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J Virol. 2017 July 15; 91(14): e00450-17.
Published online 2017 June 26. Prepublished online 2017 May 10. doi:  10.1128/JVI.00450-17
PMCID: PMC5487566

A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase

Stanley Perlman, Editor
Stanley Perlman, University of Iowa;


Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3Cpro cleavage sites at the 5′ and 3′ ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-β expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event.

IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we identified a special case of cross-order recombination between enterovirus G (order Picornavirales) and torovirus (order Nidovirales). This naturally occurring recombination event may have broad implications for other picornaviral and/or nidoviral species. Importantly, we demonstrated that the exogenous ToV-PLP gene that was inserted into the EVG genome encodes a deubiquitinase/deISGylase and potentially suppresses host cellular innate immune responses. Our results provide insights into how a gain of function through genetic recombination, in particular cross-order recombination, may improve the ability of a virus to evade host immunity.

KEYWORDS: enterovirus G, torovirus, genetic recombination, papain-like protease, deubiquitinase


Enteroviruses (EVs) belong to the order Picornavirales, family Picornaviridae (1). The EV genus includes viruses that infect humans (species A to D), bovines (species E and F), swine (species G), and nonhuman primates (species A, B, D, H, and J). Currently, EV species G (EVG) is divided into 11 types, EV-G1 to EV-G11 (2,4). Enteroviruses are small nonenveloped viruses that contain positive-strand RNA genomes of approximately 7,400 to 7,500 nucleotides (nt). Enteroviral genomes contain a single open reading frame (ORF), flanked by 5′ and 3′ untranslated regions (UTR) and a 3′ poly(A) tail. The relatively long 5′ UTR extends about 700 to 825 nt and contains secondary structural elements essential for RNA replication as well as an internal ribosomal entry site (IRES) for the initiation of translation. The 3′ UTR is considerably shorter, at 75 to 100 nt long, and contains complex cis-acting elements that are important for RNA replication (5). Genome translation generates a large polyprotein that is proteolytically processed into four structural proteins (VP1, VP2, VP3, and VP4) and seven nonstructural viral proteins (2Apro, 2B, 2C, 3A, 3B, 3Cpro, and 3Dpol) (6,8).

Enteroviruses comprise a highly diverse group of viruses characterized by high mutation and recombination rates (9,13). Recombination is considered to be a major factor that drives viral evolution (14, 15). The majority of reported recombination events involve members of the same species. Recombination usually occurs in the nonstructural genome region, most frequently in the 2A-2C portion of the enterovirus genome (16, 17). Intratypic recombinants are produced about 100 times more often than are intertypic recombinants (18). Genetic recombination between viruses from two different families/orders seems to occur much less frequently. However, in this study, we discovered a novel example of a cross-order recombinant in which an exogenous papain-like protease (PLP) gene of torovirus (order Nidovirales) naturally recombined into the 2C/3A junction of the genome of enterovirus G (order Picornavirales), generating a chimeric virus designated EVG 08/NC_USA/2015.

Torovirus belongs to the family Coronaviridae in the order Nidovirales. In previous studies, PLPs derived from nidoviruses have been reported to possess deubiquitination (DUB) activity, a mechanism to disrupt innate immune signaling and suppress host immune responses (19,29). Many key signaling proteins that regulate the expression of host cellular immune genes are dynamically modulated by protein posttranslational modifications. In particular, the covalent conjugation of ubiquitin (Ub) or Ub-like proteins, such as interferon (IFN)-stimulated gene 15 (ISG15), is a critical mechanism for the orchestration of appropriate innate immune responses (30,33). Ub conjugation (ubiquitination) and ISG15 conjugation (ISGylation) to cellular substrate proteins occur through similar mechanisms (33,35). Together with other posttranslational modifications, they fine-tune the activation, strength, and duration of the antiviral immune responses (30, 32, 33, 35,37). PLPs from several viral species of the Nidovirales function as deubiquitinases that cleave and remove Ub and Ub-like modifiers from host cell substrates (21, 24, 26, 27, 38, 39). Such PLPs possess general DUB activity toward cellular ubiquitin conjugates and also cleave the IFN-induced Ub homologue ISG15 (deISGylation) from cellular proteins.

In this study, we characterized the novel recombinant virus EVG 08/NC_USA/2015. We elucidated the DUB function of the EVG ToV-PLP protein and investigated its effect on host cell innate immune responses. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus and analyzed its ability to induce innate immune responses in infected cells. Our study reveals a novel cross-order recombination event and provides new insights into enterovirus genome plasticity and its influence on viral pathogenesis.


Identification of a naturally occurring recombinant virus, EVG 08/NC_USA/2015.

In 2015, fecal samples from neonatal pigs with diarrhea symptoms were submitted to the Kansas State Veterinary Diagnostic Laboratory (KSVDL) for diagnostic testing. Inoculation of a 0.2-μm filtered fecal slurry onto a monolayer of swine testicular (ST) cells yielded cytopathic effects in ~48 h. By using metagenomic sequencing, a novel enterovirus G genome was assembled, which contained an ~600-nt foreign gene insertion within the 2C/3A region. We observed a mixed population of foreign gene insertions by next-generation sequencing (NGS) analysis, in which two different lengths, 582 nt and 648 nt, were observed. In comparison to the 582-nt foreign gene sequence, the 648-nt insertion contains an additional 66 nt at the C terminus (data not shown). BLAST search results showed that this 66-nt sequence has little or no homology to any other sequences in GenBank. This virus was subsequently purified by using a plaque assay. Nine large plaques were selected for sequencing analysis of the foreign gene insertion region. The recombinant viruses obtained from these 9 plaques contained only the 582-nt insertion, which was determined to be stable in 10 passages in ST cells. We also directly passaged the original sample (without plaque purification) on ST cells for 10 passages and sequenced the foreign gene insertion region. Quantitative reverse transcription-PCR (RT-PCR) and sequencing results showed that about 60% of the population contained the 648-nt insertion and that about 40% of the population contained the 582-nt insertion at passage 2. However, only about 20% of the population contained the 648-nt insertion at passage 3, while no 648-nt insertion was detected at passage 4 (data not shown). These results indicate that the C-terminal 66 nt of the 648-nt insertion is not stable and may be lost through adaptation of the recombinant virus in the host cells.

