«ΜΕΛΕΤΗ ΤΗΣ ΜΟΡΙΑΚΗΣ ΠΟΙΚΙΛΟΜΟΡΦΙΑΣ ΣΤΗΝ ΠΕΡΙΟΧΗ ΤΟΥ ΠΥΡΗΝΙΚΟΥ ΑΝΤΙΓΟΝΟΥ ΤΟΥ ΙΟΥ ΤΗΣ ΗΠΑΤΙΤΙ ΑΣ Β ΣΕ ΧΡΟΝΙΟΥΣ ΑΣΥΜΠΤΩΜΑΤΙΚΟΥΣ ΦΟΡΕΙΣ ΤΟΥ ΙΟΥ»

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1 ΠΑΝΕΠΙΣΤΗΜΙΟ ΠΑΤΡΩΝ ΣΧΟΛΗ ΕΠΙΣΤΗΜΩΝ ΥΓΕΙΑΣ ΤΜΗΜΑ ΙΑΤΡΙΚΗΣ ΠΡΟΓΡΑΜΜΑ ΜΕΤΑΠΤΥΧΙΑΚΩΝ ΣΠΟΥ ΩΝ ΣΤΙΣ ΕΦΑΡΜΟΓΕΣ ΤΩΝ ΒΑΣΙΚΩΝ ΙΑΤΡΙΚΩΝ ΕΠΙΣΤΗΜΩΝ ΙΠΛΩΜΑΤΙΚΗ ΕΡΓΑΣΙΑ «ΜΕΛΕΤΗ ΤΗΣ ΜΟΡΙΑΚΗΣ ΠΟΙΚΙΛΟΜΟΡΦΙΑΣ ΣΤΗΝ ΠΕΡΙΟΧΗ ΤΟΥ ΠΥΡΗΝΙΚΟΥ ΑΝΤΙΓΟΝΟΥ ΤΟΥ ΙΟΥ ΤΗΣ ΗΠΑΤΙΤΙ ΑΣ Β ΣΕ ΧΡΟΝΙΟΥΣ ΑΣΥΜΠΤΩΜΑΤΙΚΟΥΣ ΦΟΡΕΙΣ ΤΟΥ ΙΟΥ» ΝΙΚΗΤΑΣ. ΝΙΚΗΤΑΣ (ΙΑΤΡΟΣ), Α.Μ. 474

2 Επιβλέπουσα Καθηγήτρια: Μουζάκη Α., Αναπληρώτρια Καθηγήτρια Εργαστηριακής Αιµατολογίας Αιµοδοσίας Τριµελής Εξεταστική Επιτροπή: -Φλωρδέλλης Χ., Καθηγητής Φαρµακολογίας -Μουζάκη Α., Αναπληρώτρια Καθηγήτρια Εργαστηριακής Αιµατολογίας Αιµοδοσίας -Λυγερού Ζ., Επίκουρος Καθηγήτρια Βιολογίας

3 ΕΥΧΑΡΙΣΤΙΕΣ Θα ήθελα να ευχαριστήσω θερµά, Την Αναπληρώτρια Καθηγήτρια Εργαστηριακής Αιµατολογίας Αιµοδοσίας κ. Μουζάκη Αθανασία για την πολύπλευρη βοήθεια της κατά την διεξαγωγή του ερευνητικού µου έργου και την συγγραφή της παρούσης εργασίας, δίχως την οποία όλα θα είχαν µείνει σε επίπεδο ερευνητικών ιδεών χωρίς να µετουσιωθούν σε πράξη. Τα µέλη του εργαστηρίου Αιµατολογίας Αιµοδοσίας για την πολύτιµη βοήθειά τους σε θέµατα και τεχνικές µοριακής βιολογίας. Τον Καθηγητή Φαρµακολογίας της Ιατρικής Σχολής του Πανεπιστηµίου Πατρών και Σύµβουλο Καθηγητή µου κ. Φλωρδέλλη Χριστόδουλο για την εποικοδοµητική συµβουλευτική του κατά την διάρκεια των µεταπτυχιακών µου σπουδών, την συνεργασία του και την τιµητική προς το πρόσωπό µου συµµετοχή του στην τριµελή επιτροπή. Την Λέκτορα Βιολογίας κ. Λυγερού Ζωή για την τιµητική προς το πρόσωπό µου συµµετοχή της στην τριµελή επιτροπή και την συνεργασία της. Τον Καθηγητή Παθολογίας Ηπατολογίας της Ιατρικής Σχολής του Imperial College του Λονδίνου Prof. Howard C. Thomas και τον Λέκτορα Μοριακής Ιολογίας της Ιατρικής Σχολής του Imperial College του Λονδίνου Dr. Peter Karayiannis, γιατί µου εµπιστεύθηκαν δείγµατα της Πανεπιστηµιακής Κλινικής του Νοσοκοµείου St. Mary s για την συγγραφή της παρούσης εργασίας και γενικότερα γιατί πίστεψαν και στήριξαν το ερευνητικό µου έργο κατά την σύντοµη παραµονή µου στην Μεγάλη Βρετανία.

4 Αντί Προλόγου, «Η Ιατρική αρχίζει από τον άρρωστο, συνεχίζεται µε τον άρρωστο και καταλήγει στον άρρωστο. εν έχει τόση σηµασία η διάγνωση από ποια αρρώστια έχει προσβληθεί ένας άρρωστος όση η διερεύνηση ποιον άρρωστο έχει προσβάλλει καθεµιά αρρώστια» Sir William Osler

