key: cord-0944139-b930c5tc
authors: Imai, Ryo; Nagai, Makoto; Oba, Mami; Sakaguchi, Shoichi; Ujike, Makoto; Kimura, Ruka; Kida, Moeko; Masuda, Tsuneyuki; Kuroda, Moegi; Wen, Rongduo; Li, Kaixin; Katayama, Yukie; Naoi, Yuki; Tsuchiaka, Shinobu; Omatsu, Tsutomu; Yamazato, Hiroshi; Makino, Shinji; Mizutani, Tetsuya
title: 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
date: 2019-07-22
journal: Infect Genet Evol
DOI: 10.1016/j.meegid.2019.103975
sha: 63d21b170240273cacfe787f778ecb1bd56fb5e7
doc_id: 944139
cord_uid: b930c5tc

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.

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.

Enterovirus G (EV-G) is a non-enveloped RNA virus, belonging to the family of Picornaviridae. Many types of EV-G have been identified (Anbalagan et al., 2014; Bunke et al., 2018; Zell et al., 2017) and the unique properties for reclassification of these viruses were reported (Krumbholz et al., 2002) . The viral genome is single-stranded, positivesensed RNA and consisted of the 5′ untranslated regions (5′ UTR), one open reading frame (ORF), 3′ UTR and the 3′ end poly(A) sequence. In infected cells, the polyprotein is translated from the ORF and then processed to 4 structural proteins (VP1, VP2, VP3, VP4) and 7 nonstructural proteins (2Apro, 2B, 2C, 3A, 3B, 3Cpro, 3Dpol) via viral proteinases, 2A and 3CD.

By using a metagenomics approach, we have detected the genomes of EV-G1, 2, 3, 4, 6, 9, 10 and a new type of EV-Gs in feces of pigs with or without diarrhea in Japan (Tsuchiaka et al., 2017) . Among them, 16 sequences of EV-G1 and one isolate of EV-G2 show an insertion of a papain-like cysteine protease (PLCP) gene from porcine torovirus at the 2C-3A junction sites (Tsuchiaka et al., 2017) (Fig. 1) . We call them type 1 recombinant EV-G in this study. Our previous data demonstrated high prevalence of type 1 recombinant EV-G in the EV-G population (Tsuchiaka et al., 2018) prevalence of type 1 recombinant EV-G in the EV-G population (Tsuchiaka et al., 2018) . Type 1 recombinant EV-Gs have been discovered from feces of neonatal pigs showing clinical symptoms, such as diarrhea, in the US, Belgium, Germany and South Korea (Bunke et al., 2018; Conceição-Lee and Lee, 2019; Neto et al., 2017; Knutson et al., 2017; Shang et al., 2017) , while type 1 recombinant EV-Gs identified in Japan are detected from normal as well as diarrhea pig feces (Tsuchiaka 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.

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. , 2018) . In addition to type 1 recombinant EV-G, second type of recombinant EV-G (we call type 2 recombinant EV-G in this study), which carried the PLCP gene in place of viral structural genes, has been identified in a Chinese pig farm (Wang et al., 2018) . The PLCP gene is encoded in the ORF1 of the genome of torovirus, a member of family Coronaviridae, and the order Nidovirales (Conceição-Neto et al., 2017; Knutson et al., 2017; Shang et al., 2017; Tsuchiaka et al., 2017; Wang et al., 2018) . The PLCP of nidoviruses serves as a protease to cleave viral gene 1 polyproteins to mature proteins (Mielech et al., 2014) . It also has functions of deubiquitinating and deISGylating (interferonstimulated gene15-removing) enzymes, which plays important roles in viral pathogenesis by acting as an innate immunity antagonist (Shang et al., 2017) .

In the present study, we identified a novel type 2 recombinant EV-G in pig feces in Japan. In contrast to type 2 recombinant EV-G detected with short flanking genes in the Chinese pig farm (Wang et al., 2018) , the novel type 2 recombinant EV-G had an insertion of PLCP with long flanking genes, one of which had a region showing high homology with a baculovirus gene having anti-apoptotic function.