One of the largest plaques, EVG 08/NC_USA/2015, was isolated for complete genome characterization. The full-length genome of EVG 08/NC_USA/2015 is 7,981 nt long, excluding the poly(A) tail. A long ORF of 7,098 nt, encoding a 2,365-amino-acid (aa) polyprotein precursor, is flanked by an 812-nt 5′ UTR and a 71-nt 3′ UTR. The P1, P2, and P3 regions of EVG 08/NC_USA/2015 contain 2,514 nt (838 aa), 1,734 nt (538 aa), and 2,265 nt (755 aa), respectively (Fig. 1). The full-length genome sequences of EVG 08/NC_USA/2015 show 75.4% nucleotide identity to the sequence of prototypic EVG strain PEV9 UKG/410/73 (GenBank accession no. Y14459.1) (Table 1). Phylogenetic analysis shows that the novel recombinant virus EVG 08/NC_USA/2015 is most closely related to a group of EVGs detected in 2012 in pigs in the ThanhBinh and CaoLanh areas of Vietnam (Fig. 2).

Schematic diagram of the genome organization of EVG 08/NC_USA/2015. The single ORF is flanked by a long 5′ UTR (812 nucleotides) and a short 3′ UTR (72 nucleotides), followed by a poly(A) tail. Secondary structural elements in the 5′ ...
Genome comparison of EVG 08/NC_USA/2015 and a prototype EVG strain (PEV9 UKG/410/73)c
Phylogenetic analysis of enterovirus full-length genome nucleotide sequences. The phylogenetic tree was constructed by the maximum likelihood method using the best-fitting general time-reversible model with a gamma distribution. The numbers on branches ...

Full-length genome sequencing of EVG 08/NC_USA/2015 identified a 582-nt-long foreign gene insertion at the 2C/3A junction of the enterovirus genome (Fig. 1). BLAST search results showed that this foreign gene is most homologous to the PLP gene located in the nsp3-like region of the torovirus genome (54 to 68% amino acid identity). Phylogenetic analysis further showed that this foreign PLP forms a well-supported clade with PLPs of other toroviruses, including bovine, porcine, and equine toroviruses (Fig. 3) (40, 41). We designated this foreign gene ToV-PLP. In the genome of EVG 08/NC_USA/2015, the recombinant ToV-PLP gene is flanked by two predicted 3Cpro cleavage sites, ALFQ|GPPVFR and AEFQ|GPPTFK, at its 5′ and 3′ ends, respectively (Fig. 1). This suggests that ToV-PLP may be cleaved at both ends, minimizing its potential influence on proteolytic processing and maturation of enteroviral 2C and 3A proteins.

Phylogenetic analysis of coronaviral and picornaviral papain-like proteases. The phylogenetic tree was constructed by the maximum likelihood method using the best-fitting algorithm Whelan-and-Goldman model. The numbers on branches are bootstrap values ...

Recombinant EVG ToV-PLP functions as a deubiquitinase and deISGylase to suppress host cell innate immune responses.

To obtain insights into potential functions of ToV-PLP within the context of EVG 08/NC_USA/2015, we explored the structural homology between this PLP and other viral proteases. Iterative modeling using I-TASSER (42) pinpointed sequence and structural similarities between EVG ToV-PLP and the leader protease of foot-and-mouth disease virus (FMDV-Lpro) (Fig. 4 and and5),5), although EVG ToV-PLP and FMDV-Lpro have limited homology at the sequence level (24.3% amino acid identity) (Fig. 3). A previous study pointed out sequence similarities between FMDV-Lpro and another toroviral PLP, that of Breda-1 bovine torovirus (43). Like FMDV-Lpro, EVG ToV-PLP appears to adopt a minimal papain-like fold with a characteristic arrangement of its Cys-His-Asp catalytic triad (44, 45). The Cys residue is located near the N-terminal cap of the first helix of the primarily helical N-terminal subdomain of the core domain, while the His and Asp residues are located near each other within the beta-stranded subdomain in the C-terminal half of the core domain (Fig. 5). EVG ToV-PLP also exhibits structural homology to portions of the catalytic domains of several eukaryotic deubiquitinating enzymes and ubiquitin proteases, including USP2, USP4, USP14, USP18, and USP46. Furthermore, ToV-PLP exhibits various levels of sequence and structural homology to the N- and C-terminal subdomains of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) PLPs, which are similar to those of mammalian deubiquitinating enzymes (Fig. 4 and and5).5). A major distinction is that the minimal protease domains of ToV-PLP (and FMDV-Lpro) lack the primarily β-stranded “finger domains” that bridge the N- and C-terminal subdomains, e.g., “thumb and palm domains,” in the catalytic folds of coronaviral proteases and eukaryotic deubiquitinases (46). Moreover, EVG ToV-PLP and FMDV-Lpro are structurally distinct from the PLPs/Otubain-like deubiquitinases of selected nidoviruses and arteriviruses. The latter deubiquitinases also utilize Cys-His-Asp/Asn catalytic triads but have different catalytic residue arrangements and overall topologies (38). Intriguingly, FMDV-Lpro possesses deubiquitinating activity against innate immune signaling components, including RIG-I, TRAF6, and TBK1 (47). PLPs from the SARS and MERS coronaviruses also possess DUB as well as deISGylating (ISG15-removing) activities (21, 23, 25, 48).

Amino acid sequence alignment of the EVG ToV-PLP protein with other known papain-like proteases. Shown is structure- and sequence-guided alignment of EVG ToV-PLP with the corresponding catalytic domains of the FMDV leader protease, coronaviral PLPs, and ...
Structural modeling and comparison of the EVG ToV-PLP protein with other known papain-like proteases. Structures of the homology model of the core ToV-PLP catalytic domain were compared with those of the catalytic domains of representative viral proteases ...