5 ΠΕΡΙΛΗΨΗ ΣΤΗΝ ΕΛΛΗΝΙΚΗ ΓΛΩΣΣΑ Εισαγωγή: Κατά τη διάρκεια της τελευταίας δεκαετίας παρατηρείται ένα προοδευτικώς αυξανόµενο ενδιαφέρον για την διερεύνηση της ύπαρξης ή µη συσχέτισης, ανάµεσα στην γενετική ποικιλοµορφία της περιοχής του πυρηνικού υποκινητή (Core Promoter, CP, ), της προπυρηνικής περιοχής (Precore, PC, ) και της περιοχής του κυρίως πυρηνικού αντιγόνου (Core, ) του ιού της Ηπατίτιδας Β, τόσο σε νουκλεοτιδικό όσο και σε πρωτεϊνικό επίπεδο, και στην κλινική εικόνα της Οξείας ή Χρόνιας Ηπατίτιδας Β, την πιθανότητα εξέλιξης και το ρυθµό εξέλιξης της χρόνιας HBV λοίµωξης σε Χρόνια Ενεργό Ηπατίτιδα, Κίρρωση του Ήπατος και Ηπατοκυτταρικό Καρκίνωµα (ΗΚΚ). Στόχοι Έρευνας: Λαµβάνοντας υπόψη όλες τις προηγουµένως δηµοσιευθείσες εργασίες και δεδοµένης της ολοένα και αυξανόµενης σηµασίας που αποκτά η γενετική ποικιλοµορφία της περιοχής του πυρηνικού αντιγόνου στην εξέλιξη της χρόνιας HBV λοίµωξης, προχωρήσαµε σε ανάλυση της γονιδιακής αλληλουχίας των περιοχών του κυρίως πυρηνικού υποκινητή, και της προπυρηνικής περιοχής καθώς και σε ανάλυση της γονιδιακής και πρωτεϊνικής αλληλουχίας του πυρηνικού αντιγόνου σε στελέχη του ιού που αποµονώθηκαν από 23 Χρόνιους Ασυµπτωµατικούς Φορείς (ΧΑΦ) του ιού HBV, χρησιµοποιώντας ως µάρτυρες (controls) στελέχη του ιού που αποµονώθηκαν από 4 ασθενείς µε Χρόνια Ενεργό Ηπατίτιδα Β (ΧΕΗΒ) προ της ενάρξεως οποιασδήποτε θεραπευτικής αγωγής και κλωνοποιήθηκαν. Οι στόχοι της παρούσας έρευνας συνοψίζονται ως εξής: α) Να καταγράψουµε το σύνολο των µεταλλάξεων στις υπό µελέτη περιοχές και το σύνολο των αµινοξικών αντικαταστάσεων στο πυρηνικό αντιγόνο χωρίς να εστιάσουµε µόνο σε 3-4 µεταλλάξεις οι οποίες αποτέλεσαν µεµονωµένο αντικείµενο µελέτης στην συντριπτική πλειονότητα των έως τώρα δηµοσιευµένων ερευνών, β) Να εξακριβώσουµε την συχνότητα εµφάνισης κάθε µετάλλαξης και πως αυτή διαφοροποιείται ανάλογα µε την κλινική κατάσταση και το ορολογικό προφίλ των ασθενών, τον γονότυπο, και τον υπότυπο του ιού, γ) να προτείνουµε ένα ακριβές προφίλ µεταλλάξεων και αµινοξικών αντικαταστάσεων που χαρακτηρίζει τους ΧΑΦ και να δείξουµε πως αυτό διαφοροποιείται στους ασθενείς µε ΧΕΗΒ, δ) Να ερευνήσουµε την ποσοτική αλλά και ποιοτική επίπτωση των διαπιστούµενων, στην κωδικοποιούσα το πυρηνικό αντιγόνο αλληλουχία, νουκλεοτιδικών αλλαγών επί της αµινοξικής αλληλουχίας του πυρηνικού αντιγόνου, και πως αυτή επηρεάζει την σύσταση των αντιστοίχων επί του πυρηνικού αντιγόνου αντιγονικών επίτοπων, ε) Να προτείνουµε νέες τεχνικές αποµόνωσης ιικού DNA και πολυµερισµού τµηµάτων του ιικού γονιδιώµατος σε ασθενείς µε πολύ χαµηλά επίπεδα ιαιµίας, όπως οι ΧΑΦ, στ)

6 Να επιβεβαιώσουµε ή να αντικρούσουµε τα δεδοµένα της διεθνούς βιβλιογραφίας, που αφορούν στην συχνότητα εµφάνισης µεταλλάξεων και την εντόπιση τους στις υπό µελέτη περιοχές, την διαφοροποίηση αυτών µεταξύ διαφορετικών κατηγοριών ασθενών, διαφορετικών γονότυπων και διαφορετικών υπότυπων του αυτού γονότυπου του ιού. Μέθοδοι: Το ιικό DNA αποµονώθηκε µε την αυτή τεχνική από τους ορούς 23 ΧΑΦ και 4 ασθενών µε ΧΕΗΒ. Στα δείγµατα από όλους τους ασθενείς ακολούθησε ενίσχυση της προς έρευνα περιοχής του ιικού γονιδιώµατος ( ) µε την τεχνική Semi-Nested PCR µε τις αναγκαίες τροποποιήσεις για τους ΧΑΦ. Έπειτα από εξαγωγή και καθαρισµό των προϊόντων πολυµερισµού από το πήκτωµα αγαρόζης (gel extraction) τα δείγµατα από τους ΧΑΦ εστάλησαν για άµεση αλληλούχιση (direct sequencing) ενώ τα προϊόντα πολυµερισµού από τους ασθενείς µε ΧΕΗΒ υπέστησαν υποκλωνοποίηση µε συνοδό δηµιουργία κλώνων, οι οποίοι µε την σειρά τους εστάλησαν για αλληλούχιση. Ακολούθησε επεξεργασία, διόρθωση και συγκριτική αξιολόγηση των αποτελεσµάτων της αλληλούχισης, καθώς και µετάφραση των αλληλουχιών που κωδικοποιούσαν το πυρηνικό αντιγόνο, δια µέσω των υπολογιστικών λειτουργικών συστηµάτων Bioedit, Dnasis και Prosis αντίστοιχα. Αποτελέσµατα: Η αποµόνωση του ιικού DNA κατέστη δυνατή στα 23/23 (100%) δείγµατα των ΧΑΦ και στα 4/4 (100%) δείγµατα ασθενών µε ΧΕΗΒ. Η ενίσχυση της υπό µελέτης περιοχής του ιικού γονιδιώµατος δια της τεχνικής Semi-Nested PCR, κατέστη δυνατή σε 21/23 (91.3%) δείγµατα ΧΑΦ, ποσοστό από τα υψηλότερα που έχουν ποτέ επιτευχθεί στην διεθνή βιβλιογραφία για ΧΑΦ και σε 4/4 (100%) δείγµατα ασθενών µε ΧΕΗΒ. Από τα 21 αυτά δείγµατα των ΧΑΦ στα 18 (85.7%) κατέστη δυνατή η ενίσχυση και των τριών περιοχών (CP, PC, Core) ( ) ενώ σε 2 δείγµατα (14.3%) κατέστη δυνατή η ενίσχυση µόνο του πυρηνικού υποκινητή και της προπυρηνικής περιοχής ( ). Από τα 21 στελέχη του ιού που αποµονώθηκαν από τους ΧΑΦ, στα οποία τελικώς πολυµερίστηκαν οι υπό µελέτη περιοχές, όλα έδωσαν πλήρη και απολύτως ελεύθερα επιµολύνσεων αποτελέσµατα στην αλληλούχιση. Στα 15/21 δείγµατα εκ των ΧΑΦ αποµονώθηκαν στελέχη γονότυπου D και υπότυπου ayw3, ενώ από τους λοιπούς 6 ΧΑΦ αποµονώθηκαν στελέχη γονότυπου C και υπότυπου ayr, όπως άλλωστε αναµενόταν εκ της γεωγραφικής προελεύσεως των ασθενών εκ των οποίων ελήφθησαν τα δείγµατα (Ελλάδα και Α. Ασία αντίστοιχα). Και στα 4 δείγµατα των ασθενών µε ΧΕΗΒ (controls) κατέστη δυνατή η ενίσχυση και των τριών υπό µελέτη περιοχών του γονιδιώµατος, ως ένα ενιαίο προϊόν πολυµερισµού µήκους 750bp. Έπειτα από υποκλωνοποίηση εκάστου προϊόντος πολυµερισµού µε τη χρήση του φορέα pgem-t easy αποµονώθηκαν υπό κεκαθαρµένη µορφή και εστάλησαν για αλληλούχιση 4 κλώνοι εκ του δείγµατος 1, 7 κλώνοι εκ του δείγµατος 2, 7

