A novel defective recombinant porcine enterovirus G virus carrying a porcine torovirus papain-like cysteine protease gene and a putative anti-apoptosis gene in place of viral structural protein genes

Abstract

Enterovirus G (EV-G) belongs to the family of Picornaviridae. Two types of recombinant porcine EV-Gs carrying papain-like cysteine protease (PLCP) gene of porcine torovirus, a virus in Coronaviridae, are reported. Type 1 recombinant EV-Gs are detected in pig feces in Japan, USA, and Belgium and carry the PLPC gene at the junction site of 2C/3A genes, while PLPC gene replaces the viral structural genes in type 2 recombinant EV-G detected in pig feces in a Chinese farm. We identified a novel type 2 recombinant EV-G carrying the PLCP gene with flanking sequences in place of the viral structural genes in pig feces in Japan. The ~0.3 kb-long upstream flanking sequence had no sequence homology with any proteins deposited in GenBank, while the downstream ~0.9 kb-long flanking sequence included a domain having high amino acid sequence homology with a baculoviral inhibitor of apoptosis repeat superfamily. The pig feces, where the novel type 2 recombinant EV-G was detected, also carried type 1 recombinant EV-G. The amount of type 1 and type 2 recombinant EV-G genomes was almost same in the pig feces. Although the phylogenetic analysis suggested that these two recombinant EV-Gs have independently evolved, type 1 recombinant EV-G might have served as a helper virus by providing viral structural proteins for dissemination of the type 2 recombinant EV-G.

Keywords: Enterovirus; Recombination; Torovirus.

Fig. 1

Schematic diagram of the genome organization of EV-G, type 1 and type 2 recombinant EVGs. Genome order of EV-G, type 1 and type 2 recombinant EV-G came from the EVG/Porcine/JPN/Iba464-3-1/2015/G1 (Tsuchiaka et al., 2017), EVG/Porcine/JPN/MoI2-1-1/2015/G1-PL-CP (Tsuchiaka et al., 2018), and a newly identified type 2 EV-G (EVG/Porcine/JPN/MoI2-1-2/2015/type 2) in this study, respectively.

Fig. 2

Overlapping PCR of type 2 recombinant EV-G. RNAs were re-extracted in this study from the pig feces using High Pure Viral Nucleic Acid Kit (Roche), and cDNA was synthesized with random primers using SuperScript III First-Strand Synthesis System (Invitrogen), as described previously (Tsuchiaka et al., 2018). Primers for PCR (Table 1) were designed to amplify the viral genome at approximately every 1 kb with overlap regions. PCR was performed using the Premix Taq (Takara Bio) with the following conditions: an initial denaturation at 95 °C for 2 min; followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; and a final extension at 72 °C for 5 min. Lane 1, 31F and 1191R primers; lane 2, 930F and 1978R primers; lane 3, 1725F and 2760R primers; lane 4, 2500F and 3447R primers; lane 5, 3001F and 4188R primers; lane 6, 3921F and 4961R primers; lane 7, 4835F and 5852R primers; lane 8, 5619F and 6694R primers in Table 1. The PCR products were electrophoresed using agarose gel and nucleotide sequences were confirmed by direct sequence of the PCR products, which had been purified by agarose gel electrophoresis. The three bands marked as asterisk were confirmed no relation to EV-G by direct sequencing.