To determine whether the torovirus-derived PLP domain of EVG 08/NC_USA/2015 also possesses DUB and/or deISGylation activities, we carried out cell-based DUB and deISGylation assays. HEK-293T cells were transfected with a plasmid expressing hemagglutinin (HA)-tagged ubiquitin (HA-Ub) and a plasmid expressing wild-type ToV-PLP or a catalytic-site mutant of ToV-PLP (mutations introduced into the putative catalytic triad, C1449/H1557/D1572 to A1449/A1557/A1572). An empty plasmid vector and a plasmid expressing the PLP2 domain of porcine reproductive and respiratory syndrome virus (PRRSV) were used as controls (26, 27). The expression of each PLP and HA-Ub in transfected cells was confirmed by Western blotting (Fig. 6A, bottom). In comparison to cells transfected with the empty plasmid, the expression of wild-type PLP from either ToV or PRRSV reduced the levels of ubiquitin-conjugated proteins (Fig. 6A, top, compare lanes 2 and 4 with lane 6), indicating that EVG ToV-PLP antagonizes the ubiquitination process of host cellular proteins. As we expected, the DUB activities of both ToV-PLP and PRRSV-PLP2 were abolished by mutations of the putative catalytic-triad residues. Next, we investigated the effect of ToV-PLP expression on host protein ISGylation. ISG15 conjugates were generated by transfecting HEK-293T cells with plasmids that express Flag-tagged ISG15 and ISG15-specific E1, E2, and E3 enzymes. Cells were cotransfected with the ToV-PLP expression vector. Once more, we used PRRSV-PLP2, which also possesses deISGylating activity, as a positive control (27). The coexpression of wild-type PLPs resulted in a clear decrease in the levels of ISGylated proteins (Fig. 6B, compare lanes 2 and 4 with lane 6). The deISGylation activities of the PLPs were abolished by mutations of the putative catalytic residues. This result confirmed that the ToV-PLP domain possesses deISGylation activity.

Deubiquitination and deISGylation activities of the recombinant ToV-PLP protease. (A and B) Effect of PLP expression on Ub or ISG15 conjugation. (A) HEK-293T cells were cotransfected with plasmid DNAs expressing HA-Ub and FLAG-tagged ToV-PLP or PRRSV-PLP2 ...

To further verify that the ToV-PLP domain directly targets the conjugation of the polyubiquitin chain, we carried out in vitro ubiquitin deconjugation assays using recombinant ToV-PLP purified from Escherichia coli. K48-, K63-, or M1 (linear)-linked polyubiquitin chains were incubated with serial dilutions of recombinant PLPs (Fig. 6C to toE).E). Both K48- and K63-linked polyubiquitin substrates are efficiently cleaved to monomeric ubiquitin by recombinant ToV-PLP. Interestingly, ToV-PLP also cleaves linear polyubiquitin (M1), a modification involved in NF-κB activation, among other innate immune pathways (31). In contrast, the polyubiquitin cleavage activity was totally abolished by mutations of the putative catalytic triad (Fig. 6C to toEE).

To confirm the deISGylation activity of this recombinant PLP, we carried out an in vitro proteolytic assay using an ISG15 precursor (proISG15) as a cleavage substrate (22, 49). Reactions were terminated at specific time points through the time course of the assay. Wild-type ToV-PLP cleaves the majority of proISG15 into mature forms in 10 min (Fig. 6F). In contrast, a ToV-PLP mutant that contains mutations in the putative catalytic triad exhibits no deISGylation activity (Fig. 6F).

Since ubiquitination affects innate immune gene signaling pathways (35,37), we investigated whether ToV-PLP also influences innate immune gene expression in a host cell context. We first carried out luciferase reporter assays using a luciferase reporter plasmid (p125-Luc) that expresses firefly luciferase under the control of the IFN-β promoter. HEK-293T cells were cotransfected with plasmids that express wild-type or mutant ToV-PLP, p125-Luc, and Renilla luciferase (pRL-SV40) to normalize the expression levels of the samples. As a positive control, we cotransfected cells with the reporter plasmid along with a plasmid containing the nidoviral Otubain-like deubiquitinase PRRSV-PLP2. At 24 h posttransfection, cells were infected with Sendai virus (SeV) to induce luciferase production. As we expected, the expression of PRRSV-PLP2 significantly inhibited luciferase gene expression. In contrast, a strong reporter signal was observed in cells transfected with the empty plasmid after SeV infection. Cells expressing ToV-PLP exhibited a 12-fold reduction of the IFN-β promoter-driven luciferase reporter signal (Fig. 7A). Quantitative RT-PCR results also showed that the IFN-β mRNA expression level was significantly decreased by 3-fold in ToV-PLP-transfected cells (Fig. 7B), while mutations introduced into catalytic sites of the protease increased reporter gene and IFN-β mRNA expression levels (Fig. 7A and andB).B). These data indicate that the ToV-PLP protein may function as an innate immune antagonist through its DUB/deISGylation activities.

Effect of ToV-PLP expression on IFN-β expression. (A) ToV-PLP affects reporter gene expression derived by the IFN-β promoter. HEK-293T cells were transfected with a plasmid expressing ToV-PLP, the PRRSV-PLP2 domain, or the catalytic-site ...

Growth of the ToV-PLP knockout recombinant enterovirus is attenuated in cell culture.

To explore the potential contribution of torovirus PLP recombination to the growth and fitness of enterovirus, we generated a full-length cDNA infectious clone of EVG 08/NC_USA/2015 (pEVG) (Fig. 8A). The pEVG construct contains a cytomegalovirus (CMV) promoter at the 5′ terminus of the viral genome, the 7,981-nucleotide full-length genome of EVG 08/NC_USA/2015, and a 20-nt poly(A) tail incorporated at the 3′ end of the genome (Fig. 8A). Compared to the genome sequence of the parental virus, the DNA sequence of pEVG contained 3-nt differences at the following genome positions: T14, G2029, and A7724. The T14-to-C mutation is located within the 5′ UTR, the A7724-to-G mutation is a synonymous mutation, and the G2029-to-T mutation changed the amino acid sequence from glycine to valine. To rescue the cloned virus, plasmid DNA of pEVG was transfected into BHK-21 cells, and the cell culture supernatant from the transfected cells was passaged onto ST cells at 48 h posttransfection. At 18 h postinfection (hpi), infected ST cells were stained by using PLP and VP1 protein-specific monoclonal antibodies (MAbs) (Fig. 8C). These results indicate that viable cloned virus (vEVG) was recovered from the full-length cDNA infectious clone pEVG. Sequence analysis confirmed that the vEVG genome contains C14, T2029, and G7724 mutations, which differentiate vEVG from wild-type EVG 16-08.