7 κλώνοι εκ του δείγµατος 3 και 3 κλώνοι εκ του δείγµατος 5, σύνολο δηλαδή 21 κλώνοι. Έκαστος εκ των κλώνων αυτών αντιπροσώπευε και ένα σχεδόν-είδος (quasi-species) προερχοµένου εκ του κυρίου για κάθε δείγµα στελέχους του ιού. 14/21 (67%) σχεδόν-είδη βρέθηκε ότι είναι γονότυπου D και υπότυπου ayw3 (δείγµατα 1,2 και 5) ενώ 7/21 σχεδόν είδη (όλα εκ του δείγµατος 3) είναι γονότυπου D και υπότυπου ayr. Η µετάφραση της κωδικοποιούσας το πυρηνικό αντιγόνο περιοχής κατέστη δυνατή για την συνολική έκταση του πυρηνικού αντιγόνου (πάνω από το 98%) σε 17/21 (80.95%) στελέχη από τους ΧΑΦ και σε 21/21 (100%) κλώνους από τους ασθενείς µε ΧΕΗΒ. Κατόπιν συγκριτικής αντιπαράθεσης των αλληλουχιών που ελήφθησαν από τα δείγµατα των ΧΑΦ και των ασθενών µε ΧΕΗΒ, καθεµιάς µε τους αντίστοιχους άγριους τύπους του ιού, µεταξύ τους ανά κλινική κατηγορία, µεταξύ διαφορετικών υπότυπων ανά κλινική κατηγορία και µεταξύ των δύο διαφορετικών κλινικών κατηγοριών, ελήφθησαν τα εξής αποτελέσµατα, όσον αφορά τις σηµαντικότερες από κλινικής σκοπιάς µεταλλάξεις: Οι ασθενείς µε γονότυπο D και υπότυπο ayw3 και όλοι οι ΧΑΦ είχαν σαφώς µεγαλύτερο αριθµό µεταλλάξεων σε όλες τις υπό µελέτη περιοχές, ιδιαιτέρως στην προπυρηνική (PC) περιοχή και την κυρίως πυρηνική περιοχή (Core). Η βασική προπυρηνική τερµατική µετάλλαξη G1896A αποτέλεσε και στην δική µας µελέτη την συχνότερα παρατηρούµενη (80% ΧΑΦ και 100% στους ασθενείς µε ΧΕΗΒ), ενώ αξιοσηµείωτη ήταν η παντελής απουσία της από τους στελέχη του ιού µε υπότυπο ayr. Ο γνωστός συνδυασµός µεταλλάξεων G1896A & G1899A της προπυρηνικής περιοχής βρέθηκε στο 80% των ΧΑΦ, αλλά και στο 100% των ασθενών µε ΧΕΗΒ, ενώ µόλις το 7% των ΧΑΦ παρουσίαζε µονήρη αλλαγή G1896A. Επίσης, σταθερός και αρκετά συχνά παρατηρούµενος (80% των ΧΑΦ) ήταν και ο συνδυασµός των τριών µεταλλάξεων G1896A&G1899A&C1909T, µε µικρότερη όµως συχνότητα εµφάνισης στους ΧΑΦ σε σχέση µε τους ασθενείς µε ΧΕΗΒ. Αξίζει να σηµειωθεί η ανεύρεση της µετάλλαξης A1827C στην πολύ καλά συντηρηµένη και ζωτικής σηµασίας για την αναπαραγωγική δραστηριότητα του ιού περιοχή DR1 (Direct Repeat 1) σε όλα τα δείγµατα των ΧΑΦ µε απουσία της G1896A. Εξίσου σηµαντική είναι και η διαπίστωση µόνο στους ΧΑΦ της διπλής µετάλλαξης T1846A&T1847A, η οποία προηγείται αµέσως της έναρξης του «ε» επίτοπου, της θέσεως έναρξης δηλαδή της αντιγραφής του ιικού DNA. Η κατά καιρούς συνδεόµενη µε εξέλιξη προς κεραυνοβόλο ηπατίτιδα Β µετάλλαξη G1862T, δεν βρέθηκε σε κανένα στέλεχος του ιού, όπως άλλωστε αναµενόταν. Στην περιοχή του κυρίως πυρηνικού υποκινητή, και ειδικότερα στην περιοχή του Enhancer II, όλες οι µεταλλάξεις αφορούσαν σε θέσεις δέσµευσης των µεταγραφικών παραγόντων και ήταν λιγότερες σε αριθµό και συχνότητα εµφάνισης στους ΧΑΦ. Η αλλαγή T1724C (θέση δέσµευσης του µεταγραφικού παράγοντα HNF3) ήταν 5 φορές σπανιότερη στους ΧΑΦ σε σχέση µε την ΧΕΗΒ, ενώ η αλλαγή A1725C δεν παρατηρήθηκε σε κανέναν ΧΑΦ αλλά µόνο στο 30% περίπου των ασθενών µε ΧΕΗΒ. Κοινή και για τις δυο οµάδες ασθενών αλλά 3 φορές σπανιότερη στους ΧΑΦ ήταν η αλλαγή

