Analysis of the genomic homologous recombination in Theilovirus based on complete genomes

At present, Theilovirus is considered to comprise four distinct serotypes, including Theiler's murine encephalomyelitis virus, Vilyuisk human encephalomyelitis virus, Thera virus, and Saffold virus. So far, there is no systematical study that investigated the genomic recombination of Theilovirus. The present study performed the phylogenetic and recombination analysis of Theilovirus over the complete genomes. Seven potentially significant recombination events were identified. However, according to the strains information and references related to the recombinants and their parental strains, four of the recombination events might happen non-naturally. These results will provide valuable hints for future research on evolution and antigenic variability of Theilovirus.


Introduction
Encephalomyocarditis virus (EMCV) and Theilovirus are two distinct species in the Cardiovirus genus of the family Picornaviridae [1]. The EMCVs comprise a single serotype and have a wide host range, while the Theilovirus species, probably includes four serotypes: Theiler's murine encephalomyelitis virus (TMEV), Vilyuisk human encephalomyelitis virus (VHEV), Thera virus (TRV; isolated from rats) and Saffold virus (SAFV; isolated from humans). TMEVs were originally isolated from mice and later from rats [2]. Serological studies indicated that the feral house mouse Mus musculus is the natural host for TMEV [3]. VHEV was isolated by the inoculation of mice with nasopharyngeal secretions, serum samples, feces, cerebrospinal fluid (CSF) specimens, and brain specimens from the Yakut-Evenk population, indigenous rural people in Siberia that had a chronic form of encephalitis [4]. TRV was isolated from sentinel rats housed with TMEVseropositive rats in Japan [5]. This virus has not yet been associated with disease in rats but has raised the possibility of additional clades of undiscovered theiloviruses.
SAFVs, new theiloviruses, were first isolated in California from a fecal sample from an 8-month-old infant with fever of undetermined origin [6] and then from a nasopharyngeal sample collected from a 23-month-old child in Canada in 2006 [7].
For picornaviruses, recombination is a common mechanism of evolution and antigenic variability. Although a recent report suggested that recombination happened in Cardiovirus genus [8], no study has systematically investigated the recombination among Theilovirus strains. In the present study, therefore, we systematically analyzed the available complete Theilovirus genome sequences in GenBank to elucidate the recombination among these viruses.

Sequences
The study sequences comprised all the 23 available complete genome sequences of Theilovirus from Gen-Bank dated January 2011. Sequences were firstly screened to exclude patented and artificial mutants, and then aligned in the ClustalW program [9]. The alignment was manually adjusted for the correct reading frame. Sequences showing less than 1% divergence from each other were considered as the same. The strain information of the remaining 21 Theilovirus genomes were shown in Table 1. Because there was no complete genome of VFHV in GenBank before our analysis, this virus was not analyzed in the present study.

Phylogenetic analysis
Before phylogenetic analysis, multiple-alignment was performed in the ClustalW program. Phylogenetic trees were constructed using the neighbor-joining method and evaluated using the interior branch test method with Mega 4 software [10]. Percent bootstrap support was indicated at each node. GenBank accession no. was indicated at each branch.

Results and Discussion
Based on the 21 complete Theilovirus genomes, a phylogenetic tree was constructed ( Figure 1). The taxonomy of these Theilovirus showed in the phylogenetic tree was consistent with the strain information from the original sources. From the phylogenetic tree, we can see that Theilovirus were divided into two major different genetical groups. Among the two major groups, SAFV formed a single group, while TMEV and TRV closely clustered, forming the other group. Sequence alignment indicated that TMEV strains shared 71.2%-75.3% and 67.4%-70.1% sequence identities with TRV and SAFV strains, respectively. While TRV strains showed 72.2%-74.8% sequence homologies to SAFV strain. Seven potentially significant recombination events were detected with a high degree of confidence (p value ≤ 1.3 × 10 -4 ) judged by the above-mentioned six recombination detection methods. Figure 2 indicated the 7 recombination events, where we can see that event1 included three recombinants which had the same parental strains while the other six recombination events contained six recombinants, respectively.   [7,19,20]. In this recombination event, the two parental strains were isolated in different countries, and the three daughter recombinants were distributed in different countries, which might hint that this recombination event happened long time ago and the recombinants were prevalent worldwide.
Recombination event2 identified the recombination occurred between two SAFV strains [GenBank: GU595289, GenBank:EU681179], leading to the other recombinant SAFV strain [GenBank:EU681176] (Additional File 1, Part A). However, in this recombination event, one of the parental strain [GenBank: EU681179] and the daughter strain were sequenced in the same lab [19], therefore, whether this recombination event occurred naturally or not should be verified by future studies. Additional File 1, Part B and C indicated the recombination event3 and event4, respectively, and three SAFV strains [GenBank: FJ463615, GenBank:FJ463616, GenBank:FJ463617] involved in the two recombination events were all sequenced in the same lab [21], therefore, it should be cared whether these two recombination events non-naturally occurred by sequencing error and/or contamination. The recombination event5 (Additional File 1, Part D) also contained two strains [GenBank: EU681179, GenBank: EU681178] which were isolated in the same lab [18], therefore, whether this recombination event non-naturally occurred by sequencing error and/or contamination should be elucidated by further study. Figure 4 indicated the recombination event6 that occurred between a two TMEV strains, Yale strain [GenBank:EU723238] and DA strain [GenBank: M20301] [22], which led to the recombinant TMEV strain BeAn [GenBank:M16020] which was isolated from mouse in 1987, and these three virus strains were all isolated from mouse in USA [1,22]. Figure 5 revealed the putative recombinant TMEV strain (Gen-Bank:M20301), however, the accurate parental strains has not been detected in the present study, which may due to the limited numbers of Theilovirus sequence available at present, therefore, further study should be performed to identify the accurate parental strains with the increasing number of Theilovirus genome sequences.
Recombination is a relatively common phenomenon in RNA viruses and understanding recombination will be helpful in unravelling the evolution of pathogens and drug resistance [23][24][25]. In the present study, we performed phylogenetic and recombination analyses over the full genome of Theilovirus available in Gen-Bank nowadays. Seven potentially significant recombination events were detected. However, four of the  recombination events might happen non-naturally in the lab, which should be taken into notice in the future evolutionary analysis of Theilovirus. The other three recombination events were further analyzed using other algorithms in RDP software bag and some of them were confirmed by phylogenetic analysis. The recombination phenomena of Theilovirus will also be noted in the further research because this will be one pattern of virulence factor variation in Theilovirus.

Additional material
Additional file 1: BOOTSCAN evidence for the recombination event 2, 3, 4, and 5. Analysis was based on pairwise distance, modeled with a window size 200, step size 20, and 100 Bootstrap replicates.