Construction of the EVG 08/NC_USA/2015 infectious clone and rescue of recombinant viruses. (A) Strategy for assembling the full-length cDNA infectious clone of EVG 08/NC_USA/2015. Two RT-PCR-amplified genomic fragments and a synthesized HDV ribozyme gene ...

By using the pEVG infectious clone, a PLP gene knockout mutant (vPLP-KO) was generated (Fig. 8B). Growth kinetics of the parental virus EVG 08/NC_USA/2015, the cloned virus (vEVG), and the PLP knockout mutant (vPLP-KO) were compared. Cells were infected with each of the viruses and harvested at 0, 2, 4, 6, 8, 10, and 12 hpi. These results showed that vEVG exhibits growth kinetics similar to those of the parental virus EVG 08/NC_USA/2015 (Fig. 9). Both the parental virus and the cloned virus reached titers of 103.2 focus-forming units (FFU)/ml and 103.4 FFU/ml at 12 hpi, respectively. In contrast, vPLP-KO was impaired in growth in ST cells, with an ~1-log-lower virus titer at 12 hpi (Fig. 9).

Characterization of in vitro growth properties of recombinant EVGs rescued from full-length cDNA infectious clones. Confluent ST cells were initially inoculated with the recombinant virus at an MOI of 0.01, and the cell culture supernatant was serially ...

The ToV-PLP knockout recombinant enterovirus shows a reduced ability to inhibit innate immune gene expression in infected cells.

To determine whether the knockout of ToV-PLP affects the innate immune suppression ability of the virus, we analyzed DUB and deISGylation activities of wild-type and mutant ToV-PLP knockout viruses. Compared to uninfected cells (Fig. 10A, lane 3), the amount of Ub-conjugated proteins was decreased by about 95% in both EVG 08/NC_USA/2015- and vEVG-infected cells (Fig. 10A, lanes 4 and 5). In contrast, levels of Ub-conjugated proteins were elevated 8.7-fold in vPLP-KO-infected cells, compared to cells infected with the cloned virus vEVG (Fig. 10A, lanes 5 and 6). In addition, the amount of ISG15-conjugated proteins in cells infected with vPLP-KO was elevated 47-fold compared to that in cells infected with EVG 08/NC_USA/2015 or vEVG (Fig. 10B, lanes 4 to 6). We observed no significant difference in the DUB/deISGylation abilities of parental and cloned viruses; similar levels of Ub/ISG15-conjugated host proteins were detected in cells infected with EVG 08/NC_USA/2015 and vEVG (Fig. 10).

FIG 10
Effect of ToV-PLP deletion on the deubiquitination and deISGylation abilities of enterovirus. (A) HEK-293T cells were transfected with plasmid DNAs expressing HA-Ub and then infected with the parental virus EVG 08/NC_USA/2015, the cloned virus vEVG, or ...

To extend these observations, we investigated the effect of the PLP knockout on innate immune gene expression in infected cells. ST cells were infected with EVG 08/NC_USA/2015, vEVG, or vPLP-KO, and innate immune gene expression was analyzed at 10 hpi. As expected, similar levels of IFN-β, interleukin-28B (IL-28B), and ISG15 expression were observed in cells infected with EVG 08/NC_USA/2015 and vEVG. In comparison to those in vEVG-infected cells, there were 6.6-, 5.3-, and 4.1-fold increased expression levels of type I IFN (IFN-β), type III IFN (IL-28B), and ISG15 in vPLP-KO-infected cells, respectively (Fig. 11A to toC).C). To confirm this result, we further tested innate immune gene expression levels in infected cells stimulated by SeV. EVG 08/NC_USA/2015-, vEVG-, or vPLP-KO-infected cells were stimulated with SeV at 6 hpi. An extended panel of innate immune gene expression was analyzed at 4 h poststimulation. The results consistently showed that EVG 08/NC_USA/2015 and vEVG largely suppressed the expression of both type I interferons (Fig. 11D and andE)E) and type III interferons (Fig. 11F and andG)G) as well as the expression of the selected ISGs IFN regulatory factor 7 (IRF7) (Fig. 11H) and ISG15 (Fig. 11I) to similar levels. In contrast, vPLP-KO exhibited a reduced ability to antagonize the expression of SeV-induced innate immune genes. mRNA expression levels of the type I interferons IFN-α1 and IFN-β were increased by 4-fold and 13-fold, respectively (Fig. 11D and andE),E), in vPLP-KO-infected cells compared to vEVG-infected cells. Similarly, IL-29 and IL-28B expression levels were increased by 8.3- and 12.7-fold, respectively (Fig. 11F and andG)G) in vPLP-KO-infected cells compared to vEVG-infected cells. In addition, we also observed increased expression levels of ISGs, including IRF7 (7.4-fold increase) and ISG15 (17.9-fold increase), in vPLP-KO-infected cells (Fig. 11H and andII).

FIG 11
Effect of ToV-PLP deletion on innate immune gene expression levels. ST cells were infected with the parental virus EVG 08/NC_USA/2015, the cloned virus vEVG, or the PLP knockout mutant vPLP-KO at an MOI of 3. Mock-infected cells were used as a control. ...


