A novel cardiovirus in wild rats
Cardioviruses cause severe illnesses in rodents and humans. In recent years, novel cardioviruses have been frequently found, which promoted further studies of the genetic diversity of cardioviruses. Using viral metagenomics, we genetically characterized a novel cardiovirus (named SX1) from wild rat feces. The genomic structure of SX1 shared similar features with those of the Theiler’s murine encephalomyelitis viruses, including a leader protein, four structural proteins and seven non-structural proteins. Phylogenetic analysis based on both structural proteins and non-structural proteins coding regions showed that SX1 was formed into a separate branch, being located between the branches of Theiler’s murine encephalomyelitis viruses and Thera viruses. Variable resides presented in the Ser/Thr rich domain of L protein, VP1 loops, and VP2 puffs distinguished SX1 from Theiler’s murine encephalomyelitis viruses, suggesting the different antigenicity and pathogenicity of SX1.
KeywordsCardiovirus genus Viral metagenomics Genomic structure Phylogenetic analysis
Theiler’s murine encephalomyelitis virus
Vilyuisk human encephalomyelitis virus
Cardioviruses is a genus of picornaviruses that cause severe illnesses in rodents and human [1, 2, 3, 4]. The genus Cardiovirus includes three species, namely Cardiovirus A, Cardiovirus B, and Cardiovirus C. Cardiovirus A has only one member, encephalomycarditis virus (EMCV), which causes rat encephalitis and myocarditis [5, 6]; Cardiovirus C is a novel cardiovirus identified in laboratory rats and Rattus norvegicus ; Cardiovirus B is composed of Theiler’s murine encephalomyelitis virus (TMEV), Vilyuisk human encephalomyelitis virus (VHEV), Thera virus (TRV), and Saffold virus (SAFV), where VHEV and SAFV can infect humans and cause encephalomyelitis, acute gastroenteritis and so on [1, 8, 9]. TMEV and TRV mainly infect mice and cause neurological diseases [3, 10, 11]. TMEVs were originally isolated from colony-bred mice that developed spontaneous paralysis in the early 1930s . Now it is reported that TMEVs mainly cause asymptomatic infections in mice, and in rare cases, neurological symptoms featured with early poliomyelitis or late demyelinating disease . TRVs were firstly isolated from sentinel rats housed with TMEV-seropositive rats in Japan in 2002 . In 2008, a novel isolate of TRV was detected in the feces of rats . Till now, there is no report about the association between TRV and diseases in rats. Using virus metagenomics method, we detected a novel cardiovirus in the feces of wild rats and characterized the complete genome. The novel cardiovirus was named SX1 and its genomic sequence was submitted to GenBank with accession no. MF172923.
In order to investigate rat intestinal virome, 40 intestinal content samples were collected from wild M. longicaudus rats captured by the Chinese Center for Disease Control and Prevention in Taizhou City from three districts of Taizhou City including Taixing (n = 15), Gaogang (n = 15), and Hailing (n = 10) from June to August in 2014. All of the wild rats were adults and the exact ages were unknown.
Viral metagenomics method was used to identify viral sequences in these samples. Four separate pools were randomly generated, each of which contained 10 fecal specimens. Briefly, fecal samples were suspended in Dulbecco’s phosphate-buffered saline (DPBS). After low speed centrifugation and filtration, the samples were treated with DNase and RNase to reduce levels of rat nucleic acids while viral genome within viral capsid was protected from digestion . Four libraries were then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the Miseq Illumina platform with 250 bases paired ends with a distinct molecular tag for each pool. Bioinformatics analysis was performed according to a previous study .
To investigate the prevalence of SX1 in rats, we designed nested PCR primers targeting VP2 gene of cardiovirus by RT-PCR. Primer SX1 F1 (5’-GCCCATCGCGGAGAACACCC-3′) and SX1 R1 (5’-TGTCCAGGAGCTGGTCGGGG-3′) were used for the first round of PCR, and SX1 F2 (5’-CGGGGCTTTCTCCCACGTTCG-3′) and SX2 R2 (5′- CGTTTCGGCCGTCATAGCGGT-3′) for the second round, the expected length of amplicons was 308 bp. PCR screening results showed that 3 of the 40 fecal samples were positive with positive rate of 7.5% (3/40). The three positive samples were all obtained from rats collected in Gaogang. The specific PCR products were sequenced by Sanger method. Sequence alignment showed that the three 308 bp sequences shared > 98% nucleotide identities with each other, while shared < 79% nucleotide identities with other members of the genus, indicating the prevalence of a single strain in the wild rats in this area.
In summary, we identified a novel cardiovirus in wild rats and characterized its complete genome. SX1 encodes a polyprotein including an L protein, four structural proteins and seven non-structural proteins, in addition, an L* protein is encoded 13 nucleotides downstream of the start site. Comparing with TMEVs and TRVs, many amino acids are different among them in loops of VP1 and puffs of VP2, both of those domains are vital for viral binding with host cells. The amino acid differences in those domains indicate that SX1 may have different pathogenic and antigenic properties. Phylogenetic analysis showed that the SX1 formed a separate branch that was distant from TMEVs and TRVs. The epidemiologic study suggested a single strain was prevalent in the wild rats in this area.
We thank Taizhou Center for Disease Control and Prevention to samples collection. We thank Dr. Yanjin Zhang for helpful comments on this manuscript.
This work was partly supported by the National Key Research and Development Programs of China No. 2017YFC1200201, the Natural Science Foundation of Jiangsu Province No. BK20140537 and BK20140578, the National Natural Science Foundation of China No. 31402211, the Professional Research Foundation for Advanced Talents of Jiangsu University No. 12JDG085 and 13JDG087, China Postdoctoral Foundation No. 2014 M561597, and Jiangsu Postdoctoral Foundation No. 1302057C.
Availability of data and materials
All relevant data are included in the manuscript. Nucleotide sequences are available at the GenBank depository under accession numbers: MF172923-MF192925.
YW and SY conceived the study. All authors participated in the experiments. SY wrote the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 7.Hansen TA, Mollerup S, Nguyen NP, White NE, Coghlan M, Alquezar-Planas DE, Joshi T, Jensen RH, Fridholm H, Kjartansdottir KR, et al. High diversity of picornaviruses in rats from different continents revealed by deep sequencing. Emerg Microbes Infect. 2016;5:e90.CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Carrillo-Salinas FJ, Mestre L, Mecha M, Feliu A, Del Campo R, Villarrubia N, Espejo C, Montalban X, Alvarez-Cermeno JC, Villar LM, Guaza C. Gut dysbiosis and neuroimmune responses to brain infection with Theiler's murine encephalomyelitis virus. Sci Rep. 2017;7:44377.CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, Ingersoll R, Sheppard HW, Ray SC. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol. 1999;73:152–60.PubMedPubMedCentralGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.