8 T1741G (θέση SP1 site 2) και το αυτό διαπιστώθηκε και για την-πολλάκις συσχετιζόµενη µε την εξέλιξη προς κίρρωση και ΗΚΚ-µετάλλαξη T1753C. Μεγάλης σπουδαιότητας κρίνεται η διαπίστωση της πλήρους απουσίας από τους ΧΑΦ των µεταλλάξεων G1757A (θέση SP1 site 3) & C1766G (θέση δέσµευσης του HNF 4), οι οποίες έχουν συσχετιστεί µε πολύ µεγάλη αύξηση της αναπαραγωγικής δραστηριότητας του ιού και µε ταχεία εξέλιξη προς κίρρωση και ΗΚΚ, και οι οποίες ήταν παρούσες σε µεγάλο αριθµό κλώνων ΧΕΗΒ, ανεξαρτήτως υπότυπου. Έκπληξη προκάλεσε η απουσία ανεύρεσης της πλέον συχνής διπλής µετάλλαξης του πυρηνικού υποκινητή T1762&A1764 από όλα τα δείγµατα των ΧΑΦ ενώ στους ασθενείς µε ΧΕΗΒ εµφανίστηκε στο 1/3 των δειγµάτων η αντιρρόπηση της έλλειψης αυτής από την µετάλλαξη G1766. Πολύ ενδιαφέρουσα µπορεί να χαρακτηριστεί η ανεύρεση 3 νέων µεταλλάξεων µόνο στους ΧΑΦ T1862G, C1802T, G1803T, (εντός και επί των 3 ορίων της Kunitz Domain) οι οποίες πιθανότατα να συνδέονται και µε την µειωµένη αναπαραγωγική δραστηριότητα του ιού στους ΧΑΦ. Γενικώς ο γονότυπος D και ο υπότυπος ayw3 χαρακτηρίζονταν από σχεδόν διπλάσιο αριθµό µεταλλάξεων επί του πυρηνικού υποκινητή, σε σχέση µε τον υπότυπο ayr, στον οποίο όµως παρατηρήθηκε µια σταθερή εµφάνιση της εξαιρετικά σπάνιας µετάλλαξης A1827C. Ενώ, επί του συνόλου των µεταλλάξεων, οι περισσότερες παρατηρήθηκαν εντός της κωδικοποιούσας το κυρίως πυρηνικό αντιγόνο περιοχής (Core), πλείστες εξ αυτών δεν είχαν ουδεµία ποιοτική επίπτωση επί της αµινοξικής αλληλουχίας του πυρηνικού αντιγόνου. Οι µεταλλάξεις αυτές ήταν σχεδόν διπλάσιες στους ΧΑΦ (122) σε σχέση µε τους ασθενείς µε ΧΕΗΒ (70) καθώς επίσης και στον υπότυπο ayw σε σχέση µε τον υπότυπο ayr (ο οποίος σηµειωτέον εµφάνισε την core περιοχή εξαιρετικώς συντηρηµένη στο µεγαλύτερο µέρος της). Επιπροσθέτως τα στελέχη από τους ΧΑΦ, ανεξαρτήτως γονότυπου, εµφάνισαν σηµαντικώς υψηλότερο αριθµό αµινοξικών αντικαταστάσεων επί των αντιγονικών επιτόπων των Th, CTL και B κυττάρων. Τα στελέχη από τους ΧΑΦ παρουσίασαν ιδιαιτέρως υψηλό αριθµό αµινοξικών αντικαταστάσεων στον Β κυτταρικό αντιγονικό επίτοπο στη θέση και µόνο στους ΧΑΦ διαπιστώθηκαν αµινοξικές αντικαταστάσεις επί του Β κυτταρικού αντιγονικού επίτοπου στη θέση Σηµαντικώς ελαττωµένος βρέθηκε ο αριθµός και η συχνότητα εµφάνισης των αµινοξικών αντικαταστάσεων επί των αντιγονικών επιτόπων, σε όλα τα στελέχη από τους ΧΑΦ τα οποία δεν έφεραν την προπυρηνική τερµατική µετάλλαξη G1896A. Στην δική µας έρευνα, για πρώτη φορά στην διεθνή βιβλιογραφία, βρέθηκαν οι εξής αµινοξικές αντικαταστάσεις επί των αντιγονικών επιτόπων του πυρηνικού αντιγόνου, σε στελέχη του ιού από ΧΑΦ: L6F, C7S (Th Επίτοπος), S50A, T62S, A63V (Th Επίτοπος), S78T, H80D, Q86L, A87S, I88V (B Επίτοπος), V103G, E106Q, P108Q, V115F/L (B Επίτοπος), L129I (Th Επίτοπος), T143V/A, I145L (CTL A31/AW68 Επίτοπος), L148S, R156S, T157S (B Επίτοπος).

9 Εκτός των προαναφεροµένων, κλινικώς σηµαντικών, νουκλεοτιδικών και αµινοξικών αντικαταστάσεων, σηµαντικός αριθµός πρόσθετων µεταλλάξεων παρατηρήθηκε και στις 3 υπό µελέτη περιοχές, χωρίς ωστόσο αυτές να εντοπίζονται σε λειτουργικά σηµαντικά τµήµατα του γονιδιώµατος του ιού. Πιθανότατα οι µεταλλάξεις αυτές αποτελούν επιµέρους ατοµικές διαφοροποιήσεις των στελεχών του ιού εξ εκάστου ασθενούς καθώς η συχνότητα ανεύρεσης τους δεν είναι σταθερή και υψηλή, και κατ επέκταση στερούνται πάσης κλινικής σηµασίας.

10 University of Patras School of Health Sciences Department of Medicine Postgraduate Course in Applications of Basic Medical Sciences MSc Study of Molecular variations in the Core Promoter, Precore, and Core regions of Hepatitis B virus genome, and within the antigenic epitopes of HBcAg in viral strains isolated from Asymptomatic Carriers of the Hepatitis B Virus NIKITAS NIKITAS, MD PATRAS MAY 2006

11 ABSTRACT Background Infection with HBV may lead to a wide spectrum of liver disease that ranges, in acute infection from mild self-limited to fulminant hepatitis, and in persistent infection from an ASC state to severe chronic hepatitis, cirrhosis and HCC. Several host factors are important in determining outcome, including age at infection, immune competence and MHC haplotype. Viral factors may also play an important role. Over the past decade, there has been considerable interest in whether certain genetic variants of HBV are associated with increased pathogenicity, such as the development of acute liver failure and progression of persistent infections to Chronic Active Hepatitis B, Liver Cirrhosis and Hepatocellular Carcinoma. Aims We proceeded to the sequencing of the entire CP, PC and Core regions of the HBV genome and the analysis of the Molecular variation in them in HBV isolates derived from 23 ASCs and 4 patients with CHB. 17 ASCs were Greeks (genotype D [ayw3]) and 6 were Chinese (Genotype C [ayr]) while all CHB patients were Greeks (though 3 of Genotype D ayw3 and 1 of Genotype D ayr). Our ultimate aims were the identification of all nucleotide and amino acid substitutions within the aforementioned regions and Core protein, respectively, and the demonstration of the differential presentation, distribution and frequency patterns of these substitutions and their respective combinations, in terms of clinical, virological and immunological characteristics of the patients. Materials and Methods HBV isolates derived from 23 ASCs and 4 patients with CHB. 17 ASCs were Greeks (genotype D [ayw3]) and 6 were Chinese (Genotype C [ayr]) while all CHB patients were Greeks (though 3 of Genotype D ayw3 and 1 of Genotype D ayr). HBV DNA was isolated from the sera of 23 ASCs and 4 CHB patients and the three studied regions (Core Promoter, Precore, and Core) spanning nucleotides were amplified with Semi Nested PCR, modified for ASCs. After agarose gel extraction and purification, all ASCs amplicons were sequenced directly while CHB amplicons were sub-cloned into a pgem-t vector, resulting in the production of 21 clones which were also sequenced. Finally, all the obtained sequences were corrected, edited, stored, aligned and compared by the Bioedit, Dnasis and Prosis software programmes. Results The nucleotide substitutions in all studied regions were different both in quantity and quality both in each clinical group and in each viral genotype of the same group. The precore stop codon mutation A1896 was once again the most commonly identified mutation in both clinical groups, albeit slightly less prevalent in ASCs compared to the CHB patients and absent in all strains and quasi-species of ayr subtype. The pattern of mutations and combinations of mutations in Core Promoter region was entirely different in each clinical group. The two common mutations C1724, C1753 were less prevalent in ASCs. The ASC specific mutations were G1727, G1741, G1760, G1782, T1802, T1803, C1827, A1846, A1847. CHB samples demonstrated an accumulation of mutations within the Transcription Factors binding sites, absent from all ASCs samples. Mutations within in the well preserved DR1, Kunitz domains and the region were identified only in ASCs. Double BCP mutation was not identified in any sample of both clinical groups and of both genotypes. Each clinical Group demonstrated a differential (clinically-specific) pattern of distribution of

12 amino acid substitutions within the epitopes of HBcAg. Amino acid substitutions were more in ASCs than CHB patients and the extreme epitopes contained significantly greater number of substitutions, reflecting their clinical significance. The Greek ASCs demonstrated a substantial differentiation in the distribution of amino acid substitutions within the HBcAg epitopes compared to ASCs of different national background. Conclusions There is a substantial differentiation in the presentation, distribution, and frequency patterns of nucleotide and amino acid substitutions within the Core promoter, Precore regions and Core protein respectively, not only between different clinical groups of Chronic Hepatitis B patients (ASCs and Chronic Active hepatitis B patients) but also between different viral genotypes (and subtypes) of the same clinical group. Different combinations of mutations in the Core Promoter region are responsible for the Enhancement or Attenuation of the downregulatory effect of A1896 mutation on viral replication. A combination of mutations within the Precore and Core promoter regions, and not A1896 by itself, should be considered responsible for the development of either ASC state or Progression of Chronic Hepatitis B. Greek ASCs demonstrate a entirely different pattern of Amino Acid Substitutions within the epitopes of HBcAg compared to the ASCs of different national background.