Recombination events potentially exert a major influence on multiple aspects of viral evolution and pathogenesis, including the emergence of new virus variants, changes of host ranges and tissue tropism, increases in virulence, evasion of host immunity, and resistance to antivirals (14, 15, 50). Enteroviruses are known for their high recombination rates, and recombination has been reported to contribute significantly to enteroviral genetic diversity (9,12). This is well illustrated by the example of a prototypic human enterovirus, poliovirus (PV). The discovery of PV recombination may date as far back as the 1960s (51). The widely used oral poliovirus vaccine (OPV) contains a cocktail of three attenuated serotypes, which facilitates intratypic recombination (52, 53). Recombinant PV can be easily detected in both healthy vaccinees and vaccine-associated paralytic poliomyelitis patients (52,56). The intertypic recombination of PV with human enterovirus C (HEV-C) species also appears to occur under natural conditions (16, 17, 52, 57). Furthermore, the highly recombinogenic nature of PV was associated with the emergence of pathogenic vaccine-derived viruses during the global polio eradication campaign (10,12). Recombination within animal species of simian, swine, and bovine enteroviruses has also been documented (58,60). A typical example is the emergence of swine vesicular disease virus (SVDV), which is genetically closely linked to coxsackievirus B5 (CV-B5). Their close phylogenetic relationship suggests that SVDV may have arisen from CV-B5 by recombination (61, 62). To our knowledge, all enterovirus recombinants that have been reported so far arose through recombination events within a species or genus. Our present study identified a unique cross-order recombination event between viruses of the orders Nidovirales (torovirus) and Picornavirales (enterovirus). Considering the strikingly different genome organizations and protein expression and processing mechanisms of viruses of these two orders, it is intriguing to understand how this rare recombination event occurred. Moreover, it is also important to understand how the enteroviral genome backbone was modified to overcome potential nucleotide- and protein-level incompatibilities in order to allow the insertion of the heterologous gene. Proposed factors that control the occurrence of recombination include local sequence homology, RNA secondary structural elements, coinfection, subcellular colocalization, and/or coreplication of the parental viruses in the same host (15, 50). At the PLP gene insertion site, the 2C/3A cleavage junction, the original 3C protease cleavage site was duplicated and flanks both the 5′ and 3′ ends of the PLP-coding sequence. This suggests that the “foreign” PLP may be cleaved off from the enterovirus polyprotein, thereby avoiding a disruption of the proteolytic processing of the polyprotein and preserving the functions of other enterovirus proteins. Such a recombination event may have occurred within a host animal that was simultaneously infected with enterovirus and torovirus. Notably, enterovirus and torovirus are frequently detected in swine hosts, and more importantly, both viruses are associated with enteric infection (2, 4, 60, 63,73). Our metagenomic sequencing uncovered no ToV-derived genetic material other than the ToV-PLP sequence in the sample from which we isolated EVG 08/NC_USA/2015. This suggests that the recombination event occurred prior to infection of the particular piglet from which the sample was obtained. Phylogenetic analysis shows that this novel virus is most closely related to EVGs detected in pigs from the ThanhBinh and CaoLanh areas of Vietnam in 2012. However, the viruses isolated from these Vietnamese pigs were not reported to contain the ToV-PLP gene insertion. The origin of this novel recombinant virus therefore needs to be explored further in the future.

We determined that the 582-nt ToV-PLP gene segment inserted into the enterovirus genome encodes an enzyme with deubiquitinase activity. Structural homology modeling and sequence analysis suggest that ToV-PLP may be grouped along with the leader proteases of FMDV and, potentially, ERAV (equine rhinitis A virus) into a novel class of structurally minimal PLPs with deubiquitinating and deISGylating activities (43, 47). The catalytic domains of these PLPs assume folds similar to the core papain fold, with few or no substantial added domains and relatively short loops connecting the main structural elements (76). This is intriguing given that toroviruses belong to the Coronaviridae, while FMDV and ERAV are members of the Picornaviridae. Notably, PLPs from coronaviruses other than toroviruses are substantially larger. Moreover, ToV-PLP and FMDV-Lpro do not contain the finger subdomain characteristic of other coronaviral PLPs/deubiquitinating enzymes.

The minimal nature of the ToV-PLP and FMDV-Lpro folds suggests that they may have descended from an ancestral papain-like fold that gained deubiquitination function. It is unclear whether this gain of function occurred separately in the Coronaviridae and Picornaviridae or represents a holdover from a more ancestral virus. It is noteworthy that FMDV-Lpro, in its capacity as a protease, recognizes basic motifs that also contain glycines/serines. In particular, FMDV-Lpro cleaves between a basic residue and glycine, with a strong preference for leucine at the P2 position (76). Such motifs are present not only in the FMDV polyprotein that is cleaved by the protease (77) but also in several host cell targets, including eukaryotic initiation factor 4G (eIF4G) and the IRES-binding protein Gemin5. Cleavage of these targets suppresses host cell protein synthesis and promotes the translation of viral gene products (78,80). In contrast, larger coronaviral PLPs, such as SARS-CoV PLP, recognize LXGG motifs specifically (81, 82); thus, the targets of their classical protease activity and deubiquitinating/deISGylating activity are equivalent, as ubiquitin and ISG15 terminate in RLRGG sequences. It is unclear whether ToV-PLP possesses protease sequence specificity similar to that of FMDV-Lpro. However, ToV-PLP may also potentiate viral replication and pathogenesis by acting on host translation factors or on antiviral pathways other than innate immunity.

Interestingly, ToV-PLP and FMDV-Lpro, but not PLpro of SARS-CoV and related CoVs, contain additional acidic residues located immediately C terminal to the catalytic Asp residues. These acidic motifs form a loop proximal to the catalytic triad and may help to establish proteolytic sequence specificity in these minimal PLPs. However, proteolytic target sequences may bind to the ToV-PLP and FMDV-Lpro active sites in a different manner than the extended, flexible C-terminal sequences of ubiquitin/ISG15 that are also cleaved by these enzymes.

In SARS-CoV PLpro and related coronaviral PLPs, the principal binding sites for the globular portions of ubiquitin and ISG15 are located at an interface between the finger and C-terminal (palm) subdomains (83). A second, distal site is formed primarily by residues on the N-terminal thumb subdomain, which are also not present in FMDV-Lpro and ToV-PLP. From the principal binding site, the flexible C-terminal tail of ubiquitin extends into the catalytic site, stabilized by an extensive hydrogen-bonding network with residues near the catalytic triad (83). The ubiquitin/ISG15 C termini may follow a path to the active site of ToV-PLP and FMDV-Lpro similar to that of SARS-CoV PLpro. However, it is not yet clear exactly where the principal and/or secondary binding surfaces for the globular domains of polyubiquitin and ISG15 are located on the minimal catalytic domains of ToV-PLP and FMDV-Lpro. Structural elucidation of complexes of these enzymes with ubiquitin and ISG15 is required to answer this question and to clarify the nature of polyubiquitin chain specificity and substrate preferences for these viral proteases.

