.Of all 35 OBI samples,26 showed amino acid mutation,which was in MHR region of S gene,especially in HBV α epitope.Conclusion The rate of prevalence of OBI in AnHui Province was 0.017％,there was also certain OBI infection in FuJian and JiangXi Province.In the OBI samples which were amplified S region,the positive rates of anti-HBc in three blood centers were 73.3％,93.8％,100％.B Genotype was the main HBV genotype.The mutation in MHR region of S gene,especially in HBV α epitope,may be one of the reasons to cause OBI.
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Objective@#To investigate the genetic characteristics of Lamivudine-resistant mutation patterns and HBV S gene mutants in patients with chronic hepatitis disease of different disease progression.@*Methods@#Blood samples of LAM-resistant patients with chronic hepatitis disease were collected. HBV RT gene nucleotide sequences were obtained, and then differences in drug-resistant mutation patterns, drug susceptibility and HBV S gene mutants characteristics between the two groups were analyzed.@*Results@#Forty-seven chronic hepatitis B (CHB) patients and 16 HBV-related liver cirrhosis (LC)/HBV-related hepatocellular carcinoma (HCC) patients were included in this study. M204I single point mutation and L180M+ M204I/V were the most common pattern during patients with chronic hepatitis disease (35/63, 55.56%). The numbers of resistant to three nucleos(t)ide analogues in LC/HCC group was higher than CHB group’s (62.50% vs 34.04%, P=0.046). In HBV S gene, more immune associated HBsAg-escape mutations were detected in LC/HCC group than that in CHB group (62.50% vs 31.91%, P=0.031). I126T/V and G145A (for LCC/HCC group, 60%), I126S/T and S117T (for CHB group, 46.67%) were showed as the most common form for HBsAg escape mutations in the two groups. The two groups both detected RT mutations concomitantly with stop codon mutations in S gene (rtA181T/sW172* and rtM204I/sW196*).@*Conclusions@#Different characteristics in Lamivudine-resistant mutations and associated HBV S gene mutants were found in patients with chronic hepatitis disease of different disease progression, and LC/HCC patients exhibit more multi-drug resistant variants and immune associated HBsAg-escape mutants than CHB patients.
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In order to understand the porcine epidemic diarrhea virus (PEDV) origin and variant characteristics in Liaoning province,diagnosed by PCR,separated by Vero cell,and identified by cell pathological observation,RT-PCR and S gene sequence analysis,1 PEDV strains (LN-2015-1) was successfully isolated from a pig farm of Liaoning province.Analysis of S gene sequence showed that compared withCV777 strain,there were the longest 9 bp insertion,6 bp deletion and 13 bp continuous mutation in addition to point mutation.There also were the longest 3 AA insert,2 AA deletion,and 3 AA or more continuous mutation.The epitope analysis showed that there were 16AA mutations in the 5 epitope regions.Homology analysis show that it had the highest sequence similarity of 99.2％ with HB-HA2015 strain,higher sequence similarity of 98.5％-98.8％ with the domestic and foreign representative strains isolated since 2010,and lower sequence similarity of 93.2％-95.6％ with the traditional strain isolated before 2010;the phylogenetic analysis showed that LN-2015-1 was clustered into the same group with home and abroad variation strain in recent years,and formed a small subgroup with HB-HA2015 at the same time.The evolutionary distance was far from the traditional strains.
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PURPOSE: The aim of this study is to investigate the molecular characteristics of occult hepatitis B virus (HBV) infection in ‘anti-HBc alone’ subjects. MATERIALS AND METHODS: Twenty-four patients with ‘anti-HBc alone’ and 20 control patients diagnosed with HBV were analyzed regarding S and pre-S gene mutations. All specimens were analyzed for HBs Ag, anti-HBc, and anti-HBs. For specimens with an anti-HBc alone, quantitative analysis of HBV DNA, as well as sequencing and mutation analysis of S and pre-S genes, were performed. RESULTS: A total 24 were analyzed for the S gene, and 14 were analyzed for the pre-S gene through sequencing. A total of 20 control patients were analyzed for S and pre-S gene simultaneously. Nineteen point mutations of the major hydrophilic region were found in six of 24 patients. Among them, three mutations, S114T, P127S/T, M133T, were detected in common. Only one mutation was found in five subjects of the control group; this mutation was not found in the occult HBV infection group, however. Pre-S mutations were detected in 10 patients, and mutations of site aa58–aa100 were detected in 9 patients. A mutation on D114E was simultaneously detected. Although five mutations from the control group were found at the same location (aa58–aa100), no mutations of occult HBV infection were detected. CONCLUSION: The prevalence of occult HBV infection is not low among ‘anti-HBc alone’ subjects. Variable mutations in the S gene and pre-S gene were associated with the occurrence of occult HBV infection. Further larger scale studies are required to determine the significance of newly detected mutations.