13 Contents Pages 1. INTRODUCTION Epidemiology of Hepatitis B Virus Infection Clinical Features of Hepatitis B Molecular Biology of HBV General Features Genome of HBV Molecular Biology of the Precore/Core gene and the Core Promoter region Background in Molecular Variation in the Precore, Core Promoter and Core regions of HBV and their association with clinical course of Hepatitis B Introduction Precore Variants Core Promoter Variants Core Gene/Protein Variants AIMS MATERIALS AND METHODS HBV DNA extraction from patients sera Amplification of the studied regions of HBV genome Gel extraction of amplicons Direct Sequencing and analysis of the obtained nucleotide sequences Sub-cloning of the amplicons derived from CHB patients Plasmid Extraction Sequencing of quasi-species derived from CHB patients RESULTS General Asymptomatic Carriers Chronic Hepatitis B Patients Comparative analysis of Nucleotide substitutions identified in ASCs and CHB patients Amino Acid substitutions in Core Protein HBcAg Epitopes Comparative analysis of Amino Acid substitutions identified in Greek ASCs and Greek CHB patients of Genotype D (ayw3 subtype) Comparative analysis of Amino Acid substitutions identified in Greek ASCs in our study and in ASCs of different national background in previous studies DISCUSSION APPENDIX REFERENCES

14

15 1. INTRODUCTION 1.1 Epidemiology of Hepatitis B Virus Infection Worldwide, Hepatitis B Virus (HBV) is the most common among those hepatitis viruses that cause chronic infections of the liver in humans and represents a global public health problem. Chronic Hepatitis caused by HBV may progress to cirrhosis and death from liver failure, and chronic HBV infection is the major cause of Hepatocellular Carcinoma (HCC) worldwide [1, 2]. Acute Hepatitis B usually runs a self-limited course in adult subjects. Fulminant hepatitis occurs in 1-2% of acute infections, resulting in the death of most of these patients. About 15-40% of chronically infected subjects will develop complications, leading to an estimated 520,000-1,200,000 deaths each year due to acute and chronic hepatitis, cirrhosis and liver cancer [3, 4, 5, 6, 7, 8]. HBV causes 60-80% of the world s HCCs [9, 10] and therefore it has been classified by the International Agency for Research on Cancer as Carcinogenic to humans [10]. More than 2 billion people have been infected worldwide and, of these, 360 million suffer from chronic HBV infection. The incidence of HBV infection and patterns of transmission vary greatly throughout the world in different population subgroups [Fig.1]. Prevalence of anti-hbs and anti-hbc are much higher than those of chronic carriers-hepatitis B surface antigen positives in all populations. Endemicity of infection is considered high in those parts of the world where at least 8% of the population is HBsAg positive. In these areas, 70-90% of the population have serological evidence of previous HBV infection [11]. In high endemic areas such as Asia, Sub-Saharan Africa and the Pacific, carrier rates for HBsAg range from 8% to 25% and anti-hbs prevalence from 60% to

16 85%. Thus, exposure to HBV in high endemic areas, measured serologically, may approach 100% [12,13]. Fig.1: The Worldwide Prevalence of HBsAg carriers In areas with an intermediate pattern of HBV infection (eastern and central Europe, Mediterranean, Middle East, India and Amazon basin), the prevalence of HBsAg positivity ranges from 1% to 8%, serological evidence of past infection is found in 10-60% of the population and the lifetime risk of becoming HBV infected is estimated to be 20-60% [14, 15]. In most developed parts of the world, the prevalence of chronic HBV infection is < 1%, and the overall infection rate is 5-7% [15]. Overall, approximately 45% of the global population live in areas of high chronic HBV prevalence [16, 13]. Genetic analysis has allowed us to classify HBV into seven distinct genotypes (A-G) that have different geographical distributions and associations with different risk groups for infection. Genotype A is predominantly found in north-western Europe, North America and Central Africa. Genotypes B and C are found predominantly in eastern and south-eastern Asia including China and Japan. Genotype D is mainly found in Mediterranean area and the vast majority of

17 Greek patients are infected by that genotype. Genotype E is predominant in Western Africa and the most divergent genotype F is found exclusively among indigenous populations in central and South America [17, 18]. Genotype G has been identified in a few samples in USA and France. Worldwide, genotypes B, C and D are predominant, with an estimated 240 million subjects infected with genotypes B/C and 40 million infected with genotype D [19-21, 22, 23, 24-28]. Although, at the beginning of the 3 rd millennium, Hepatitis B remains a major public health problem globally, data on the decrease of prevalence of HBV markers are accumulating in countries where routine hepatitis B vaccination programmes were implemented. Furthermore, the better hygiene and the intense AIDS campaign which addresses the dangers of promiscuity and of shared syringes and needles, contribute to the globally-observed reduction tendency in Hepatitis B prevalence [37]. These data suggest that by preventing infections in children through universal immunization programmes, the number of HBeAgpositive individuals declines rapidly over time, because of the inherent clearance of the HBeAg in older persons, and this will lead to an ongoing and significant change in the global epidemiology of hepatitis B [29]. 1.2 Clinical features of Hepatitis B The incubation period of HBV infection is long, ranging from 45 to 160 days (average 120 days). The virus os found in highest rates in blood and serous exudates [30, 31]. Although HBsAg has been detected in a wide variety of body fluids, only serum, semen and saliva have been demonstrated to be infectious [32, 33], while breast milk and urine remain controversial. Presence of HBeAg in serum correlates with high titres of HBV and greater infectivity [34, 35]. However, HBV strains that have mutations in the pre-core region of the viral genome that prevent expression of HBeAg (HBeAg-negative/anti-HBe positive variants) also have been associated with transmission [36]. In high carriage rate areas, infection is acquired by passage from the mother to the neonate [37]. The

18 risk of perinatal HBV transmission is greatest for infants born to women who are HBeAg-positive and ranges from 70% to 90% at 6 months of age. About 90% of these children remain chronically infected [38]. The risk of perinatal infection among infants born to HBeAg-negative mothers ranges from 10% to 40% with 40% to 70% of these infected infants remaining chronically infected [38, 39]. In high endemic areas, the transmission is also horizontal through kissing, shared utensils such as toothbrushes and razors and injections [40, 41, 42]. Contact in preschool day care centres is possible. Sexual contacts in the family are at risk [43]. Infection among homosexuals is related to the duration of homosexual activity, number of sexual contacts and anal contact [44]. Blood transfusion continues to cause hepatitis B in countries where donor blood is not screened. Transmission is more likely with blood from paid donors than from volunteer blood [37]. Opportunities for parenteral infection include the use of non-sterile instruments for dental treatment, ear piercing and manicures, neurological examination, prophylactic inoculations, subcutaneous injections, acupuncture and tattooing [37]. Parenteral drug abusers develop hepatitis from using shared, non-sterile equipment. The mortality may be very high in this group. Hospital staff in contact with patients, and especially patient s blood, usually has a higher carrier rate than the general community. This applies particularly to staff on renal dialysis or oncology units. Spread from a health care worker is usually through a surgeon, performing complex invasive procedures [37,45]. The clinical course of acute hepatitis B may be anicteric and subclinical episodes must be extremely frequent. The non-icteric case is more liable to become chronic than the icteric one. The usual clinical attack diagnosed in the adults tends to be more severe than but similar (as it concerns the overall clinical picture) to those of hepatitis A or C. The self-limited, benign icteric disease usually lasts less than 4 months. Jaundice rarely exceeds 4 weeks. Occasionally, a prolonged benign course is marked by increased serum