Ubiquitination and ISGylation play important roles in the regulation of host antiviral immune responses (30,35). In previous studies, PLPs of nidoviruses were shown to disrupt innate immune signaling pathways through their deubiquitination/deISGylation activities, which have been proposed to be associated with viral pathogenicity (21,29, 39). In our study, the novel ToV-PLP robustly disassembles both K48- and K63-linked polyubiquitins as well as linear polyubiquitin. These polyubiquitins are the primary ubiquitin species that regulate specific innate immune signaling pathways. ToV-PLP overexpression in cell culture also reduced the levels of ISG15-conjugated cellular proteins, and we further confirmed its deISGylation activity using in vitro assays. The biological significance of these DUB and deISGylation activities was further supported by the fact that ToV-PLP is able to inhibit the mRNA expression of IFN-β. When we introduced a targeted deletion to knock out ToV-PLP expression, a recombinant mutant virus was rescued in cell culture, suggesting that ToV-PLP is not absolutely essential for viral replication. However, these mutants exhibited reduced DUB/deISGylation activity and enhanced the expression levels of representative innate immune genes in infected swine cells.

Like most enteroviral infections, EVG infection is generally considered to be asymptomatic, with limited evidence to support its association with clinical diseases (60, 71, 72, 84). However, in the present study, this unique enterovirus strain (EVG 08/NC_USA/2015) was isolated from a fecal sample of a piglet experiencing diarrhea. The acquisition of a foreign innate immune antagonist, ToV-PLP, may explain the pathogenicity in the natural host. Further in vivo characterization of this emerging chimeric virus is needed to fulfill Koch's postulates and evaluate the contribution of exogenous ToV-PLP in the pathogenesis of EVG in animals.


Cells and viruses.

ST, BHK-21, and HEK-293T cells were cultured in minimum essential medium (MEM) (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO), antibiotics (100 U/ml of penicillin [Gibco, Carlsbad, CA] and 100 μg/ml of streptomycin [Gibco, Carlsbad, CA]), and 0.25 μg/ml amphotericin B (Fungizone; Gibco, Carlsbad, CA) at 37°C with 5% CO2. Infected ST or HEK-293T cells were maintained in 2% horse serum (HyClone, Logan, UT) at 37°C with 5% CO2.

The EVG-positive fecal sample was obtained from a piglet diarrhea case submitted to KSVDL in 2015. EVG was initially identified by metagenomics, and the virus was subsequently isolated after inoculation into ST cells. The isolated virus was plaque purified and designated EVG 08/NC_USA/2015. The recombinant viruses vEVG (cloned virus) and vPLP-KO (PLP knockout virus) were rescued from BHK-21 cells transfected with plasmid DNA of EVG full-length cDNA infectious clones. Rescued recombinant viruses were passaged on ST cells. For both parental and recombinant viruses, passage 2 viruses on ST cells were used for subsequent experiments. The SeV Cantell strain, cultured in embryonated chicken eggs, was used to stimulate type I IFN responses. As a control, PRRSV strain SD95-21 was used for the expression of the PRRSV-PLP2 domain (26, 27).

Sequence analysis.

Fecal samples submitted to KSVDL were subjected to metagenomic sequencing as previously described (85). Sequence reads were mapped to the host Sus scrofa genome, and unmapped reads were assembled de novo by using CLC Genomics. Contigs were identified by BLASTN. A majority of the reads mapped to a 7,492-bp contig that contained a 6,561-bp open reading frame, which was incomplete at the 3′ end. The genome sequence was completed with GeneRacer (Invitrogen) and Sanger sequencing. The complete genome sequence of EVG 08/NC_USA/2015 was aligned to those of representative members of the genus Enterovirus by using the ClustalW algorithm in MEGA 7.0. Phylogeny was inferred by using the maximum likelihood algorithm, using the best-fitting model with a gamma distribution. Tree topology was assessed by using 500 bootstrap replicates.

PLP structural analysis.

Disorder prediction of the ToV-PLP sequence using GlobPlot (86) suggested that PLP is comprised primarily of a single core domain with less-ordered flanking regions. Homology modeling of EVG ToV-PLP was carried out by using I-TASSER (42), without explicit specification of any template models. Structural predictions of ToV-PLP input sequences were conducted with different boundaries in order to reduce the impact of disordered sequences at the N and C termini and to help define the core domain of the protease. The removal of ~10 N-terminal and ~20 C-terminal residues resulted in consistent predictions, with I-TASSER C scores of −0.8 to −0.9 and high structural homology to the leader protease of foot-and-mouth disease virus, with 23 to 24% sequence identity over ~87 to 89% coverage of the input sequences (44, 45). The majority of other structurally homologous templates identified by I-TASSER were eukaryotic ubiquitin-specific proteases/deubiquitinating enzymes (18 to 25% sequence identity over 78 to 94% coverage). The final structural model was further optimized, and its geometry was corrected by using ModRefiner (87). Structural figures were generated by using PyMOL (PyMOL Molecular Graphics System, version 1.7, Schrödinger, LLC).


The plasmid expressing ToV-PLP was constructed by RT-PCR amplification of the region spanning nt 5061 to 5642 of the EVG 08/NC_USA/2015 genome, while the plasmid expressing PRRSV-PLP2 was constructed by RT-PCR amplification of the region spanning nt 1340 to 2218 of the PRRSV SD95-21 genome (GenBank accession no. KC469618.1). For protein expression in mammalian cells, the PCR products were cloned into a eukaryotic expression vector, pFLAG-CMV-24 (Sigma-Aldrich, St. Louis, MO), and designated pFLAG-ToV-PLP and pFLAG-PRRSV-PLP2, respectively. For protein expression in E. coli, the PCR products were cloned into the prokaryotic expression vector pDB-His-GST-TEV (88) to yield plasmid pDB-His-GST-PLP. Specific mutations in the catalytic sites of the protease (C1449/H1557/D1572 to A1449/A1557/A1572) were introduced by overlapping extension PCR as described previously (89), and the resulting plasmid is designated pDB-His-GST-PLP-C/H/D>A. Plasmid p3xFLAG-ISG15 for the expression of FLAG-tagged ISG15 was constructed as described previously (27), while plasmid pcDNA3.1(+)-HA-Ub for the expression of HA-tagged ubiquitin was provided by Domenico Tortorella (Mount Sinai School of Medicine, NY) (26).