Fig. 3

Amino acid alignment of unique region 2 and BIR family. (A) Junction sites of unique region 1-PLCP-unique region 2-N-terminal truncated 2A of EVG/Porcine/JPN/MoI2-1-2/2015/type 2. (B) Amino acid sequence of amino acids at 180-240 of unique region 2 of the newly identified type 2 EV-G is shown in top and is aligned with other BIR family protein. moi212uniq2 represents the newly identified type 2 EV-G. The accession number of protein sequences from GenBank used in this figure is indicated as XP_020140715.1: E3 ubiquitin-protein ligase XIAP isoform X3 of Microcebus murinus, XP_008068909.1: E3 ubiquitin-protein ligase XIAP of Carlito syrichta, XP_023363885.1: E3 ubiquitin-protein ligase XIAP of Otolemur garnettii, NP_001164796.1: E3 ubiquitin-protein ligase XIAP of Oryctolagus cuniculus, XP_022417552.1: E3 ubiquitin-protein ligase XIAP isoform X1 of Delphinapterus leucas, XP_007111017.1: E3 ubiquitin-protein ligase XIAP isoform X1 of Physeter catodon, XP_020935024.1: E3 ubiquitin-protein ligase XIAP isoform X1 of Sus scrofa, XP_020040963.1: E3 ubiquitin-protein ligase XIAP of Castor canadensis, AAB58376.1: X-linked inhibitor of apoptosis of Mus musculus, XP_023590042.1: E3 ubiquitin-protein ligase XIAP isoform X2 of Trichechus manatus latirostris, XP_013003432.1: E3 ubiquitin-protein ligase XIAP of Cavia porcellus, XP_021484438.1: E3 ubiquitin-protein ligase XIAP of Meriones unguiculatus, OWJ99336.1: XIAP of Cervus elaphus hippelaphus, XP_004480589.1: E3 ubiquitin-protein ligase XIAP of Dasypus novemcinctus, XP_023055645.1: E3 ubiquitin-protein ligase XIAP of Piliocolobus tephrosceles, NP_001271387.1: E3 ubiquitin-protein ligase XIAP of Canis lupus familiaris. Dots indicates different amino acids from moi212uniq2.

Fig. 4

Phylogenetic trees of EVG/Porcine/JPN/MoI2-1-2/2015/type 2 with enterovirus G strains from GenBank database based on nucleotide sequences of PLCP (A), 2A (B), 2B (C), 2C (D), 3C (E) and 3D (F). The trees were constructed using Neighbor-joining method in MEGA 7.0.14 and bootstrap test (n-1000). The genetic distance was calculated using Kimura's two parameter model. The scale bar indicates nucleotide substitutions per site.

Fig. 4

Phylogenetic trees of EVG/Porcine/JPN/MoI2-1-2/2015/type 2 with enterovirus G strains from GenBank database based on nucleotide sequences of PLCP (A), 2A (B), 2B (C), 2C (D), 3C (E) and 3D (F). The trees were constructed using Neighbor-joining method in MEGA 7.0.14 and bootstrap test (n-1000). The genetic distance was calculated using Kimura's two parameter model. The scale bar indicates nucleotide substitutions per site.

Fig. 4

Phylogenetic trees of EVG/Porcine/JPN/MoI2-1-2/2015/type 2 with enterovirus G strains from GenBank database based on nucleotide sequences of PLCP (A), 2A (B), 2B (C), 2C (D), 3C (E) and 3D (F). The trees were constructed using Neighbor-joining method in MEGA 7.0.14 and bootstrap test (n-1000). The genetic distance was calculated using Kimura's two parameter model. The scale bar indicates nucleotide substitutions per site.

Fig. 4

Phylogenetic trees of EVG/Porcine/JPN/MoI2-1-2/2015/type 2 with enterovirus G strains from GenBank database based on nucleotide sequences of PLCP (A), 2A (B), 2B (C), 2C (D), 3C (E) and 3D (F). The trees were constructed using Neighbor-joining method in MEGA 7.0.14 and bootstrap test (n-1000). The genetic distance was calculated using Kimura's two parameter model. The scale bar indicates nucleotide substitutions per site.

Fig. 4

Phylogenetic trees of EVG/Porcine/JPN/MoI2-1-2/2015/type 2 with enterovirus G strains from GenBank database based on nucleotide sequences of PLCP (A), 2A (B), 2B (C), 2C (D), 3C (E) and 3D (F). The trees were constructed using Neighbor-joining method in MEGA 7.0.14 and bootstrap test (n-1000). The genetic distance was calculated using Kimura's two parameter model. The scale bar indicates nucleotide substitutions per site.

Fig. 4

Phylogenetic trees of EVG/Porcine/JPN/MoI2-1-2/2015/type 2 with enterovirus G strains from GenBank database based on nucleotide sequences of PLCP (A), 2A (B), 2B (C), 2C (D), 3C (E) and 3D (F). The trees were constructed using Neighbor-joining method in MEGA 7.0.14 and bootstrap test (n-1000). The genetic distance was calculated using Kimura's two parameter model. The scale bar indicates nucleotide substitutions per site.