19 transaminase values for more than 100 days. Relapses are rare and the cholestatic hepatitis with prolonged deep jaundice is unusual. Fulminant course of hepatitis B and subacute hepatic necrosis are also rare. Hepatitis B-associated extrahepatic manifestations have also been observed including Immune complex-mediated small and medium-sized arteritis, glomerulonephritis Polymyalgia Rheumatica, Guillain-Barre syndrome and Myocarditis [37, 46, 47, 48, 49, 50, 51]. The markers for serological diagnosis of hepatitis B and their clinical significance are shown on table 1. (Table 1) Significance of serological markers in Hepatitis B Marker Significance HBsAg Acute or Chronic Hepatitis B carriage IgM Anti- HBc Acute Hepatitis B (high titre)/ Chronic Hepatitis (low titreflares) IgG Anti-HBc Past exposure to Hepatitis B (with negative HBsAg) Anti-HBs Immune to Hepatitis B HBeAg Acute Hepatitis B. Persistence means infectious state HBV DNA Continued infectious state the best marker of disease activity Exposure to HBV can have different results [Fig.2]. Some of the infected subjects are immune and have no clinical attack (the Nil group). In others acute attack develops varying from anicteric to Fulminant. Previously normal individuals usually clear the surface antigen from the serum within 4-6 weeks from the onset of symptoms resulting in complete recovery (90% of the infected). The rest 10% of patients with acute hepatitis B evolves to the chronic course of the disease. 90% of the latter group will develop chronic active hepatitis B resulting in chronic hepatic failure, cirrhosis or Hepatocellular carcinoma. The rest 10% of patients with chronic hepatitis B are characterized as Chronic Healthy, Asymptomatic, Inactive or Normal Hepatitis B Virus Carriers. This very interesting group of patients may show changes on liver biopsy ranging from non-specific minimal abnormalities through to chronic hepatitis and cirrhosis.

20 CLINICAL SEQUELAE OF ACUTE HBV INFECTION 90% ACUTE HEPATITIS B CONVALESCENCE 10% CHRONIC HBV INFECTION 90% 10% CHRONIC ACTIVE HEPATITIS B ASYMPTOMATIC CARRIERS 100% 1.6% CHRONIC HEPATIC FAILURE (50-65%) LIVER CIRRHOSIS (30-40%) HEPATOCELLULAR CARCINOMA (5-10%) Fig.2: Clinical Sequelae of Acute and Chronic HBV infection

21 The extent of the changes is not reflected by serum biochemical tests and may only be revealed by liver biopsy. The carrier presenting by chance is likely to have minor hepatic changes compared to the patient presenting to a gastroenterology department where more serious liver disease is likely. By definition, ASCs of HBV have no symptoms or clinical signs of either acute or chronic Hepatitis B, their AST/ALT levels are normal, they have very low levels of viraemia (very low HBV DNA levels), most of them are HBeAg (-) and Anti-HBe (+) and all of them are characterized by a benign clinical course of HBV infection as only 1.6% of them develop Chronic Active Hepatitis and its associated complications. In a survey of patients found to be HBsAg positive at blood donation (healthy carriers of HBV), 95% had near normal liver biopsies and only 1.6% proceeded to chronic active hepatitis or cirrhosis. Furthermore 90% of them were serum HBeAg negative and Anti-HBe positive [37, 52]. 1.3 MOLECULAR BIOLOGY OF HBV General features Hepatitis B Virus (HBV) was the first human hepatitis virus from which the proteins and genome were identified and characterized. In 1963, Blumberg and colleagues, in a search of polymorphic serum proteins, discovered a previously unknown antigen in the blood of an Australian Aborigine (Australia Antigen). Four years later, it was recognized that the appearance of this antigen was related to type B hepatitis [53]. Using immune electron microscopy, Dane and co-workers eventually discovered virus-like particles that carried this antigen on their surface, in the serum of hepatitis B patients [54], and these particles were considered to be the HBV. A search for an HBV-like agent in woodchucks, which had been observed to develop liver cancer, resulted in the subsequent discovery of Woodchuck

22 hepatitis Virus (WHV) [55]. Also, a similar virus was found in seemingly healthy ground squirrels (GSHV), and arctic ground squirrels [56], which are both distantly related to marmots. In primates, hepadnaviruses could be isolated from Old World and New World monkeys including chimpanzees, orang-utans, gorillas, gibbons, and woolly monkeys [57]. More distantly related hepadnaviruses were found in Pekin ducks (Duck Hepatitis B Virus, DHBV) [58] and in other avian species [59,60,61,62]. HBV and its relatives through the animal kingdom comprise a family termed Hepadnaviridae, the name derived from their hepatotropism and DNA genome [63]. A highlight was the discovery of reverse transcription of viral RNA into DNA within the core particles of DHBV as an essential step of the viral genome replication [64]. Thus hepadnaviruses, badnaviruses and caulimoviruses, which also use a similar type of replication, are also termed pararetroviruses in contrast to orthoretroviruses, which have an RNA genome Genome of HBV Using electron microscopy, the DNA of HBV was shown to be circular, partially double-stranded and 3200 nucleotides long [65]. Complete HBV genomes are between 3182 and 3221 bases long [66] and the numbering of the bases starts in most publications (as it starts in this thesis) at the cleavage site for the restriction enzyme EcoRI or at homologous sites if a particular genome type does not have such an EcoRI site [Fig]. The endogenous DNA polymerase reaction is possible, because one of the DNA strands is incomplete [67, 68]. The remaining gap is partially filled by the viral DNA polymerase if the dntps are added in vitro. In vivo, HBV particles are obviously secreted from the infected cell before the completion of the double strand. Thus, the incomplete plusstrand has a defined 5 end but a variable 3 end [Fig.3]. The structure of the hepadnaviral genome does not abide by the usual classification criteria for viruses [69] in several respects. It contains both DNA and RNA, and its genome

23 contains partially single-stranded and even triple-stranded DNA. These unusual features are a direct consequence of its replication mechanism. All genomes of mammalian hepadnaviruses contain four ORFs, which are encoded by the same DNA strand [Fig.4]. The polarity of the viral nucleic acid strands is defined in a way that mrna has plus polarity. Thus, the protein-encoding DNA strand of the HBV genome, which is transcribed to into mrna, has minus polarity. Additional ORFs can be identified on the minus- and plus- strand, but they are not conserved, and are probably of minor importance, or generated by statistical sequence variations [29]. Fig.3: The Genome of Hepatitis B Virus

