Karataş Eylem, Erensoy Selda, Akarca Ulus Salih, Sertöz Rüçhan
Ege University Faculty of Medicine, Department of Medical Microbiology, Izmir, Turkey.
Ege University Faculty of Medicine, Department of Physiology, Izmir, Turkey.
Mikrobiyol Bul. 2018 Jan;52(1):23-34. doi: 10.5578/mb.61909.
Mutations in preS and S gene regions of hepatitis B virus genome may cause immune escape and diagnostic escape HBV mutants. The aim of this study was to determine preS1, preS2 and S gene regions of HBV from HBV infected patient groups by sequence analysis and contribute to the relevant literature. Nucleic acid sequence analysis of preS and S genes of HBV PCR products from 56 archived plasma samples sent to Ege University Faculty of Medicine Medical Microbiology Department Molecular Virology laboratory, for HBV tests were determined by chain termination reaction. Amino acid (aa) sequences were compared with the reference sequences obtained from GenBank. Plasma samples belonged to four groups of patients: A- Chronic HBV infected patients with typical HBV serological profiles (22 samples), B- HBV infected patients with atypical HBV serological profiles (26 samples), C- HBV re-infected patients after liver transplantation (5 samples), D- Seroconversion phase following acute HBV infection (3 samples). One of two vaccine escape mutant samples was also diagnostic escape mutant; the other diagnostic escape mutant was isolated from anti-HBc positive sample. All of the sequences were determined as genotype D. HBsAg subtypes were determined as; two ayw1, six ayw3, two mix, 46 ayw2. Among the 304 codons analysed between preS 33rd and S 162nd amino acids; aa variants were determinedin 105 codons (34.5%). Sequences can be found in GenBank with accession numbers FJ001941-FJ001996. At least one aa variation was detected in 48 of 56 samples (85.7%). The amino acid variants were as follows; PreS1: A33T, A39T, P41K, D44del, D50N, T51P, D54N, L65P/M, F67L, W77T, A81S, Q82E, I84T, L85I/M, Q86H/T, L88S, A90T/V, N91K/del, A95P, S96A, T97I/A, N98K, Q100K, S101T, S109T, P110S, N114D/E, PreS2: M1V, Q2R, S5H, F8S, H9Q, Q13L, D14N, R16K, R18K, G19S/D, F22L/S, S28T, G30E, N33T, V39A, P41H/L, I42T/L, I45T, F46Y, S47L, R48K, I49T, D51V/G, P52L, A53V, L54R/G, N55K; S gene: E2D, I4F, F8L, G10A, V14A, F20S, L22del, R24K, P29L, Q30K, N40S, F41del, G44E, T45L, T46P, V47A, L49R, Q54R, P56L, S64F, P70A, M75I, C76Y, R79H, I81T, F83C, L88P, L94S, Y100F, Q101H/R, M103L, L104F, L109I/M, I110L, G112S/R, S113N/P, S114A/del, T115I, T116N, T118A/K, P120A/T, T123A, in "a" determinant; T126I, Q129H/R, T131N, M133T, Y134N, S136Y, S143L/M/T, D144E, G145A/R. Deletions were also found in all three preS/S gene regions. The highest number of aa variations were detectedin the isolated anti-HBc positive sample (in 24 codons), followed by liver transplant group (8-13 codons). Point mutation was detected in the preS2/S promoter CCAAT box. Major hydrophilic region (MHR) variants were determined in 41.1% of 56 samples. The highest number of MHR variants belonged to atypical HBV serological profile group (group B; 61.5%) and liver transplantation group with HBV re-infection (all C group). Among the diagnostic escape and immune escape mutant (anti-HBs positive) samples, reported MHR and "a" determinant mutations were detected. In conclusion, the study population carries HBV preS/S variants; MHR and "a" determinant variant rates are high among diagnostic or immune escape mutants. It is important to evaluate the mutant detection performance of HBsAg tests.
乙型肝炎病毒基因组前S和S基因区域的突变可能导致免疫逃逸和诊断逃逸的HBV突变体。本研究的目的是通过序列分析确定HBV感染患者组中HBV的前S1、前S2和S基因区域，并为相关文献提供资料。对送至伊兹密尔大学医学院医学微生物学系分子病毒学实验室进行HBV检测的56份存档血浆样本的HBV PCR产物的前S和S基因进行核酸序列分析，通过链终止反应确定序列。将氨基酸（aa）序列与从GenBank获得的参考序列进行比较。血浆样本属于四组患者：A组——具有典型HBV血清学特征的慢性HBV感染患者（22份样本），B组——具有非典型HBV血清学特征的HBV感染患者（26份样本），C组——肝移植后HBV再感染患者（5份样本），D组——急性HBV感染后的血清转换期患者（3份样本）。两个疫苗逃逸突变体样本中的一个也是诊断逃逸突变体；另一个诊断逃逸突变体是从抗-HBc阳性样本中分离出来的。所有序列均确定为D基因型。HBsAg亚型确定为：两个ayw1、六个ayw3、两个混合型、46个ayw2。在分析的前S第33位和S第162位氨基酸之间的304个密码子中；在105个密码子（34.5%）中确定了aa变异。序列可在GenBank中找到，登录号为FJ001941-FJ001996。56份样本中的48份（85.7%）检测到至少一个aa变异。氨基酸变异如下：前S1：A33T、A39T、P41K、D44del、D50N、T51P、D54N、L65P/M、F67L、W77T、A81S、Q82E、I84T、L85I/M、Q86H/T、L88S、A90T/V、N91K/del、A95P、S96A、T97I/A/N、N98K、Q100K、S101T、S109T、P110S、N114D/E；前S2：M1V、Q2R、S5H、F8S、H9Q、Q13L、D14N、R16K、R18K、G19S/D、F22L/S、S28T、G30E、N33T、V39A、P41H/L、I42T/L、I45T、F46Y、S47L、R48K、I49T、D51V/G、P52L、A53V、L54R/G、N55K；S基因：E2D、I4F、F8L、G10A、V14A、F20S、L22del、R24K、P29L、Q30K、N40S、F41del、G44E、T45L、T46P、V47A、L49R、Q54R、P56L、S64F、P70A、M75I、C76Y、R79H、I81T、F83C、L88P、L94S、Y100F、Q101H/R、M103L、L104F、L109I/M、I110L、G112S/R、S113N/P、S114A/del、T115I、T116N、T118A/K、P120A/T、T123A，在“a”决定簇中；T126I、Q129H/R、T131N、M133T、Y134N、S136Y、S143L/M/T、D144E、G145A/R。在所有三个前S/S基因区域也发现了缺失。在分离的抗-HBc阳性样本中检测到的aa变异数量最多（24个密码子），其次是肝移植组（8-13个密码子）。在前S2/S启动子CCAAT框中检测到点突变。在56份样本的41.1%中确定了主要亲水区域（MHR）变异。MHR变异数量最多的属于非典型HBV血清学特征组（B组；61.5%）和HBV再感染的肝移植组（所有C组）。在诊断逃逸和免疫逃逸突变体（抗-HBs阳性）样本中，检测到了报道的MHR和“a”决定簇突变。总之，研究人群携带HBV前S/S变异体；在诊断或免疫逃逸突变体中，MHR和“a”决定簇变异率较高。评估HBsAg检测的突变体检测性能很重要。