MAb 115-5 (anti-VP1) and MAb 129-28 (anti-PLP) were generated by immunizing BALB/c mice with VP1 and PLP recombinant proteins, respectively. Detailed experimental procedures for MAb production were described previously (74, 75). MAb 140-68 against PRRSV-PLP2 was produced in our previous study (74). The anti-FLAG M2 MAb (Sigma-Aldrich, St. Louis, MO) was used for the detection of Flag-tagged proteins. ISGylated cellular proteins were detected by anti-Flag M2 MAb (Sigma-Aldrich, St. Louis, MO) or anti-ISG15 MAb F-9 (Santa Cruz, Dallas, TX), while HA-Ub-conjugated cellular proteins were detected by anti-HA MAb 16B12 (Abcam, Cambridge, MA). Additionally, an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (PAb) (Santa Cruz, Dallas, TX) was used to detect the expression of the GAPDH housekeeping gene.

Recombinant ToV-PLP expression and purification.

His–glutathione S-transferase (GST)-tagged EVG ToV-PLP and its catalytic-site mutant were expressed in E. coli as previously described (90). Plasmid pDB-His-GST-PLP or pDB-His-GST-PLP-C/H/D>A was transformed into E. coli BL21(DE3) cells and cultured in 2× YT medium (Fisher Scientific, Pittsburgh, PA) at 37°C. Recombinant protein expression was induced overnight at 25°C with 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Recombinant proteins were purified with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Valencia, CA) under native conditions according to the manufacturer's instructions. The protein concentration was determined by the absorbance at 280 nm.

Western blotting.

Western blotting was performed by using a modified method described previously (89). Briefly, cells were lysed by using Pierce IP lysis buffer (Thermo Fisher Scientific, Carlsbad, CA) and then centrifuged at 12,000 rpm for 10 min to clean up cell debris. Cell lysates were mixed with 4× Laemmli sample buffer (Bio-Rad, Hercules, CA), denatured at 95°C for 10 min, and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred onto a nitrocellulose membrane (GE Healthcare Bio-Sciences, Pittsburgh, PA). The membrane was blocked with 5% nonfat milk in PBST (phosphate-buffered saline [PBS] supplemented with 0.05% Tween 20) at room temperature (RT) for 1 h, followed by incubation with the appropriate primary antibody at RT for 1 h. After three washes with PBST, the membrane was incubated with IRDye 800CW goat anti-mouse IgG(H+L) and/or IRDye 680RD goat anti-rabbit IgG(H+L) (Li-Cor Biosciences, Lincoln, NE) for another 45 min at RT. Specific protein bands were visualized by using an Odyssey Fc imaging system (Li-Cor Biosciences, Lincoln, NE).

Deubiquitination assays.

To determine the DUB activity of ToV-PLP in an in vitro expression system, HEK-293T cells were cotransfected with 0.5 μg plasmid DNA of pcDNA3.1(+)-HA-Ub with 0.5 μg pFLAG-ToV-PLP, pFLAG-PRRSV-PLP2, or its catalytic-site mutant. Cells cotransfected with plasmid DNA of pcDNA3.1(+)-HA-Ub and the empty vector were used as positive controls, while cells transfected with 1 μg plasmid DNA of the empty vector only were used as negative controls. At 36 h posttransfection, cell lysates were harvested and subjected to Western blot analysis. The Ub-conjugated host cellular proteins were detected by using anti-HA MAb. The expression of ToV-PLP and PRRSV-PLP2 was detected by using an anti-FLAG M2 MAb. The expression of the housekeeping gene GAPDH was detected by a PAb as a loading control.

To determine the DUB activity of ToV-PLP in the context of viral infection, HEK-293T cells were initially transfected with 0.5 μg plasmid DNA of pcDNA3.1-HA-Ub or the empty plasmid vector as the control. After 24 h posttransfection, cells were infected with wild-type EVG 08/NC_USA/2015, the cloned virus vEVG, or the ToV-PLP knockout recombinant virus at a multiplicity of infection (MOI) of 3 (see below for the construction of recombinant EVG mutants). After 10 hpi, host protein ubiquitination was detected by Western blotting as described above, while the expression of ToV-PLP was detected by anti-PLP MAb 129-28.

The polyubiquitin chain cleavage activity of ToV-PLP was determined under cell-free conditions as described previously (28, 49, 91). Briefly, 2.5 μg of K48-, K63-, or M1-linked polyubiquitin chains (Boston Biochem, Cambridge, MA) was incubated with serial dilutions (1 μg, 0.5 μg, and 0.25 μg) of purified recombinant GST-PLP or its catalytic-site mutant (GST-PLP C/H/D>A) in a final volume of 10 μl assay buffer (50 mM Tris, 5 mM MgCl2, 2 mM dithiothreitol [DTT] [pH 7.5]) for 2 h at 37°C. The reaction was terminated with 4× loading buffer (Bio-Rad, Hercules, CA) at 37°C for 20 min. Cleavage products were separated by SDS-PAGE and visualized by Coomassie brilliant blue staining.

DeISGylation assays.

To investigate the deISGylation activity of ToV-PLP in an in vitro expression system, HEK-293T cells cultured in 24-well plates were transfected with 0.5 μg of plasmid DNA expressing ToV-PLP or PRRSV-PLP2, together with 0.15 μg of pCAGGS-HA-UbE1L (E1), 0.1 μg of p3xFLAG-UbcH8 (E2), 0.25 μg of pcDNA-TAP-HA-HERC5 (E3), and 0.25 μg of p3xFLAG-ISG15 plasmid DNAs. Cells transfected with plasmid DNA of the empty vector were used as a control. At 36 h posttransfection, cell lysates were harvested and subjected to Western blot analysis. The ISG15-conjugated host cellular proteins were detected by using anti-ISG15 MAb F-9. The expression of ToV-PLP and PRRSV-PLP2 was detected by MAb 129-28 and MAb 140-68, respectively. The expression of GAPDH was detected by a specific PAb as a loading control.

To determine the deISGylation activity of ToV-PLP in the context of viral infection, HEK-293T cells were initially transfected with plasmid DNAs of ISGylation machinery (0.15 μg of pCAGGS-HA-UbE1L [E1], 0.1 μg of p3xFLAG-UbcH8 [E2], 0.25 μg of pcDNA-TAP-HA-HERC5 [E3], and 0.25 μg of p3xFLAG-ISG15). Cells transfected with plasmid DNA of the empty vector were used as a control. After 24 h posttransfection, cells were infected with wild-type EVG 08/NC_USA/2015, the cloned virus vEVG, or the vPLP-KO mutant at an MOI of 3 (see below for the construction of recombinant EVG mutants). At 48 hpi, cell lysates were harvested and subjected to Western blot analysis. Anti-Flag M2 MAb was used to visualize ISGylated host proteins, while the expression of ToV-PLP was detected by MAb 129-28.

