Research paper
Whole genome analysis of porcine astroviruses detected in Japanese pigs reveals genetic diversity and possible intra-genotypic recombination

https://doi.org/10.1016/j.meegid.2017.02.008Get rights and content

Highlights

  • Nearly complete genome of Japanese porcine astroviruses were sequenced and analyzed.

  • They were classified into four genotypes.

  • Multiple possible intra-genotype recombination events were observed.

  • Recombination processes may play an important role in the evolution of porcine astroviruses.

Abstract

Porcine astroviruses (PoAstVs) are ubiquitous enteric virus of pigs that are distributed in several countries throughout the world. Since PoAstVs are detected in apparent healthy pigs, the clinical significance of infection is unknown. However, AstVs have recently been associated with a severe neurological disorder in animals, including humans, and zoonotic potential has been suggested. To date, little is known about the epidemiology of PoAstVs among the pig population in Japan. In this report, we present an analysis of nearly complete genomes of 36 PoAstVs detected by a metagenomics approach in the feces of Japanese pigs. Based on a phylogenetic analysis and pairwise sequence comparison, 10, 5, 15, and 6 sequences were classified as PoAstV2, PoAstV3, PoAstV4, and PoAstV5, respectively. Co-infection with two or three strains was found in individual fecal samples from eight pigs. The phylogenetic trees of ORF1a, ORF1b, and ORF2 of PoAstV2 and PoAstV4 showed differences in their topologies. The PoAstV3 and PoAstV5 strains shared high sequence identities within each genotype in all ORFs; however, one PoAstV3 strain and one PoAstV5 strain showed considerable sequence divergence from the other PoAstV3 and PoAstV5 strains, respectively, in ORF2. Recombination analysis using whole genomes revealed evidence of multiple possible intra-genotype recombination events in PoAstV2 and PoAstV4, suggesting that recombination might have contributed to the genetic diversity and played an important role in the evolution of Japanese PoAstVs.

Introduction

Astroviruses (AstVs) have been found in the feces of large range animals throughout the world and are known as cause of gastroenteritis in human (De Benedictis et al., 2011). Their association with enteric disease in mammals other than humans has not been well documented, and the clinical significance of these infections is not well understood. For porcine AstVs (PoAstVs), there is one report that an experimental oral infection of 4-day-old pigs resulted in mild diarrhea (Shimizu et al., 1990); however, PoAstVs are commonly found in the feces of apparently healthy pigs (De Benedictis et al., 2011). In recent years, non-enteric AstV infections in mammals, such as encephalitis in humans (Brown et al., 2015, Cordey et al., 2016, Frémond et al., 2015, Naccache et al., 2015, Quan et al., 2010, Sato et al., 2016, Wunderli et al., 2011), minks (Blomström et al., 2010), and cattle (Bouzalas et al., 2014, Li et al., 2013, Schlottau et al., 2016, Seuberlich et al., 2016), as well as isolation of AstVs from the brains of piglets suffering from congenital tremors (Blomström et al., 2014), and from nasal swabs of piglets with acute respiratory disease (Padmanabhan and Hause, 2016) have been reported. This suggests that the clinical importance and impact of AstVs are increasing.

AstVs have non-enveloped positive-sense, single-stranded RNA of about 6.4–7.3 kb that contains three overlapping open reading frames (ORFs) (Méndez and Arias, 2013). A ribosomal frame shift mediated by ribosomal slippage and a hairpin structure results in translation of ORF1ab (Jiang et al., 1993). ORF1a and ORF1b encode nonstructural proteins, a serine protease, and an RNA-dependent RNA polymerase, respectively (Koonin, 1991). ORF2, encoding the viral capsid protein, is translated from a subgenomic RNA (Méndez et al., 2013). High sequence diversity was found in ORF2; ORF1b is the least divergent (Strain et al., 2008). The N-terminal half of AstV ORF2 encodes the particle assembly domain that is conserved among AstVs, whereas the C-terminal half forms the hypervariable receptor-interaction domain (Krishna, 2005). Because this hypervariable domain is believed to form the capsid spike and to contain neutralizing epitopes (Dong et al., 2011), ORF2 is used for presumption of AstV serotypes by PCR with subsequent sequencing (Matsui et al., 1998).