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Humans , DNA , Hepatitis B virus , Point Mutation , PrevalenceABSTRACT
O presente estudo teve como objetivo descrever a ocorrência de otite parasitária causada por Rhabditis blumi em bovinos leiteiros de raça Gir de uma fazenda da região Norte do Brasil. Foram coletadas amostras de 42 bovinos por swab e lavado dos condutos auditivos externos (CAE). Ao exame clínico, 9,5% (4/42) dos bovinos apresentavam sintomatologia nervosa, como leve a moderada rotação da cabeça, apatia, flacidez dos lábios e ptose palpebral unilateral, alopecia das regiões da cabeça e cupim, causados pelo desconforto e prurido da região auricular, alteração na mastigação e acúmulo de alimento na cavidade oral. Adicionalmente, 71,4% (30/42) dos abovinos mostraram a presença do parasita no cerúmen dos condutos auditivos. À análise microscópica do material do saco conjuntival foi observado presença do parasita em 90% (9/10) dos bovinos avaliados. Os 30 bovinos positivos para Rhabditis spp. foram distribuídos aleatoriamente em três grupos de 10 animais: (G1) Bovinos controle, (G2) Bovinos tratados com ivermectina 1% pour on e (G3) Bovinos submetidos a lavado dos condutos auditivos externos (CAE). Cada tratamento foi repetido três vezes com intervalo de sete dias. No G1 os 10 bovinos mantiveram-se infectados durante todo o estudo. No G2 20% dos bovinos foram negativos após os dois primeiros tratamentos, porém, mostraram-se positivos na terceira avaliação. No G3 todos os bovinos mantiveram-se positivos, sendo observada apenas diminuição da carga parasitária. A identificação por análise molecular por meio de fragmentos amplificados da expansão D2/D3 do 28S rDNA confirmou a presença apenas da espécie Rhabditis blumi nos animais. Baseado nas observações clínicas, morfológicas e moleculares pode-se relatar o primeiro caso de R. blumi em bovinos da raça Gir no Estado do Pará, através da compra de animais oriundos de áreas onde a otite parasitária tem sido diagnosticada, principalmente de Minas Gerais, para formar animais mestiços (Gir x Holandês). Desta forma ressalta-se a importância do exame clínico prévio dos animais a serem transferidos para outras propriedades ou regiões. Este relato também parece ser o primeiro sobre a presença de R. blumi no saco conjuntival de bovinos. O tratamento com ivermectina no G2 não surtiu melhora clínica dos bovinos.(AU)
This study aimed to describe the occurrence of parasitic otitis caused by Rhabditis blumi in dairy cattle of the Gir race from a farm in northern Brazil. Forty-two samples were collected from cattle by swab washed from the external auditory canal (EAC). On clinical examination, in 71.4% (30/42) of the cattle the parasite was found in the cerumen of the ear canal, along with alopecia of head and hump caused by discomfort and itching of the auricular region. At microscopic analysis of material from the conjunctival sac the parasite was found in 90% (9/10) of the evaluated cattle. In addition, 9.5% (4/42) of the cattle showed nervous symptoms, such as mild to moderate rotation of the head, apathy, flaccid lips and unilateral ptosis, change in chewing and food accumulation in the oral cavity. Thirty cattle positive for Rhabditis spp. were randomly divided into three groups of 10 animals each: (G1) Cattle Control, (G2) Cattle treated with ivermectin 1% pour-on, and (G3) Cattle undergoing wash of the external auditory canal (EAC). Each treatment was repeated three times with intervals of seven days. In G1, 10 cattle remained infected throughout the study. In G2, 20% of the cattle were negative after the first two treatments, however were positive at the third evaluation. In G3, all cattle remained positive, but with decrease in parasite load. Identification by molecular analysis of amplified fragments through the expansion D2/D3 28S rDNA confirmed the presence of only Rhabditis blumi. Based on clinical, morphologic and molecular examination, it appears to be the first report of the occurrence of R. blumi infection in Gir cattle in the State of Pará, due to the purchase of cattle from areas where parasitic otitis has been diagnosed, as from Minas Gerais, to produce crossbred animals (Gir x Holstein). This emphasizes the importance of prior clinical examination by the veterinary service in order to transfer only healthy animals to other properties or regions. This appears also to be the first report on R. blumi infection of the conjunctival sac in cattle. Treatment with ivermectin in G2 did not produce clinical improvement.(AU)
