A24 Phylogeographic analysis of hepatitis A virus in Russia
Authors: Koreshova A , Bazykin G , Neverov A 
Published: August 2019

Abstract
Hepatitis A virus (HAV) is a positive-stranded RNA virus, a member of Picornaviridae, and a representative of genus Hepatovirus. It is unique among picornaviruses with regards to its hepatotropism, structure, and life cycle. HAV is spread via the fecal-oral route as a non-enveloped particle, while, in the blood the virus circulates in an envelope formed from the host cell membrane. HAV causes acute hepatitis in adults and is usually asymptomatic in children <6 years of age. The clinical features include fever, malaise, anorexia, nausea, abdominal discomfort, dark urine, and jaundice, all of which usually last >2 months. There is no evidence of chronic liver disease or persistent infection following acute infection. Due to its mode of transmission, HAV prevails in areas with low hygiene standards but does not give rise to epidemics because most people are infected at an early age and derive a life-long immunity. Thus, HAV infection has more impact on countries with higher socio-economical level where it is mostly registered as an outbreak in adults, which is the case in Russia. One feature distinguishing HAV from other picornaviruses is its remarkably slow mutation rate. HAV genotyping is typically carried out using highly variable regions VP1/2A and 2C/3A. Recently, it was shown that resolution provided by short fragments is not enough for reliable results. Unfortunately, previous research in HAV phylogeography was carried out only on these short sequences and did not include Russia or CIS territories. HAV comprises six genotypes, of which I and III are most frequent in humans and are both divided into A and B subgenotypes. Preliminary phylodynamic analysis of 80 highly variable region sequences (carried out by A. Neverov) has shown a particular pattern of geographical distribution of HAV genotypes in Russia. There are only two subgenotypes widely spread: IA predominates in the European part of Russia, and IIIA is found mainly in the Asian part. However, the history of HAV spread in Russia remains unclear. We hypothesized that IIIA subgenotype originated from India, while IA subgenotype came later from Europe and is still expanding. The Central Research Institute of Epidemiology kindly provided us with the unique collection of HAV isolates obtained from more than 30 subjects of the Russian Federation, as well as a number of isolates obtained from CIS countries. Samples (>500 isolates) were collected from 1999 to 2015 and characterized by one or both of the two most variable fragments of HAV genome (VP1/2A and 2C regions). The dataset includes 145 unique sequences of 2C/3A region, length ∼650 bp, and 243 sequences of VP1/2A region, length ∼400 bp. For each sample, date and location of collection are indicated. Whole-genome sequences of HAV from GenBank database were also used. They were aligned with MUSCLE, and the target 2C/3A and VP1/2A fragments were extracted. Partial HAV sequences from GenBank were not added to the analysis due to too little overlap with our sequences. Initial phylogeographic analysis was carried out in BEAST. Results were checked with the Tracer program, and the Spread3 package was used to visualize the results of the phylogeographic analysis in continuous space [16]. The BEAST output supports the hypothesis that IIIA subgenotype originated from India, whereas the situation with the IA subgenotype remains unclear. The reason for this might be either poor sampling of the Mediterranean area and Middle East in our analysis or low precision provided with variable fragments. The next step is to obtain full-genome sequences of approximately 100 of our samples to increase resolution and make use of hundreds of partial sequences of HAV genomes available in GenBank.