Hepatit B Virüsü (HBV) Genotip D ile Enfekte Hasta Gruplarında HBV preS1, preS2 ve S Gen Bölgelerinin Analizi
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Tarih
2018
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info:eu-repo/semantics/openAccess
Özet
Hepatit B virüsü (HBV)’nün preS ve S gen bölgelerinde oluşan mutasyonlar; immün ve tanısal kaçak mutantlara neden olabilmektedir. Bu çalışmada, HBV ile enfekte olgu gruplarında preS1, preS2 ve S gen bölgelerinde dizi analizi ile varyant ve mutasyonların araştırılması ve bu konudaki literatüre katkıda bulunulması amaçlanmıştır. Ege Üniversitesi Tıp Fakültesi Tıbbi Mikrobiyoloji Anabilim Dalı Moleküler Viroloji laboratuvarına HBV testleri için gönderilmiş ve arşivlenmiş 56 plazma örneğinden elde edilen HBV DNA PCR ürünlerinin preS ve S nükleik asit dizi analizi; zincir sonlandırma reaksiyonu ile gerçekleştirilmiştir. İncelenen plazma örnekleri: A- Alışılmış HBV serolojik profiline sahip (22 örnek), B- Alışılmış dışı HBV serolojik profile sahip kronik HBV enfeksiyonu (26 örnek), C- Karaciğer transplantasyonu sonrası reenfeksiyon (5 örnek), D- Serokonversiyon dönemindeki akut HBV enfeksiyonu (3 örnek) olan dört hasta grubundan elde edilmiştir. Çalışma grubundaki iki aşı kaçak örneğin biri tanısal kaçak mutant; diğeri ise anti-HBc pozitif örnekten izole edilen tanısal kaçak mutant örneğinden oluşmuştur. Elde edilen amino asit (aa) dizileri GenBank’tan alınan referans dizilerle karşılaştırılmıştır. Hepsi genotip D olarak saptanan örneklerin ikisi ayw1, altısı ayw3, ikisi karışık, kalan 46 örnek ayw2 HBsAg alt tipleri olarak tanımlanmıştır PreS1 33. ve S 162. aa arasında değerlendirmeye alınan 304 kodonun 105 (%34.5)’inde aa değişikliği saptanmıştır. Bu dizilere GenBank FJ001941-FJ001996 numaraları kullanılarak ulaşılabilmektedir. Elli altı örnekten 48 (%85.7)’inde en az bir aa değişikliği tespit edilmiştir. PreS1’de 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’de 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 gen bölgesinde 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, “a” determinantında T126I, Q129H/R, T131N, M133T, Y134N, S136Y, S143L/M/T, D144E, G145A/R aa varyantları saptanmıştır. Üç gen bölgesinde de delesyonlar tespit edilmiştir. En yüksek aa değişikliği izole anti-HBc pozitifliği olan (24 kodonda) örnekte, ardından karaciğer transplantasyonu sonrası re-enfeksiyon olan hasta grubunda (8- 13 kodonda) saptanmıştır. PreS2/S promotor CCAAT kutusunda da nokta mutasyon tespit edilmiştir. Elli altı örneğin %41.1’inde majör hidrofilik bölgede (MHR), %23.2’sinde “a” determinantında aa değişikliği saptanmıştır. MHR’de en yüksek aa değişikliği olan örnekler alışılmış dışı HBV serolojik profili olan (B grubu; %61.5) ve karaciğer transplantasyonu sonrası re-enfeksiyon olan hasta grubunda (C grubunun hepsi) gözlenmiştir. Tanısal ve immün (anti-HBs pozitif) kaçak örneklerde MHR ve “a” determinantında bildirilmiş mutasyonlar saptanmıştır. Sonuç olarak, çalışılan popülasyonda preS/S mutasyonları bulunmakta ve tanısal veya immün kaçak olarak düşünülen örneklerde MHR ve “a” determinant bölgelerinde aa değişiklik oranı yüksek seviyede tespit edilmiştir. Bu çalışma, HBsAg testlerinin kullanımında mutant saptama özelliklerinin değerlendirilmesinin önemli olduğu göstermektedir.
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), BHBV 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 mutati
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), BHBV 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 mutati
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Mikrobiyoloji Bülteni
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52
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