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Animals , Cattle , Ivermectin/therapeutic use , Otitis/diagnosis , Otitis/parasitology , Otitis/therapy , Rhabditoidea/parasitology , Genome , Parasitic Diseases/diagnosisABSTRACT
Abstract Background The mechanism underlying the coexistence of hepatitis B surface antigen and antibodies to HBsAg in chronic hepatitis B patients remains unknown. Aims This research aimed to determine the clinical and virological features of the rare pattern. Methods A total of 32 chronic hepatitis B patients infected by HBV genotype C were included: 15 carrying both HBsAg and anti-HBs (group I) and 17 solely positive for HBsAg (group II). S gene and reverse transcriptase region sequences were amplified, sequenced and compared with the reference sequences. Results The amino acid variability within major hydrophilic region, especially the “a” determinant region, and within reverse transcriptase for regions overlapping the major hydrophilic region in group I is significantly higher than those in group II. Mutation sI126S/T within the “a” determinant was the most frequent change, and only patients from group I had the sQ129R, sG130N, sF134I, sG145R amino acid changes, which are known to alter immunogenicity. Conclusions In chronic patients, the concurrent HBsAg/anti-HBs serological profile is associated with an increased aa variability in several key areas of HBV genome. Additional research on these genetic mutants are needed to clarify their biological significance for viral persistence.
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Adult , Female , Humans , Male , Young Adult , Hepatitis B Antibodies/blood , Hepatitis B Surface Antigens/blood , Hepatitis B virus/genetics , Hepatitis B, Chronic/genetics , Hepatitis B, Chronic/immunology , RNA-Directed DNA Polymerase/genetics , Viral Envelope Proteins/genetics , China , DNA, Viral , Genotype , Hepatitis B virus/immunology , Mutation , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Objective To analyze the characteristics of newly added N-glycosylation mutation in major hydrophilic region (MHR) of HBV S gene in patients with coexistence of HBsAg and antiHBs, and reveal the generation mechanism and clinical implications of the coexistence. Methods HBV S genes from 284 patients with HBsAg+antiHBs and 314 patients with single HBsAg were amplified respectively for sequence analysis. A chronic hepatitis B (CHB) patient with HBsAg+antiHBs in MHR was found to harbor a novel double N-glycosylation mutation and selected for further study. Recombinant vectors harboring the novel mutant or control PreS/S genes were constructed and transfected in HepG2 cells respectively for phenotypic analysis, and the effects of the mutations on HBV duplication and antigenicity were investigated. Results The detection rate of MHR N-glycosylation mutation was significantly higher in HBsAg+antiHBs group than in single HBsAg group (11.3% vs. 2.9%, P<0.01, respectively). In HBsAg+antiHBs cohort, the proportion of hepatocellular carcinoma (HCC) patients accounted for 46.9%(15/32) in patients with N-glycosylation mutation at the time of testing; by contrast, the number was 22.6%(57/252) in patients with non-N-glycosylation mutation (P<0.01). N-glycosylation mutational pattern of the novel strain was s116-118TST → NST+s131-133TSM → NST concomitant with sP120 deletion+G145D mutation. The novel mutants accounted for 98.0%, 2.0% and 2.5%, respectively, of viral clones in three sequential serum samples. Mutants with single N-glycosylation mutation s130-132GTS→NSS without sP120 deletion+G145D were detected in sample 2, accounting for 17.6% of viral clones. Compared to the wild-type, the novel mutant had an increase of 31% in replication capacity, but a decrease of 99% in HBsAg level. Immunofluorescence showed that elimination of the two additional N-glycosylation mutations only partly restored HBsAg detection by antiHBs, suggesting that sP120 deletion+G145D mutation also attenuated HBsAg antigenicity. Conclusions Additional N-glycosylation mutation in MHR of HBV S gene is associated with coexisting HBsAg+antiHBs, and the two parameters together might be a better risk factor for HCC occurrence. Combination of two additional N-glycosylation mutation, sP120 deletion and sG145D mutation may co-play a role in silence of HBsAg antigenicity.
