RESUMEN

Occult hepatitis B virus infection (OBI) is characterized by the presence of HBV DNA in the absence of detectable HBsAg. OBI is an important risk factor for cirrhosis and hepatocellular carcinoma, but its pathogenesis has not been fully elucidated. Mutations in the HBV preS/S genes can lead to impaired secretion of either HBsAg or S-protein resulting in the accumulation of defective viruses or S protein in cells. In our previous work, the M133S mutation was present in the HBV S gene of maintenance hemodialysis (MHD) patients with OBI. In this study, we investigated the potential role of amino acid substitutions in S proteins in S protein production and secretion through the construction of mutant S gene plasmids, structural prediction, transcriptome sequencing analysis, and in vitro functional studies. Protein structure prediction showed that the S protein M133S mutant exhibited hydrophilic modifications, with greater aggregation and accumulation of the entire structure within the membrane phospholipid bilayer. Differential gene enrichment analysis of transcriptome sequencing data showed that differentially expressed genes were mainly concentrated in protein processing in the endoplasmic reticulum (ER). The expression of heat shock family proteins and ER chaperone molecules was significantly increased in the wild-type and mutant groups, whereas the expression of mitochondria-associated proteins was decreased. Immunofluorescence staining and protein blotting showed that the endoplasmic reticulum-associated protein PDI, the autophagy marker LC3, and the lysosome-associated protein LAMP2 co-localized with the S proteins in the wild-type and mutant strains, and their expression was increased. The mitochondria-associated TOMM20 protein was also co-expressed with the S protein, but expression was significantly reduced in the mutant. The M133S mutation in the S gene is expressed as a defective and misfolded protein that accumulates in the endoplasmic reticulum causing secretion-impaired endoplasmic reticulum stress, which in turn triggers mitochondrial autophagy and recruits lysosomes to fuse with the autophagosome, leading to mitochondrial clearance. This study preliminarily demonstrated that the mutation of M133S in the S gene can cause OBI and is associated with disease progression, providing a theoretical basis for the diagnosis and treatment of OBI.

Asunto(s)

Estrés del Retículo Endoplásmico , Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B , Hepatitis B , Mitofagia , Diálisis Renal , Humanos , Mitofagia/genética , Hepatitis B/virología , Hepatitis B/genética , Hepatitis B/metabolismo , Hepatitis B/complicaciones , Virus de la Hepatitis B/genética , Estrés del Retículo Endoplásmico/genética , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Masculino , Mutación , Femenino , Persona de Mediana Edad , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , Sustitución de Aminoácidos , Adulto

RESUMEN

【Objective】 To analyze the correlation of HBV serological characteristics between non-reproducible reactivity (NRR) samples and occult hepatitis B virus infection (OBI) samples for blood screening. 【Methods】 A total of 144 samples with negative ELISA (HBV, HCV and HIV test) results and reactive nucleic acid tests(NAT) were collected from January 2021 to January 2023 in Anhui Blood Center, including 92 reactive samples by TMA method (combined ID-NAT) and 52 HBV DNA reactive samples by PCR method (ID-NAT). Supplementary differential testing and ID-NAT by PCR were performed on the reactive samples of the combined ID-NAT, samples that were non-reactive by both differential testing and ID-NAT by PCR were included in the NRR group, and samples that were reactive for HBV DNA detected by either method were included in the OBI group. Supplemented with HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc tests, the differences in serological pattern and positive rate between NRR samples and OBI samples were analyzed. 【Results】 A total of 53 samples were negative for differential testing and ID-NAT and were included in the NRR group, 91 samples were detected as HBV DNA reactive in either method and were included in the OBI group. HBsAg and HBeAg were not detected by serological testing in either group. The detection rates of anti-HBs, anti-HBc and anti-HBe in the NRR group and the OBI group were 64.15% vs 47.25%, 86.79% vs 94.51%, 35.85% vs 52.75%, respectively. Comparison of serological patterns between the two groups: the most frequent pattern in the NRR group was anti-HBs (+ ) and anti-HBc (+ ) (32.08%), and the most frequent pattern in the OBI group was anti-HBe (+ ) and anti-HBc (+ ) (37.36%). 【Conclusion】 There were differences in some of the test results between the NRR samples and the OBI samples in HBV serological testing, and higher anti-HBc positive rate in the NRR samples suggests a higher possibility of HBV infection.

