ABSTRACT
The purpose of this study was to analyze sequences of hepatitis A virus (HAV) Ia and Ib genotypes from Bulgarian patients to investigate the molecular epidemiology of HAV genotype I during the years 2012 to 2014. Around 105 serum samples were collected by the Department of Virology of the National Center of Infectious and Parasitic Diseases in Bulgaria. The sequenced region encompassed the VP1/2A region of HAV genome. The sequences obtained from the samples were 103. For the phylogenetic analyses, 5 datasets were built to investigate the viral gene in/out flow among distinct HAV subpopulations in different geographic areas and to build a Bayesian dated tree, Bayesian phylogenetic and migration pattern analyses were performed. HAV Ib Bulgarian sequences mostly grouped into a single clade. This indicates that the Bulgarian epidemic is partially compartmentalized. It originated from a limited number of viruses and then spread through fecal-oral local transmission. HAV Ia Bulgarian sequences were intermixed with European sequences, suggesting that an Ia epidemic is not restricted to Bulgaria but can affect other European countries. The time-scaled phylogeny reconstruction showed the root of the tree dating in 2008 for genotype Ib and in 1999 for genotype Ia with a second epidemic entrance in 2003. The Bayesian skyline plot for genotype Ib showed a slow but continuous growth, sustained by fecal-oral route transmission. For genotype Ia, there was an exponential growth followed by a plateau, which suggests better infection control. Bidirectional viral flow for Ib genotype, involving different Bulgarian areas, was observed, whereas a unidirectional flow from Sofia to Ihtiman for genotype Ia was highlighted, suggesting the fecal-oral transmission route for Ia.
Subject(s)
Genetic Variation , Hepatitis A Virus, Human/genetics , Hepatitis A/epidemiology , Bayes Theorem , Bulgaria/epidemiology , Genotype , Hepatitis A/virology , Humans , Molecular EpidemiologyABSTRACT
BACKGROUND: Hepatitis A virus (HAV) infection is endemic in Eastern European and Balkan region countries. In 2012, Bulgaria showed the highest rate (67.13 cases per 100,000) in Europe. Nevertheless, HAV genotypes and strains circulating in this country have never been described. The present study reports the molecular characterization of HAV from 105 patients from Bulgaria. METHODS: Anti-HAV IgM positive serum samples collected in 2012-2014 from different towns and villages in Bulgaria were analysed by nested RT-PCR, sequencing of the VP1/2A region and phylogenetic analysis; the results were analysed together with patient and geographical data. RESULTS: Phylogenetic analysis revealed two main sequence groups corresponding to the IA (78/105, 74%) and IB (27/105, 26%) sub-genotypes. In the IA group, a major and a minor cluster were observed (62 and 16 sequences, respectively). Most sequences from the major cluster (44/62, 71%) belonged to either of two strains, termed "strain 1" and "strain 2", differing only for a single specific nucleotide; the remaining sequences (18/62, 29%) showed few (1 to 4) nucleotide variations respect to strain 1 and 2. Strain 2 is identical to the strain previously responsible for an outbreak in the Czech Republic in 2008 and a large multi-country European outbreak caused by contaminated mixed frozen berries in 2013. Most sequences of the IA minor cluster and the IB group were detected in large/medium centers (LMCs). Overall, sequences from the IA major cluster were more frequent in small centers (SCs), but strain 1 and strain 2 showed an opposite relative frequency in SCs and LMCs (strain 1 more frequent in SCs, strain 2 in LMCs). CONCLUSIONS: Genotype IA predominated in Bulgaria in 2012-2014 and phylogenetic analysis identified a major cluster of highly related or identical IA sequences, representing 59% of the analysed cases; these isolates were mostly detected in SCs, in which HAV shows higher endemicity than in LMCs. The distribution of viral sequences suggests the existence of some differences between the transmission routes in SCs and LMCs. Molecular characterization of an increased number of isolates from Bulgaria, regularly collected over time, will be useful to explore specific transmission routes and plan appropriate preventing measures.