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Objective To analyze the conding region of hantanvirus S gene and predict the structure of nucleoprotein for diagnostic antigen study.Methods RT-PCR was used to amplify the S gene of hantanvirus Hunan03 strain after designing specific primers.The amplification product was cloned into pGM-T vector and then the recombinant vector was transformed into E.coli TOP10,gene sequencing was carried out after blue-white selection and PCR screening for positive clones.The database of NCBI and Swiss-Prot/TrEMBL were used to predict and analyze the structure,biological characteristics and protein structures of S gene.Results The amplification product was about 1290 bp,the pGM-T/S vector was constructed and successfully sequenced,the whole length of the open reading frame (ORF) was composed of 1290 nucleotide residues,among them the GC content was 44.11％ and the AT content was 55.89％,it was composed of 429 amino acids (20 kinds),the accession number of the sequence submitted to GenBank was JN712306,its homology of nucleotides to the 76-118 strain was 83％ and the homology of amino acids was 98％,ten nonspecific variation sites were found.The grand average of hydropathicity was-0.405.There were three transmembrane domains and four non transmembrane domains in the secondary structure of nucleoprotein including 55％ of helix structure,6.1％ of sheet structure and 38.9％ of loop structure.Conclusion The bioinformatics analysis of Hunan03 strain S gene might be important for provide the substructure data to reveal the significance of S gene characteristics on hemorrhagic fever renal syndrome (HFRS) prevention and control.
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Objective To study the complexity of S region qusispecies in various disease stages of chronic hepatitis B virus(HBV) infection and its relation to disease activity. Methods Serum samples were obtained from 112 patients with chronic hepatitis B virus infection;22 with chronic carries(ASC),30 with chronic mild or moderate hepatitis(CH),60 with fulminant hepatitis failure(FHF). HBV qusispecies populations were separated by the single strand conformation polymorphism (SSCP) method targeted the S region and DNA sequencing analysis. Results The number of SSCP bands detected in the patients with ASC、CH and FHF was 1.45?0.13,3.70?0.22 and 5.93?0.24, respectively. There was a statistically significant difference in the number of quasispecies among various disease stages ( P
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Objective To construct a DNA vaccine capable of expressing S gene of hepatitis B virus(HBV) and evaluate the expression of recombinant S gene in vitro and in vivo. Methods A cloned S X gene fragment was inserted into a eukaryote expression vector to construct a recombinant plasmid. The S gene was transcribed in vitro and expressed in a transfected cell line, and the efficiency of HBsAg in eliciting anti HBs was evaluated in mice. Results The expression of S gene was confirmed by Northern blotting, Western blotting, and ELISA(for both antigen and antibody detection). Conclusions The recombination and expression of S gene is achieved successfully in vitro.
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Objective To investigate the inhibitory effects of exogenous WAF 1 S gene on human laryngeal cancer Hep 2 cell line, and to explore the potential use of WAF 1 S in gene therapy for laryngeal cancer. Methods A eukaryotic expression vector containing 2 1kb human full length WAF 1 S cDNA was transfected into human laryngeal cancer Hep 2 cell line by using lipofectamine. Expression of exogenous WAF 1 S gene was detected by dot blot hybridization. By using Western blot and confocal microscope, expression of p21 protein was quantitatively analyzed in situ . The growth state of transfected Hep 2 cell was determined by flow cytometry and MTT. Results It was found by dot blot hybridization that WAF 1 S gene could express in Hep 2 cell. The expression of the exogenous p21 gene in Hep 2 cells was markedly higher than that in the control group. It was confirmed with flow cytometry that WAF 1 S gene could induce apoptosis of laryngeal cancer Hep 2 cell line, and the progression of cell cycle was arrested at G 1 phase. Conclusion Laryngeal cancer cells could be arrested at G 1 /S phase and the growth of the cells could be significantly suppressed by exogenous WAF 1 S gene