Comparative Analysis of Novel Strains of Porcine Astrovirus Type 3 in the USA

Figures

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  Figure 1. Phylogenic tree of selected full-genome neurotropic Mamastrovirus strains and 20 novel full-genome PoAstV3 strains identified in this study (bold). All novel PoAstV3 strains identified in this study clustered with PoAstV3 strains previously described. Nucleotide sequences were aligned using ClustalO webserver under default settings. The phylogenic tree was generated using the neighbor-joining algorithm using MEGAX and rendered with iTOL.

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  Figure 2. Consensus 5′UTR secondary RNA structure of USA PoAstV3 (a). Percent identity of 34 PoAstV3 5′UTR and 5′UTR of strain GER/L00919-K17/2014 (b). Darker blue tone indicates homologous bases. Note the inclusion of 5′UTR of strain GER/L00919-K17/2014 with initial 5′ GC bases only present in this strain. GenBank accession within parentheses. The start codon is included on both secondary structure (a) and percent identity plot (light blue box). Sequences identified in this study are denoted by a black box.

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  Figure 3. Secondary structure of the frameshift signal of PoAstV3 (a). The structure is composed of a stem motif with a conserved loop and bulge. The secondary structure of the frameshift signal of human astrovirus 2 (GenBank accession L13745) was modeled for comparison (b).

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  Figure 4. Sequence alignment of the 3′UTR region of PoAstV3 strains. Sequences with an incomplete 3′UTR region were omitted. Note insertion-deletion (IN/DEL) region present between bases 48–79 in USA and Spanish sequences and absent in Hungarian and Japanese strains, and GER/L00919-K17/2014. Stop codons UAG, UAA and UGA, in amber, ochre, and opal boxes, respectively. Dark blue indicates higher sequence percent identity. Poly (a) tail regions were excluded. Sequences were aligned using ClustalO webserver under default settings. Sequences identified in this study are denoted by a black box.

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  Figure 5. Secondary structure of the consensus PoAstV3 3‘UTR region of USA (a), Spanish (b), Hungarian (c) strains, strain GER/L00919-K17/2014 (d) and consensus Japanese strain (e). Secondary structures of (a–e) are illustrated without (left) and with pseudoknotted interaction (right). Green, yellow and blue bases indicate stems, interior loops, and hairpins, respectively. Red and orange bases indicate multiloops, and 5′ and 3′ unpaired regions, respectively.

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  Figure 6. Putative protein binding sites of the 3′UTR of PoAstV3. RNA binding sites for serine/arginine-rich splicing factors 5 (SRSF5 [UCAGA, blue bases]), heterogeneous nuclear ribonucleoprotein E2 (hnRNPE2 [UUAG/UUAU, green bases]) and polypyrimidine tract-binding protein (PTB [UCUU, red bases]). UAG and UGA stop codons in amber and opal boxes, respectively. Sequences identified in this study are denoted by a black box.

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  Figure 7. Secondary structure of 3′UTR of PoAstV3 and RNA binding sites for serine/arginine-rich splicing factor 5 (SRSF5 [UCAGA, green bases]), heterogeneous nuclear ribonucleoprotein E2 (hnRNPE2 [UUAG/UUAU, blue bases]) and polypyrimidine tract-binding protein (PTB [UCUU, red bases]). USA consensus sequence (a), strain ESP/B333/2017 (b), Hungarian consensus sequence (c), GER/L00919-K17/2014 (d) and JPN/Bu2-5/2014 (e). UAG stop codon (amber bases).

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  Figure 8. Capsid protein percent identity plot of USA PoAstV3 from this study. Blue and white areas indicate areas of higher and lower homology, respectively. Amino acid homology of linear epitopes and probable antigens of USA PoAstV3 strains identified in this study.