AstVs are the Astroviridae family, which is divided into two genera: Mamastrovirus (MAstVs) and Avastrovirus. The International Committee on Taxonomy of Viruses (ICTV) classified AstV into six MAstV species based on their host species in the Ninth ICTV Report. However, because of the many recently discovered MAstVs, the Astroviridae Study Group updated the taxonomy based not only on host range but also on genetic differences (mean amino acid (aa) genetic distances between and within genotypes range between 0.368–0.781, and 0–0.318, respectively) in the complete ORF2 sequence. To date, following these standardized criteria, 33 and 7 distinct species of MAstV (Mamastrovirus 1–33) and Avastrovirus, respectively, have been proposed (Guix et al., 2013).

PoAstV was first identified in the 1980s by electron microscopy of pig feces (Bridger, 1980, Shirai et al., 1985), and PoAstV was first isolated in 1990 using a porcine cell line (Shimizu et al., 1990). After the advent of genetic diagnostics for AstV, PoAstVs has been reported in several countries throughout the world (De Benedictis et al., 2011). Presently, five genotypes of PoAstV (PoAstV1-PoAstV5) are recognized (Xiao et al., 2013). In Japan, although PoAstVs were detected in the 1980s, only two sequences of PoAstV1 have been reported (Shan et al., 2012, Wang et al., 2001). Furthermore, only a few whole genome sequences of PoAstV are available in the DDBJ/EMBL/GenBank database. Therefore, to contribute to the whole genome sequence data available for PoAstVs, we used a metagenomics approach to sequence and analyze nearly complete genomes of PoAstVs from Japanese pigs. A broad diversity of genotype (PoAstV2, PoAstV3, PoAstV4, PoAstV5) was detected from diseased and healthy pigs and phylogenetic analysis and recombination analysis revealed multiple possible recombination events between PoAstVs.

Section snippets

Fecal samples, viral RNA extraction, and deep sequencing

A total of 145 fecal samples were collected from 38 pig farms in the mainland of Japan in 2014–2015 and were taken from 2 to 120-day-old pigs that were clinically healthy (73 samples) or had diarrhea (72 samples). Samples from single pigs (124 samples) or 2–3 pigs (pooled, 21 samples) were diluted 1:9 (w/v) in sterile phosphate buffered saline and stored in a  80 °C freezer until use. Total RNA was extracted from the supernatants of diluted fecal samples using TRIzol LS Reagent (Life

Complete or nearly complete sequencing of the PoAstV coding sequence

Contigs were generated from trimmed sequence reads and generated contigs were evaluated by map reads to reference command in CLC Genomics Workbench with strictest parameter setting (mismatch cost, 2; insertion cost, 3; deletion cost, 3; length function, 1.0; and similarity function, 1.0), and 5′ and 3′ sequences with insufficient read depth (< 3) were omitted. Only contigs that showed sequence similarities with AstVs and that were longer than 5930 nt were used in this study. Thirty-six AstV

Discussion

In the present study, 10 strains (5 PoAstV2, 2 PoAstV3, and 3 PoAstV4) were isolated from diarrheic pigs, whereas 26 strains (5 PoAstV2, 3 PoAstV3, 12 PoAstV4, and 6 PoAstV5) were identified from pigs without diarrhea. Since PoAstVs are ubiquitously distributed in apparently healthy pig, the clinical significance of PoAstV infection has not been completely clarified (De Benedictis et al., 2011). The present result indicated that there seems to be no clear association of PoAstV infection and

Acknowledgements

This work was supported by JSPS KAKENHI grant number 15K07718 and grants from the Ministry of Health, Labor, and Welfare of Japan and the Technology Research Partnership for Sustainable Development (SATREPS).

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