FILODINAMICA E FILOGEOGRAFIA DEI PRINCIPALI SOTTOTIPI DI HCV NEI BALCANI OCCIDENTALI

ABSTRACT Hepatitis C virus (HCV) is the leading cause of liver disease and causes both acute and chronic infection. 130-170 million people are chronically infected worldwide. It is characterized by an high degree of genetic heterogeneity due to the lack of proofreading activities of viral RNA polymerase. HCV is classified into 7 genotypes, which are subdivided into 67 epidemiologically diverse subtypes. The main 7 HCV genotypes vary in their geographical distribution and their level of genetic diversity. Genotype 1, 2 , 3 e 4 are endemics because circulating for a long time in restricted regions of the world. While subtype 1a, 1b, 3a, 2a, 2c and 4d are epidemics because they have spread rapidly in the last century through blood, unsafe medical injections and injection drug use. The aim of this study was to investigate HCV sub/genotype distribution in Montenegro e Albania and estimate the phylodynamics and phylogeography of the most prevalent subtype in order to reconstruct the origin and diffusion of the virus on a local regional scale, using a Bayesian approach. To this aim, we analyzed the NS5B gene sequences of a total 162 HCV-positive Montenegrin patients and 67 Albanian subjects HCV chronic infected. The Montenegrin patients are stratified into two populations: intra-venus drug users (IVDUs) and the general population. Our study showed that the prevalent HCV subtype in Montenegrin general population was 1b (45%) followed by 4d (19%), 3a (17%), 1a (16%) and 2a/c (1%). While in the IVDU Montenegrin population, the most prevalent subtype was 3a (47%), followed by 1a (25%), 4d (20%) and 1b (8%). The subtype 3a was common among subjects who used injecting drug (47%), while the subtype 1b was common related with iatrogenic transmission (53.3%). Likewise the 67 Albanian patients was stratified into two populations: intra-venus drug users (IVDUs) and the general population. The main subtype in the general population was 1b (60%) followed by 2a/c (27%), 3a and 1a (4%) while genotype 4 was present in the 4%. In the IVDU Albanian population the prevalent subtype was 3a (67%), followed by 1a (25%) and 4d (8%). In order to estimate the phylodinamics and phylogeography of HCV subtypes, three separate datasets were prepared that include 227 sequence of HCV 1b, 271 of HCV 3a and 106 of HCV 4d. The phylogeographical analysis of HCV-1b revealed two main clades: clade A included sequence from Montenegro, Albania, Germany, Italy, Greece and Cyprus; clade B included the majority of the Asian isolates (Turkey and Uzbekistan). The origin of the tree root is dated back to the year 1912 in Cyprus (state probability, sp= 0.78). The analysis of population dynamics of entire dataset shows an exponential growth of the 1b epidemics between 1960-1980, after the curve reached a plateau that still continuing. Phylogeographic analysis of HCV-3a showed that the most probable location of the tree root was Pakistan (sp=0.57). The tree showed many significant clade. Clade A and B comprise Asian and East European isolates (Bulgaria, Azerbaijan, Cyprus, Estonia) (pp=0.86 and 0.98); the clade C included 21 Pakistan isolates (pp=0.88); clade D, E, F and G included Montenegrin isolates. The time of most recent common ancestor (tMRCA) of the tree root went back to about 144 years ago, the clade A and B origin in Azerbaijan about 22 years ago (1992). The four Montenegrin clade dated back between 6-17 year ago corresponding to 1997-2007. The phylodinamics analysis of 54 Montenegrin and Albanian sequence revealed segregation between the two locations. The tree showed many recent Montenegrin clade dating back 10-11 years ago. The skyline plot suggest that subtype 3a was present in the population in the 1980, after occur a biphasic exponential growth between 1980-1990 and mid of XXI century. Phylogeographic analysis of HCV-4d shows two main significant clade: clade I included Turkish isolates; whereas the clade II included European sequences (Italian, Montenegrin, Albanian, French and Dutch). The Montenegrin isolates segregates in an unique significant clade. The root had a tMRCA of 125 years ago corresponding to 1888; the Turkish clade had a tMRCA of 48 years ago (1966) whereas the European clade had an origin of 65 years ago (1948). The Montenegrin sequences had a tMRCA of 50 years ago dating back 1964. This study suggests that HCV in Western Balkans present an high heterogeneity regarding subtype distribution through different transmission routes. In particular, HCV-1b penetrated in the XX century in the Aegean area and spread in Europe through two entrance; one in the Eastern Europe and the other in the Eastern Mediterranean probably related to unsafe iatrogenic procedure in the area after the 1950s. HCV-3a showed an old origin about 144 years ago in Pakistan but a recent increase in Central Asia and Eastern Europe about 20 years ago with rapid spread in Montenegro among IVDUs living in this area. HCV 4d entered in Europe probably through Italy in 1930 and spread in Western Balkans in the 1960s showing a clear segregation of the infection in Turkey. Nevertheless, further studies an larger group of samples are needed to clarify the phylodynamics of HCV-4d. In conclusion, the Western Balkans are an area of encounter of various ethnic groups where the virus can be spread and diversified