24 Fig.4: The Open Reading Frames of HBV Genome and the resulting peptides The genomes of Hepadnaviruses seem to have evolved toward minimal length. Thus, the genome is not much longer than its longest ORF P, which encodes the viral DNA polymerase and its accessory functions [29]. ORF S is completely located within the ORF P. ORF C and X overlap partially with the ORF P. Furthermore, HBV encodes more than one protein from one ORF by using internal AUG codons in an ORF as additional start sites for protein biosynthesis. Thus, nested sets of proteins with different amino ends and a common carboxyl ends are synthesized. ORF S encodes the three co-terminal HBs proteins [70]. ORF C encodes the HBc and HBe proteins [71]. The ORF X encodes a protein with a transcriptional transactivating function [72] but it may also encode more than one protein [73]. Furthermore the numerous genetic

25 elements that regulate transcription of the viral DNA into RNA, processing of the RNA and translation into protein are placed within coding regions. Fig.5 shows the three types of virus-associated particles present in the blood of HBV infected persons. Using electron microscopy after negative staining, the virus is spherical with a diameter of nm, showing a double-shelled structure. The outer protein shell (viral envelope) is formed by the HBs proteins [29, 74]. The inner protein shell, referred to as the core particle or viral capsid, is composed of HBc protein (core or capsid protein) and encloses a complex of viral polymerase and DNA, which is often positively stained. Morphologically, there are two different forms of capsids. They consists of 180 or 240 identical core proteins, which form capsids with a T-3 and T=4 symmetry. In cryoelectron microscopy they show diameters of 32 and 36 nm [75, 76], but there is no functional difference known to be related to the symmetry. Fig.5: Three types of HBV-associated particles

26 The sera of highly viraemic carriers contain large numbers of non-infectious particles composed of excessive HBs protein (HBsAg). Most abundant are spherical particles of nm, which are secreted in ,000-fold excess to the virus, typically reaching numbers of up to 10¹³/mL. Less numerous (up to 10¹¹/mL or 1µg/mL) are the filamentous (or tubular) particles of approximately 20 nm diameter and variable length. Sera from low-viraemic HBsAg carriers contain up to 10¹²/mL HBs spheres and few or no HBs filaments. Neither of these subviral structures contains the capsid and HBV DNA and therefore they are not infective [29]. SHBs (small surface protein) is the most abundant polypeptide in all three HBV-associated particles, whereas MHBs (middle-sized surface protein) is a minor component in all three. LHBs (largest surface protein) is more prevalent than MHBs in virions and filaments, but less prevalent in HBs spheres. It appears that the proportion of the LHBs determines the morphology of the HBs particles [77]. HBsAg particles have surfaces that are antigenically complex and this had led to the recognition of antigenic determinants. A common determinant is a [Fig.6]. The other sub-determinants are designated d, y, w, r. The four major determinants are therefore adw, adr, ayw, and ayr. They breed true and they are very helpful epidemiologically [37].

27 Fig.6: HBV a determinant of HBsAg Molecular Biology of the Precore/Core gene and the core promoter region The precore/core (PC/C) ORF has two in frame initiation (AUG) codons, which lead to the synthesis of two co-terminal proteins. Initiation of translation at the first AUG results in the synthesis of the precore/core protein, the precursor of the HBeAg [Fig.7], while the second initiation codon is utilized for the synthesis of the core protein (HBcAg). The two proteins are synthesized from different mrnas known as the precore mrna and pg mrna, respectively. Once synthesized, the precore/core protein is targeted to the endoplasmic reticulum (ER) by a 19 amino acid signal peptide at its amino-terminus [78]. Cleavage of this peptide releases the remainder of the protein into the ER

28 lumen, where further processing of the carboxy-terminus results in the removal of about 34 amino acids from this end. What remains is HBeAg, which is released from the cell. HBeAg retains 10 amino acids from the precore region and shares the remaining 149 with HBcAg. The finding of antigenic epitopes that are different between the two proteins, even though they share most of their amino acid sequence, is explained by conformational changes in HBeAg related to the 10 amino acid precore peptide at its amino-terminus and the retained carboxy-terminus arginine-rich domain of HBcAg [29]. HBeAg is conserved within all members of the Hepadnaviridae family. It is secreted from the infected cell but is not part of the virion structure and does not have any known role in replication. In the neonate born to an HBV-infected mother, it has been suggested that HBeAg crosses the placenta and induces tolerance [79, 80].

29 Fig.7&9: The Core Promoter, Precore and Core Regions of the HBV Genome and their Functional Domains HBeAg may also have an immunomodulatory function during infection in adult life. Low molecular weight soluble proteins, such as HBeAg, induce a state of immune non-responsiveness, at the T-helper cells level. HBeAg-induced tolerance results in T-cell non-response to HBcAg, as well as HBeAg-derived epitopes, so that elimination of HBV infected cells does not occur. In transgenic mice, this non-response to nucleocapsid proteins can be broken by the injection of peptides bearing single amino acid substitutions. If such substitutions occur spontaneously during persistent infection, they may account for spontaneous

30 HBeAg/anti-HBe seroconversion [81]. HBeAg probably also influences the host response by down-regulating cytokine (IFN-β) production [82]. HBcAg forms the nucleocapsid of the virus, which has icosahedral symmetry, as demonstrated by cryoelectron microscopy [83]. The core protein dimerizes readily, and the dimmers assemble spontaneously into particles that simultaneously encapsidate the pgrna with a copy of the viral polymerase. The arginine-rich motifs at the carboxy-terminus of the HBcAg are responsible for binding the pgrna, and also the newly synthesized DNA strands of the virus. Encapsidation is triggered by the binding of the polymerase to a stem loop structure at the 5 end of the pgrna known as the encapsidation signal or ε [Fig.8], with strict requirements of structure and sequence [84]. This structure, which is formed by the precore region sequences, is in turn recognized and bound by the polymerase. The latter uses the side bulge of ε for the synthesis of a short primer for minus-strand DNA synthesis. Thus, any variation in this region may affect the stability of the signal and compromise virus viability. Fig.8: DR1 Domain and the Encapsidation Signal with the most frequent mutations identified within them

31 The core promoter (CP) of the HBV genome has a pivotal role in the replication and morphogenesis of the virus. It directs initiation of transcription for the synthesis of both the precore and pgrnas. The CP consists of the Basic Core Promoter (BCP) and upstream regulatory sequences, containing both positive and negative regulatory elements that modulate promoter activity [85]. The BCP overlaps with the 3 end of the X ORF and the 5 end of the precore region, and contains cis-acting elements that can independently direct transcription of the precore mrna and pgrna, both of which are about 3.5kb in length [Fig.9] [86]. The pgrna starts at the 3 to the precore AUG and is translated into the core and polymerase proteins, but in addition serves as a template for the synthesis of the negative DNA strand of the virus by reverse transcription after encapsidation within the core particle [87]. The precore transcript, which is slightly longer than the pgrna and is initiated upstream of the precore start codon, is the template for the translation of the precore/core protein that is proteolytically processed to produce HBeAg, as already described. The enhancer II (EnII) element regulates the activity of the CP and partially overlaps with it and its upstream regulatory sequences. This whole region contains in addition nucleotide motifs that constitute transcription factor binding sites, imparting at the same time liver cell specificity for optimal function of these elements [88, 89, 90]. This interaction between the cis-acting elements with ubiquitous and liver-specific transcription factors is absolutely necessary for liver specific expression from the CP. Moreover, the interaction between these trans-acting factors and the cis-acting elements allows the virus to co-ordinately or differentially regulate the transcription of the two mrnas [91].