RESUMEN

OBJECTIVE: To determine the actual hepatitis B virus (HBV) infection rate, occult HBV infection (OBI) rate, and molecular evolutionary characteristics of the OBI virus S gene in the adolescent population living in rural and pastoral areas of Xinjiang Province. METHODS: A cross-sectional questionnaire survey was conducted among the adolescent population living in the farming and herding areas. Venous blood samples (3-5 mL) were collected from eligible students in three central schools located in Banfanggou Township, Shuixigou Village, and Miaolgou Village, all in Urumqi County, in the nine-year compulsory system. Clustersampling in a population was adopted, and informed consent was obtained from the participating students. All serum samples were qualitatively tested for hepatitis B surface antigen (HBsAg) by electrochemiluminescence. Subsequently, the HBV S gene was amplified by nested polymerase chain reaction (PCR), and the positive PCR products were purified; the target gene sequences were then amplified. Molecular evolutionary characterization of the target gene sequences was performed using MEGA 11software. RESULTS: Overall, 1712 subjects were enrolled. The HBsAg carrier rate and OBI infection rate were 1.93% (33/1712) and 6.13% (103/1679), respectively. HBsAg (-) samples included 103 OBI strains, of which B-genotype strains accounted for 80.58% (83/103; 1 case of ayw1 serotype and 82 cases of adw2 serotype), C-genotype strains accounted for 14.56% (15/103; 1 case of adw2 serotype and 14 cases of adrq+serotype), and D-genotype strains accounted for 4.85% (5/103; 1 case of adw2 serotype and 4 cases of ayw2 serotype). Mutations were detected in the "a" determinant region of the following genes: P127S, G130R, and N146S (B-genotype OBI strains); T126I and T143S (C-genotype OBI strains); T126I, P127S, F134Y, and T143S (D-genotype OBI strains). CONCLUSION: A certain proportion of young people are infected with OBI strains. The B-genotype of OBI strains is the possible dominant genotype. OBI strains have amino acid mutations in the "a" determinant region, and they are likely to undergo a change in their antigenicity and immunogenicity. More attention must be paid to prevent problems due to OBI.

Asunto(s)

Antígenos de Superficie de la Hepatitis B , Hepatitis B , Adolescente , Humanos , Estudios Transversales , ADN Viral/genética , Genotipo , Hepatitis B/epidemiología , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Población Rural , China

RESUMEN

Background: All Chinese blood centers have implemented mini pool (MP) HBV nucleic acid testing (NAT) together with HBsAg ELISA in routine donor screening since 2015. The prevalence of occult hepatitis B virus infection (OBI) in donors from different regions varies, and the molecular characterization of the HBV DNA and clinical outcomes of these OBIs remain largely unexplored. Methods: Blood donations from Heyuan city in Southern China were screened by HBsAg ELISA and HBV MP8 NAT. Donations with HBsAg-/HBV DNA+ were collected for this study. Molecular characterizations of HBV DNAs were further analyzed by various DNA amplification assays including quantitative PCR (qPCR) and nested PCR, amplifying the basic core and pre-core promoter regions (BCP/PC). The HBsAg (S) region from HBV DNA was isolated by high-volume nucleic acid extraction. Notable mutations were identified by comparison to the HBV reference sequences. The clinical outcomes of the donors with OBIs were further followed for nearly 3 years. Results: Seventy OBIs from 44,592 donations (0.15%) that we identified and reported previously were enrolled for this current study. HBV sequences were obtained from 44/70 OBIs, and genotyping analysis showed that 42/44 (95.2%) OBIs were genotype B, and 2/44 (4.8%) were genotype C. Interestingly, mutation analysis revealed that various mutations including M133L/T, F134L, P142L, V168A, R169H, S174N, L175S, and V177A of HBV DNA affecting HBsAg detection were observed in genotype B OBIs. Two notable mutations, T47K and L53S, were identified in genotype C OBIs. Follow-up studies showed that 3/31 (9.7%) OBIs converted to HBsAg+ as chronic infections while 1/31 (3.2%) HBV DNA was undetectable (classified as recovery) and 27/31 (87.1%) remained as OBIs. Conclusion: Various notable mutations affecting HBsAg detection were observed in blood donors with OBIs in Heyuan city of Southern China. Follow-up studies showed that most OBIs remained as OBIs with fluctuating or low viral loads. Higher sensitive HBV ID NAT is recommended for donor screening to further reduce the transmission risk of OBIs.

Asunto(s)

Hepatitis B Crónica , Hepatitis B , Donantes de Sangre , ADN Viral/genética , Hepatitis B/diagnóstico , Hepatitis B/epidemiología , Hepatitis B/prevención & control , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Humanos , Mutación