Our previous metagenomics analyses of RNAs extracted from pig feces obtained from Japanese pig farms have identified various types of EV-Gs and type 1 recombinant EV-G (Tsuchiaka et al., 2018) . We performed further analyses using the same metagenomics data and RNA samples. Subsequent metagenomics analysis of the RNAs of the nondiarrheal pig feces obtained from a pig farm in Tottori prefecture, Japan, led to discovery of a novel type 2 recombinant EV-G genome (EVG/Porcine/JPN/MoI2-1-2/2015: GenBank with accession number LC316774). This fecal sample also contains type 1 recombinant EV-G (EVG/Porcine/JPN/MoI2-1-1/2015/G1-Type 1) (Tsuchiaka et al., 2018) . Imai, et al. Infection, Genetics and Evolution 75 (2019) 103975 The metagenomic reads were assembled without gaps, revealing the presence of full-length type 1 and type 2 recombinant EV-G RNAs in the pig fecal sample (Supple Fig. 1A and B) . We performed long RT-PCR to amplify the viral genome approximately every 1 kb with overlapping regions. Overlapping PCR products were successfully obtained and direct sequencing analysis of the gel-purified PCR products showed that the nucleotide sequences of the PCR products were the same as those obtained in metagenomics analysis (Fig. 2) . By the results of conventional RT-PCR (data not shown) and SYBR-green real-time RT-PCR using primers in Table 1 (average Ct-value 25.0066 for type 1, 26.5738 for type 2), we also found that the fecal sample contained similar amounts of the type 1 and type 2 recombinant viruses, which implying that type 2 recombinant EV-G was not a minor population compared with type 1 recombinant EV-G in the sample. These results strongly suggested that virus particles carrying type 2 recombinant genome indeed existed in the fecal sample and our data were not an artifact of metagenomics analysis.

The new type 2 recombinant EV-G genome was approximately 6.7 kb-long and had the following gene order: 5′ UTR -unique region 1 -PLCP -unique region 2 -2A -2B -2C -3A -3B -3C -3D -3′ UTR (Fig. 1) . The new type 2 recombinant EV-G genome carried a single ORF, suggesting that translation of the viral polyprotein starts at the translational start codon (ATG) at the 5′ end of the unique region 1 and ends at the stop codon (TAA) of 3D gene. In contrast to the reported type 2 recombinant EV-G genome in China, which carries the PLCP gene of pig torovirus in place of viral structural genes (Wang et al., 2018) , the newly identified type 2 recombinant EV-G carried the PLCP gene from pig torovirus plus~0.3 kb-long upstream and~0.9 kb-long downstream flanking sequences (Fig. 3A ). In addition, the newly identified type 2 recombinant EV-G lacked 11 amino acids at the Nterminal of 2A protein. We noted that the same 11 amino acids are also deleted in type 2 recombinant EV-G detected in the pig farm in China (Wang et al., 2018) . In contrast, the N-terminal truncation of the 2A proteinase gene did not occur in type 1 recombinant and non-recombinant EV-Gs, implying that the truncated 2A may be a specific characteristic feature among type 2 recombinant EV-Gs. Furthermore, type 2 recombinant EV-Gs lacked GxCG motif in 2A, which is considered to form part of the active site of the protease and is conserved among enteroviruses (Gorbalenya et al., 1989) . In contrast all type 1 recombinant EV-Gs carry this motif in 2A (data not shown). An internal region (amino acids 180-240) of the 304 amino acid-long unique region 2 of the newly identified type 2 recombinant EV-G showed high amino acid homology with genes of baculoviral inhibitor of apoptosis repeat (BIR) superfamily by a multiple sequence alignment using Clustal W (Thompson et al., 1994) in MEGA 7 (Kumar et al., 2016) (Fig. 3B) . In contrast, unique region 1 and other regions in unique region 2 showed no significant amino acid homologies with any known proteins.