32 1.4 Background in molecular variations in the precore, core promoter, and core regions of HBV and their association with clinical course of Hepatitis B Introduction Although HBV is a DNA virus, it replicates through an RNA intermediate (pregenomic RNA) which is reverse transcribed, thus rendering it liable to a high mutation rate [87,92]. As a consequence, HBV exhibits a higher mutation rate than would otherwise be expected for a DNA virus, amounting to 2x10-4 base substitutions per site per year [93]. However, the potential for variation is constrained by the compact genomic organization of the virus, featuring overlapping open reading frames (ORFs) and the existence of regulatory elements of transcription, replication and encapsidation within these ORFs. Any nucleotide change that leads to loss of function of either protein will be lethal or deleterious to the virus and therefore lost (negative selection). However, individual point mutations frequently have no phenotypic effect, either because of the redundancy of the genetic code (the nucleotide change may not result in an amino acid change) or because the change does not affect higher level structures of the RNA or protein. Some of these phenotypically silent mutations may become incorporated into the quasi-species pool [94]. Although a lot of these mutations would be deleterious to the virus, as a result of constraints imposed by the overlapping ORFs, some would be advantageous, either offering a replication advantage, or facilitating immune escape [95]. The major force for evolutionary change within a virus population is positive selection by the host immune response.

33 1.4.2 Precore variants The most common substitution encountered in the Precore (PC) region (nucleotides ) is the G1896A substitution converting codon 28 W (TGG) to a stop codon (TAG) which prohibits the translation of functional PC region products and abolishes the production of the HBeAg [95,96,96,98]. This mutation is present in virtually all HBeAg (-) samples, whereas most of the HBeAg (+) samples do not have mutations leading to amino acid changes in the PC region [99]. It is the most prevalent mutation amongst the HBV mutants ever isolated and studied [100]. It is found to arise spontaneously around the time of seroconversion from the HBeAg to the corresponding antibody (Anti- HBe) or during the Interferon therapy [101,102]. The A1896 mutation may be primarily accompanied by another G to A change at position 1899, implying that a second selection pressure is exerted on PC after emergence of A1896 [102,103]. A1897, seen less often, converts TGG to TGA, also a translational stop codon [29]. The run of four Gs between nucleotide positions is therefore a mutational hot spot and all four can be substituted by A. Two of these, A1898 and A1899 are of unknown significance but are strongly linked with T1856 and A1896, respectively. These associations are likely to be related to encapsidation signal secondary structure requirement, but whether they have clinical significance remains unclear [29,104,105,106]. The PC stop codon mutation variant was initially detected in Anti-HBe carriers in Mediterranean [101] and previous studies from Greece reported a high frequency of PC mutants among circulating HBV strains, where the stop codon mutation was detected in 97% of isolates of genotype D [107], and generally in 85% of HBeAg(-) isolates of all genotypes. Although the PC stop codon variant was initially detected in Mediterranean, it is now supposed to have wide geographical distribution and has been detected in a median of 60% of HBeAg (-) patients worldwide [101,98]. The respective percentages of PC stop codon variants in HBeAg isolates from different geographical regions are: 92% in the

34 Mediterranean, 50% in Asia-Pacific, 35% in Africa and 24% in the USA and Northern Europe [98]. The aforementioned observed discrepancies in frequency of the PC stop codon variants in HBeAg (-) isolates from different parts of the world reflect an association between emergence of the PC variant and the HBV genotype. Due to the requirement of base pairing at the pre-genomic RNA level the presence of A1896 mutant is thought to be restricted to genotypes that have a T1858 as in the case of genotypes B, C, D and E. Thus, these genotypes favour the emergence and selection of the A1896 mutants. The absence of this mutation in genotypes A and F is similarly correlated to the presence of C at nucleotide 1858 [101,108]. These explain high prevalence of A1896 in the Mediterranean basin and in Asia where the predominant HBV genotypes are B, C, and D and its low prevalence in North America and Europe where genotype A is commonly found [108,101,96,95,100,109]. Although there had been strong epidemiological evidence, linking A1896 with Fulminant Hepatitis B (FHB) [29], results from recent studies clearly documented the absence of an association of the PC mutants with FHB [101], suggesting that further research is warranted. An association of a A1838G mutant, changing the amino acid I to V, with FHB has been proposed [110]. Incidence of the PC stop mutation during acute, non-fulminant hepatitis B is presently unclear [29]. An interesting relationship between age, HBeAg positivity, disease status and circulation of PC stop codon mutants/wild-type HBV emerged from several studies in Chronic Hepatitis B patients (CHB) [102]. HBeAg positivity, along with circulation of the PC wild-type HBV was strongly associated with chronic liver disease in both paediatric and adult patients. In both age groups, Anti-HBe positivity was associated with ASC state [102]. In many studies, it was found that mean serum HBV DNA levels in patients with only PC mutants were found to be significantly lower than that in patients with Core Promoter (CP) or both CP and PC region mutations. Chronic HBV carriers with only PC mutations had less HBV DNA load and less disease activity [96,102]. Thus, emergence of

35 A1896 mutation prior to the mutations of the CP might control the replication of HBV which leads to the remission of Hepatitis [29,96,102]. Lower viral replication and the emergence of PC mutations prior to CP ones might lead to decrease of hepatic inflammation both to ASCs and CHB patients [96,102]. Under these conditions, however, the virus sometimes regains the capacity to replicate, possibly due to the emergence of various mutations in the CP region that regulate transcription of pgrna and HBeAg production, or due to emergence of multiple mutations in the T-cell epitopes of Core protein [109,97]. On the contrary, other studies showed that A1896 mutation did not alter the replicating capacity of the virus [102], and furthermore it did not result in any difference in viraemia levels or the clinical course of CHB patients. There also were no significant differences in ALT levels and subsequently in disease severity and progression between patients with and without the PC stop codon mutation [101,111]. In conclusion, variable prevalence of PC mutants has been reported in different countries and their association with viraemia levels and severity of liver disease was either confirmed or contradicted [112]. Although the PC mutation has been detected more often among Liver Cirrhosis (LC) patients, the role of this mutation in progression of fibrosis seems less than that of the Basic Core Promoter (BCP) mutations [113]. Finally, stop codon mutation in the PC region has also been reported during the reactivation of HBV as a complication among cancer patients undergoing cytotoxic chemotherapy [114]. Studies, sequencing the whole PC region, without focusing only on screening for the predominant PC nucleotide 1896 mutation alone, have shown that other mutations were distributed over the entire PC region and mutations at nucleotides accounted for 23% of the total mutations in the PC region [101]. Furthermore, mutations at nucleotides ( ) and in the PC initiation codon ( ), as well as conversion of the second PC codon to a termination codon, and rare insertions or deletions in the PC region, were identified, mainly in the absence of the PC stop codon mutation, implying