RESUMEN

Background: Occult hepatitis B virus infection (OBI) is defined as undetectable serum hepatitis B surface antigen (HBsAg) with detectable HBV-DNA in the serum or liver. Patients with maintenance hemodialysis (MHD) are at a high risk of OBI. The prevalence of OBI in MHD patients in China is not well evaluated. In this study, we aim to assess the prevalence of OBI in MHD patients in Sichuan Province, Southwest of China and investigate the mutations in the "a" determinant of HBsAg. Methods: A total of 330 patients undergoing MHD at Sichuan Provincial People's Hospital were enrolled. Serum samples were collected for ELISA assay to test the serological markers of HBV infection, real-time PCR assay to identify the presence of HBV-DNA, and nested PCR plus sequencing analysis to investigate the gene mutations. Results: In a total of 330 MHD patients, we found that the prevalence of OBI was 4.2% (7/165) in the test group, 2.1% (7/330) in the overall dialysis cohort. After a follow-up study of 7 MHD patients with OBI for 2 years, 2 (isolated HBcAb+) of them were still detectable for HBV-DNA. By sequencing analysis, we revealed mutations at the "a" determinant of HBsAg, including Q129R, T131N, M133S, F134L and D144E. The Q129R and M133S mutations were first reported. Conclusions: Our study clarifies the prevalence of OBI in MHD patients in Sichuan Province(4.2% in the test group, 2.1% in the overall dialysis cohort), and demonstrate the mutations of Q129R and M133S in the "a" determinant of HBsAg for the first time.

Asunto(s)

Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B/epidemiología , Diálisis Renal/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Análisis Mutacional de ADN , ADN Viral/sangre , ADN Viral/genética , ADN Viral/aislamiento & purificación , Femenino , Estudios de Seguimiento , Hepatitis B/sangre , Hepatitis B/diagnóstico , Hepatitis B/virología , Antígenos de Superficie de la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Humanos , Masculino , Persona de Mediana Edad , Mutación , Prevalencia , Reacción en Cadena en Tiempo Real de la Polimerasa , Pruebas Serológicas

RESUMEN

BACKGROUND: We sought to clarify the prevalence of occult hepatitis B virus (HBV) infection (OBI) and to determine whether OBI affects the surgical outcomes in curatively resected Japanese patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). METHODS: A total of 257 patients with HCV-related HCC who underwent curative surgical resection were enrolled. All enrolled patients were serologically negative for HBV surface antigen and positive for HCV antibody. DNA was extracted from formalin-fixed paraffin-embedded liver tissue. OBI was determined by the HBV-DNA amplification of at least two different sets of primers by TaqMan real-time polymerase chain reaction. Surgical outcomes were evaluated according to overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS). RESULTS: OBI was identified in 15 of the 257 (5.8%) cases. In the multivariate analyses, the factors significantly correlated with OS were BMI >25 (P=0.0416), portal vein invasion (P=0.0065), and multiple tumors (P=0.0064). The only factor significantly correlated with DSS was T-stage (P=0.0275). The factors significantly correlated with DFS were liver fibrosis (P=0.0017) and T-stage (P=0.0001). The status of OBI did not show any significant correlation with OS, DSS or DFS, but a weak association with DSS (P=0.0603) was observed. CONCLUSIONS: The prevalence of OBI was 5.8% in 257 cases of HCV-related HCC. Although a weak association between DSS and OBI was observed, and statistical analyses were limited by small number of OBI cases, no significant correlation between OBI and surgical outcomes was detected.

RESUMEN

Objective To analyze the mutation of PreS-S region in occult hepatitis B virus(OHBV) in HBV infected persons with positive HBsAb and investigate the biological mechanisms of the special infectious model.Methods A total of 38 HB-sAb positive OBI serum samples were amplified by Nested PCR and sequenced,HBV genotype and serotype were determined.The amino acid sequences of OHBV were compared to the corresponding sequence of wild-type strains of similar genotype obtained from the GenBank database.Results PreS-S segment of 11 samples were obtained and 8 samples were sequenced successfully.Among which,5 were genotype C and 3 were genotype B.Genotype B were all serotype adw,while genotype C were 1 adw and 4 adr.The mutation rates of PreS-S region,the immunoreactive area and the major hydrophilic region (MHR) were higher in OHBV than the wild-type strains (2.6％ vs 0.8％,x2 =40.23,3.2％ vs 0.3％,x2 =52.13,3.6％ vs 0.6％,x2 =13.25,all P＜0.01) and the substitutions of I126T,Q129R,M133T,F134I,D144E,G145K in α determinant were found in OBI samples.The mutation rate of amino acids in PreS-S region was higher in genotype C than genotype B (3.5％ vs 1.2％,x2--15.98,P＜0.01),meanwhile,the mutation rates in MHR,α determinant and immunoreactive region were higher in genotype C too,but no statistical significance was attained (4.7％ vs 1.7 ％,x2 =2.96,3.6 ％ vs 2.9％,x2 =0.25,4.1％ vs 2.3％,x2 =3.59,all P ＞0.05).Conclusion Mutations in PreS-S region,especially in immunoepitope,might change the virus'immunogenicity leading to escape from immune response and cause OBI with HBsAb positive.

RESUMEN

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.