To clarify the close relationship between type 1 and type 2 R. Imai, et al. Infection, Genetics and Evolution 75 (2019) 103975 recombinant EV-Gs, Nucleotide sequences were first subjected to a multiple sequence alignment using Clustal W (Thompson et al., 1994) in Mega 7 (Kumar et al., 2016) , and then, evolutionary distance was estimated with Tamura-Nei model (PLCP) (Tamura and Nei, 1993) and General Time Reversible model (2A, 2B, 2C, 3C, 3D) (Nei and Kumar, 2000) . Phylogenetic trees were generated by the maximum-likehood method (T92 + G model or GTR + G + I model) and analyzed with 1000 replicates for bootstrap using MEGA 7. Phylogenetic analysis showed that 2A, 2B, 2C, 3C, 3D genes of the newly identified type 2 EV-G made different clusters from known non-recombination EV-Gs and type 1 recombinant EV-Gs ( Fig. 4A to E), including type 1 recombinant EV-G, which co-existed with the new type 2 recombinant type 2 in the same feces; type 1 (shown in blue) and type 2 (shown in red) were found in the same feces collected in this study. Interestingly, PLCP of type 1 and type 2 recombinant EV-Gs belong to same cluster (Fig. 4F) . These data suggest that the type 2 recombinant EV-G was not directly derived from known type 1 recombinant EV-Gs nor non-recombinant EV-Gs, and the origin of PLCP of each type was same torovirus. We also noted that the new type 2 recombinant EV-G had the 247 nt-long 3′ UTR (Fig. 1) , which was substantially longer than 40 to 165 nt-long 3′ UTR of most of picornaviruses, supporting the notion that the new type 2 recombinant EV-G has evolved independently from type 1 recombinant EV-Gs or non-recombinant EV-Gs.

In the present study, we reported the genome of a novel type 2 recombinant EV-G from pig feces in Japan. The newly identified type 2 recombinant EV-G lacked the genes for structural proteins, while it carried most of the genes encoding viral nonstructural proteins. Accordingly, this defective recombinant EV-G required helper virus, which should provide viral structural proteins for dissemination, and underwent RNA replication in the absence of helper virus. Because the type 1 recombinant EV-G was detected in the same feces sample as the new type 2 recombinant EV-G, this type 1 recombinant EV-G, which belongs to different subtype, might have served as the helper virus. Otherwise, in this study, we might detect genome of type 2 recombinant EV-G in the dead host cells, not in the viral particles. In this case, it is possibility that toroviruses infected persistently in the porcine cells which facilitates type 2 recombination frequently with circulating EV-Gs and genome of type 2 recombinant EV-Gs was not packaged into viral particle.

Some RNA viruses acquire a new function, e.g., inhibition of the host immune functions, by gaining a new gene via RNA-RNA recombination (Simon-Loriere and Holmes, 2011). Enteroviruses are genetically and antigenically highly variable due to recombination within as well as between serotypes (Perez-Losada et al., 2015; Simmonds and Welch, 2006) . Poliovirus and coxsackievirus undergo RNA recombination, with higher efficiency for non-homologous RNA recombination than homologous RNA recombination, in cell cultures, suggesting that non-homologous RNA recombination may be a transient and intermediate step for the generation and selection of the fittest homologous recombinants (Lukashev, 2010) . Identification of two different types of recombinant EV-G implied that non-homologous recombination between picornavirus RNA and non-picornavirus genes drives evolution of R. Imai, et al. Infection, Genetics and Evolution 75 (2019) 103975 picornaviruses.

Although both type 1 and type 2 recombinant EV-Gs carry the PLCP gene derived from torovirus, location of the PLCP gene in the genome of the two recombinant EV-Gs differ (see Fig. 1A ). As PLCP is known to have deISGylation activity (Shang et al., 2017) , retention of torovirus PLCP gene in the two different recombinant EV-G types imply that the PLCP gene may suppress host innate immune functions, facilitating survival of these recombinant EV-Gs. The cluster of PLCP was same between recombinant EV-G type 1 and the newly identified recombinant EV-G type 2 (Fig. 4F ), suggesting that these two different types of EV-Gs acquired the PLCP by RNA recombination from same subtypes of toroviruses.

Unlike most of recombinant EV-Gs, the newly identified type 2 recombinant EV-G carried the unique region 2, which had a domain showing extensive amino acid homology with the BIR superfamily. The baculoviral inhibitor of apoptosis (IAP) protein facilitates viral replication by preventing apoptosis (Clem and Miller, 1994; Holmblat et al., 2014) . Generally, IAP proteins contain one to three BIR domains, which play an important role in the anti-apoptotic function. IAPs have a RING domain, which relates E3 ubiquitin ligase activity for ubiquitination of target proteins degradation, at the C-terminal region (Crook and Clem, 1993; Morizane et al., 2005; Ni et al., 2015; Vaux and Silke, 2005) . Possibly, the BIR-like domain and the PLCP gene inhibited apoptosis and host innate immune function, respectively, leading to efficient replication of the newly identified type 2 recombinant EV-G.

