Recombination analysis based on the HAstV-2 and HAstV-4 complete genomes

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Highlights

  • Full genomes of four HAstV strains from Novosibirsk were sequenced.

  • Analysis of the recombination events has detected potential breakpoints.

  • The evolutionary chronology of HAstV genotypes has been reconstructed.

Abstract

Complete genome sequences of previously unstudied human astrovirus subgenotypes – HAstV-2a and HAstV-2c – and two isolates of a rare genotype HAstV-4 have been determined. These isolates were recovered from fecal samples of young children hospitalized with acute intestinal infections in Novosibirsk (Russia). Three of the four sequenced isolates (HAstV-2a, HAstV-2c, and HAstV-4) are recombinants. It has been shown that all known HAstV-2 genomes have emerged via recombination; the HAstV-1 and HAstV-4 genotypes contain both recombinant and non-recombinant isolates; and all HAstV-3, HAstV-5, and HAstV-6 whole-genome sequences display no reliable signs of recombination. The average mutation accumulation rate has been determined based on an extended ORF2 fragment and amounts to 1.0 × 10−3 substitutions per site per year. The evolutionary chronology of current HAstV genotypes has been reconstructed.

Introduction

Human astrovirus (HAstV), a member of the Astroviridae family, is a rapidly evolving virus with an ss(+) RNA genome. The HAstV genome RNA with a length of about 6.8 kb contains three overlapping open reading frames (ORFs) – ORF1a, ORF1b, and ORF2, encoding serine protease, RNA-dependent RNA polymerase, and capsid protein precursor, respectively (De Benedictis et al., 2011, Mendez and Arias, 2007). The 5′-end is linked to a VPg protein and the 3′-end has a poly(A) tract (Al Mutairy et al., 2005, Fuentes et al., 2012, Mendez and Arias, 2007). Being an agent causing acute gastroenteritis in children and adults, HAstV is spread in all regions around the world (De Grazia et al., 2011, Gabbay et al., 2007, Jeong et al., 2012). Currently, eight HAstV genotypes are known, which are identified by a direct sequencing of an ORF2 fragment; these genotypes differ in their abundance (Belliot et al., 1997, Noel et al., 1995). HAstV-1 is the most abundant, while the remaining genotypes are rather rare. Considerable genetic variation within HAstV-1, HAstV-2, and HAstV-4 genotypes has determined their subdivision into subgenotypes (De Grazia et al., 2011, Gabbay et al., 2007, Martella et al., 2013). However, the genotyping based on a single small ORF2 fragment is unable to give any information about the variation in other regions of the virus genome. Thus, a detailed molecular biological study of this virus requires that whole genomes of various HAstV isolates are sequenced.

A high rate of HAstV evolution is determined by both a high rate of mutation accumulation in the genome, amounting to (2–4) × 10−3 substitutions per site per year (Babkin et al., 2012), and the ability of genome RNA to recombine (Simmonds, 2006, Strain et al., 2008, Ulloa and Gutierrez, 2010). The phylogenies of several HAstV strains based on fragments of various genome regions may significantly differ (Belliot et al., 1997, Walter et al., 2001). Recent analysis of some fragments of HAstV-2c and HAstV-2d genomes has demonstrated their recombinant nature (De Grazia et al., 2012, Wolfaardt et al., 2011). Whole-genome HAstV sequences, including the sequences of rare genotypes, allow for search of recombination sites with high probability. Babkin et al. have studied recombination using whole-genome sequences of HAstV strains to find the regions suitable for estimating mutation accumulation rate in the genomes of this virus (Babkin et al., 2012). In this work, we have studied the role of recombination in the evolution of this virus. In addition, we revised the data on the mutation accumulation rate in HAstV genomes using extended genome fragments and detailed the evolutionary chronology of the current HAstV genotypes.

Section snippets

Viral nucleic acid extraction and reverse transcription-PCR (RT-PCR)

Virus RNA was extracted from 100 μl of 10% fecal suspension using a RIBO-sorb kit (Interlabservis, Russia) according to the manufacturer’s protocol. The cDNA was synthesized with the help of a REVERTA-L v50 kit (Interlabservis, Russia). The HAstV was detected with an AmpliSens Astrovirus-Eph kit (Interlabservis, Russia). The HAstV-positive specimens were genotyped by RT-PCR and nucleotide sequencing of a 413-bp fragment within the capsid protein precursor region (Noel et al., 1995).

Genome sequencing

Genomes were

Whole-genome sequencing

Since 2003, detection and genotyping of various viruses in clinical fecal samples from young children were performed in Novosibirsk. The detected HAstV isolates have been genotyped by sequencing the PCR fragments from ORF2 according Noel et al., 1995. Isolates HAstV-2a Rus-Nsc05-430, HAstV-2c Rus-Nsc06-1029, HAstV-4 Rus-Nsc10-N358, and HAstV-4 Rus-Nsc05-623 recovered in 2005, 2006, 2010, and 2005, respectively, were selected for whole-genome sequencing (Fig. 1).

Totally, 29 original primers were

Discussion

Earlier phylogenetic analysis of HAstV sequences has shown that the phylogenies of several HAstV-1, HAstV-2, and HAstV-4 strains based on fragments of different ORFs display significant differences (Babkin et al., 2012, Belliot et al., 1997, De Grazia et al., 2012, Pativada et al., 2013, Strain et al., 2008, Walter et al., 2001, Wolfaardt et al., 2011). Thus, a better understanding of the HAstV evolution requires analysis of the recombination events in whole-genome sequences of these HAstV

Acknowledgment

This work was supported in part by the Russian Foundation for Basic Research (project no. 13-04-00554а).

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