Whole Genome Sequence Analysis of Porcine Astroviruses Reveals Novel Genetically Diverse Strains Circulating in East African Smallholder Pig Farms

Abstract

Astroviruses (AstVs) are widely distributed and are associated with gastroenteritis in human and animals. The knowledge of the genetic diversity and epidemiology of AstVs in Africa is limited. This study aimed to characterize astroviruses in asymptomatic smallholder piglets in Kenya and Uganda. Twenty-four samples were randomly selected from a total of 446 piglets aged below 6 months that were initially collected for rotavirus study and sequenced for whole genome analysis. Thirteen (13/24) samples had contigs with high identity to genus Mamastrovirus. Analysis of seven strains with complete (or near complete) AstV genome revealed variable nucleotide and amino acid sequence identities with known porcine astrovirus (PoAstV) strains. The U083 and K321 strains had nucleotide sequence identities ranging from 66.4 to 75.4% with the known PoAstV2 strains; U460 strain had nucleotide sequence identities of 57.0 to 65.1% regarding the known PoAstV3; and K062, K366, K451, and K456 strains had nucleotide sequence identities of 63.5 to 80% with the known PoAstV4 strains. The low sequence identities (<90%) indicate that novel genotypes of PoAstVs are circulating in the study area. Recombination analysis using whole genomes revealed evidence of multiple recombination events in PoAstV4, suggesting that recombination might have contributed to the observed genetic diversity. Linear antigen epitope prediction and a comparative analysis of capsid protein of our field strains identified potential candidate epitopes that could help in the design of immuno-diagnostic tools and a subunit vaccine. These findings provide new insights into the molecular epidemiology of porcine astroviruses in East Africa.

Keywords: East Africa; glycosylation; linear antigenic epitopes; porcine astroviruses; recombination; whole genome sequences.

Figure 1

Phylogenetic analysis based on the complete (nearly complete, U460) nucleotide sequences of the full-length genome of East African PoAstVs (bold) and representative AstVs of humans and other species in the GenBank. Multiple sequence alignments were performed using the ClustalO program. The evolutionary history was inferred by using the maximum likelihood method based on the general time reversible model in MEGA X [33]. The scale bar is given in numbers of substitutions per site. Phylogeny was inferred following 1000 bootstrap replications, and the node values show percentage bootstrap support.

Figure 2

Phylogenetic tree based on the nucleotide (a) and amino acid (b) sequences of the capsid proteins (ORF2) of the East African astrovirus field strains and the known astroviruses in the GenBank. Multiple sequence alignments were performed using the ClustalO program. The evolutionary history was inferred by using the maximum likelihood method on the GTR model for nucleotide and neighbor-joining method using the p-distance substitution model for amino acid sequences in MEGA X [33]. The scale bar is given in numbers of substitutions per site. Phylogeny was inferred following 1000 bootstrap replications, and the node values show percentage bootstrap support.

Figure 3

Recombination analysis of newly discovered full-length astrovirus genomes: (a) Predicted recombination event has likely occurred in the ORF1a–ORF1b junction and covers nearly the entire RdRp region. K062 strain is recombinant. (b) Predicted recombination event has likely occurred in RdRp–ORF2 overlap and covers almost the entire capsid region (ORF2); K366 is the recombinant. (c) Predicted recombination event has occurred in the 3′ end of the capsid region ORF2; K456 strain is recombinant.

Figure 4

Capsid protein structures and conformational B-cell epitopes of PoAstV protein predicted by Ellipro from 3D structure template. Yellow balls are the residues of predicted peptides and white sticks are the non-epitope residues of protein. Each epitope is predicted with residue number and position mentioned.