One unanswered question in this study was whether the protein region translated from the non-picornaviral genes of the newly identified type 2 recombinant EV-G undergoes protein processing. Coronavirus PLCP cleaves the viral gene 1 polyproteins through recognition of a LXGG motif, while PLCP of arterivirus, another nidovirus, recognizes sequence LIGG, TTGG or PSGG (Mielech et al., 2014) . However, cleavage specificity of torovirus PLCP has not need identified. Accordingly, it is unclear whether the PLCP in the newly identified type 2 recombinant EV-G cleaves any regions in the protein region translated from the inserted foreign genes. Enterovirus 2A and 3CD cleave Y/G pair and Q/G pair, respectively (Earle et al., 1988) , while these pairs were absent at junctions at unique region 1/PLCP, PLCP/unique region 2 and unique region2/truncated 2A as well as within the putative polyprotein translated from the non-picornavirus sequences. Moreover, the absence of GxCG motif conserved in chymotrypsin-like protease in 2A of type 2 recombinant EV-Gs suggests that the 2A proteinase function of the type 2 recombinant EV-G 2A was most probably defective. As enterovirus 2A induces cleavage in host proteins, absence of biologically active 2A potentially affect host environment, including translational status, and possibly affect viral gene expression. Taken together, it is unlikely that the protein region translated from the inserted foreign genes undergo processing by the truncated 2A and 3CD. If the torovirus PLCP does not induce a cleavage(s) into the polyprotein of the newly identified type 2 recombinant EV-G, a protein consisted of unique region 1, PLPC, unique region 2 and N-terminal truncated 2A, R. Imai, et al. Infection, Genetics and Evolution 75 (2019) 103975 might have been accumulated in cells infected with the newly identified type 2 recombinant EV-G. If so, testing the deISGylation function and anti-apoptotic function of this putative protein would provide a clue as to a possible reason(s) for retention of these nonpicornavirus sequences in the type 2 recombinant EV-G. Alternatively, the putative polyprotein with the torovirus PLCP and the BIR domain may have another biological function that is important for replication of the newly identified type 2 recombinant EV-G.

First identification and characterization of porcine enterovirus G in the United States

High genetic diversity of porcine diversity of porcine enterovirus G in Schleswig-Holstein

Control of programmed cell death by the baculovirus genes p35 and IAP

Identification of an enterovirus recombinant with a torovirus-like gene insertion during a diarrhea outbreak in fattening pigs

An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif

The complete nucleotide sequence of a bovine enterovirus

Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. a distinct protein superfamily with a common structural fold

Nonhomologous recombination between defective poliovirus and coxsackievirus genomes suggests a new model of genetic plasticity for picornaviruses

A porcine enterovirus G associated with enteric disease contains a novel papain-like cysteine protease

Sequencing of porcine enterovirus group II and III reveals unique features of both virus groups

MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets

First detection of novel enterovirus G recombining a torovirus papain-like protease gene associated with diarrhea in swine in South Korea

Recombination among picornaviruses

Nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deISGylating activities

X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO

Molecular Evolution and Phylogenetics

The ubiquitin ligase ability of IAPs regulates apoptosis

Recombination in viruses: mechanisms, methods of study, and evolutionary consequences

A naturally occurring recombinant enterovirus expresses a torovirus deubiquitinase

Frequency and dynamics of recombination within different species of human enteroviruses

Why do RNA viruses recombine?

Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees

Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice

Identification of a novel bovine enterovirus possessing highly divergent amino acid sequences in capsid protein

Genetic diversity and recombination of enterovirus G strains in Japanese pigs: high prevalence of strains carrying a papain-like cysteine protease sequence in the enterovirus G population

IAPs, RINGs and ubiquitylation

Full-length and defective enterovirus G genomes with distinct torovirus protease insertions are highly prevalent on a Chinese pig farm

ICTV report C. ICTV virus taxonomy profile: picornaviridae

For type 1 amplification: type 1 Lf and type 1 Lr. For type 2 amplification: type 2 Lf and type 2 Lr.

This study is supported by funding including Japan Society for the promotion of science KAKENHI 15K07718, and operating cost from Global Innovation Research of Tokyo University of Agriculture and Technology. We thank Dr. Jayne K. Makino for proofreading of the manuscript.

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.meegid.2019.103975.