ToV-PLP cleavage activity toward the ISG15 precursor (proISG15) substrate was determined by using a cell-free assay as described previously (22, 49). Briefly, 2.5 μg of the proISG15 (Boston Biochem, Cambridge, MA) substrate was mixed with 40 nM purified recombinant GST-PLP or GST-PLP C/H/D>A in a final volume of 10 μl assay buffer and incubated at 37°C. The catalytic reaction was stopped at different time points (1 min, 2 min, 5 min, and 10 min) by the addition of 3.5 μl of 4× Laemmli sample buffer (Bio-Rad, Hercules, CA), and the mixture was incubated at 37°C for 20 min. Proteins were separated in an SDS-polyacrylamide gradient gel (Thermo Fisher Scientific, Carlsbad, CA) and stained with Coomassie brilliant blue.

Luciferase assay.

HEK-293T cells (0.5 × 105 cells/ml) seeded into 24-well plates were cotransfected with 1 μg plasmid DNA expressing a PLP (ToV-PLP, PRRSV-PLP2, or their corresponding mutants), 0.5 μg firefly luciferase reporter plasmid p125-Luc, and 30 ng Renilla luciferase expression plasmid pRL-SV40. Cells transfected with the empty plasmid vector and p125-Luc/pRL-SV40 were used as the control. Transfection was performed by using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. At 24 h posttransfection, cells were mock treated or stimulated with SeV at 100 hemagglutination (HA) units/ml for 16 h. Subsequently, cells were lysed and analyzed for reporter gene expression by using a dual-luciferase reporter system (Promega, Madison, WI) according to the manufacturer's instructions. Firefly and Renilla luciferase activities were measured by using a FLUOstar Omega microplate reader (BMG Labtech, Cary, NC). The relative luciferase activity value for each sample was defined as the ratio of firefly luciferase activity to Renilla luciferase activity.

Real-time RT-PCR for detection of innate immune gene expression.

Cell lysates collected from virus-infected cells or plasmid DNA-transfected cells were used for analysis of immune gene expression. Cellular total RNA was extracted by using an SV Total RNA isolation kit (Promega, Madison, WI) according to the manufacturer's instructions. First-strand cDNA was generated with a SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific, Carlsbad, CA). PCR was prepared with TaqMan Fast Advanced master mix (Thermo Fisher Scientific, Carlsbad, CA) and specific primer and probe sets for IFNA1, IFNB1, IL28B, IL29, IRF7, and ISG15 (Thermo Fisher Scientific, Carlsbad, CA). The housekeeping gene GAPDH was used as an internal control for PCR in ST cells, while the TBP (TATA box-binding protein) housekeeping gene was used as an internal control for PCR in HEK-293T cells. Reactions were performed on a CFX96 real-time PCR system (Bio-Rad, Hercules, CA). Relative quantification of target gene expression was performed by using cycle threshold (CT) values (92), and the results for each treatment were compared with the value for the control culture. Mean values were obtained from three repeated experiments.

Construction of full-length cDNA clones of EVG 08/NC_USA/2015.

The full-length genome sequence of purified virus was obtained by next-generation sequencing. The 3′-terminal genomic sequences were determined by using a GeneRacer core kit (Invitrogen, Carlsbad, CA). The strategy for the construction of the full-length cDNA clone is illustrated in Fig. 6. The pACYC177 vector containing a CMV promoter was generated by inserting the CMV sequence into the SphI and XbaI restriction enzyme sites, two viral genomic fragments were amplified with Phusion High-Fidelity DNA polymerase (New England BioLabs, Ipswich, MA), and the hepatitis delta virus (HDV) ribozyme element was synthesized by Integrated DNA Technologies (Coralville, IA) (89). The assembly of these DNA fragments was performed by using a NEBuilder HiFi DNA Assembly cloning kit (New England BioLabs, Ipswich, MA). To construct the PLP knockout mutant, the upstream and downstream regions of PLP were amplified and assembled by using a NEBuilder HiFi DNA Assembly cloning kit.

Recovery of recombinant viruses from full-length cDNA infectious clones.

BHK-21 cells seeded into 6-well plate were transfected with the full-length cDNA infectious clone of the wild-type virus or its mutant. Transfection was conducted by using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. At 48 h posttransfection, the cell culture supernatant was transferred to ST cells. After another 48 h of incubation, the cell culture supernatant was harvested from ST cells, and cells were fixed with 4% paraformaldehyde (pH 7.2) for 15 min. Fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min and then blocked with 1% bovine serum albumin (BSA) in PBS for 30 min. Cells were then incubated with the primary anti-VP1 MAb or anti-PLP MAb for 1 h at 37°C. Alexa Fluor 488 AffiniPure donkey anti-mouse IgG(H+L) (Jackson Immuno Research, West Grove, PA) was used as the secondary antibody. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Carlsbad, CA). Immunofluorescent signals were visualized with the Evos FL cell imaging system (Thermo Fisher Scientific, Carlsbad, CA).

In vitro growth characterization of recombinant viruses.

Passage 2 of recombinant viruses in ST cells was used to characterize in vitro growth properties. Confluent ST cells were inoculated with the recombinant virus at an MOI of 0.01. The cell culture supernatant was serially harvested at 0, 2, 4, 6, 8, 10, and 12 hpi. The virus titer was measured by a microtitration assay using ST cells in 96-well plates and calculated as FFU per milliliter by using a method described previously (26).

Statistical analysis.

All the data are shown as mean values with standard deviations and were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test using programs in GraphPad Prism 6 (GraphPad, La Jolla, CA). Significant differences are indicated by asterisks in the figure legends.

Accession number(s).

The genome sequence of EVG 08/NC_USA/2015 determined in this study was submitted to GenBank under accession no. KY761948.


This project was supported by an Agriculture and Food Research Initiative competitive grant (no. 2013-07173) from the USDA National Institute of Food and Agriculture and the Kansas State Veterinary Diagnostic Laboratory and Kansas State University research startup fund.


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