Anelloviruses, which are all single-strand circular DNA viruses, have extremely heterogeneous genetic features. To date, the family Anelloviridae is divided into 11 genus, and the most dominated species include torque teno virus (TTV), torque teno mini virus (TTMV) and torque teno midi virus (TTMDV), belonging to the genus Alphatorquevirus, Betatorquevirus and Gammatorquevirus, respectively. Most of these viruses are detected in the body fluid from the majority of human populations, although their disease associations are still elusive (de Oliveira, 2015; Focosi et al., 2016). TTMV possesses non-enveloped virion of about 30 nm diameters and a single circular genome with its size ranging from 2.8 to 2.9 kb. The genome consists of a coding region of three major overlapping open reading frames (ORFs) and a few untranslated regions. ORF1 encodes the largest protein, which is considered to play a role in the replication of the circular genome (Dinakaran et al., 2014).
TT viruses have been suspected as potential causative agent for many human diseases, including hepatitis, respiratory diseases, periodontitis, hematological and autoimmune disorders, although there are no direct evidence (Mi et al., 2014; Zhang et al., 2016). Thus, further expanding the viral diversity of the TT viruses and examining their potential threats to human beings is essential to public health. Because of the high genetic diversity and the absence of available methodology for virus isolation, the discovery of new TT virus is inefficient using normal PCR methods. The development of deep sequencing technology make the viral metagenomics a powerful tool to detect uncharacterized viruses, which became popular in the discovery of clinical or zoonotic viral agents (Rosario et al., 2012). Here, we report a novel TTMV species discovered in an encephalitis case without further evidence to the disease association.
The patient was a five-year-old boy with clinical symptoms of fever, headache, cough, vomiting and drowsiness, and had no medical history. In the routine blood test, an increased percentage of neutrophils (76.20%) was detected, while the analysis of cerebrospinal fluid (CSF) (including WBC, glucose, chloride and protein) did not show any abnormal features (Supplementary Table S1). MRI scan was performed on the patient and the result clearly revealed hyperintense lesions in the right parietal cortex (Figure 1A). The results of serum and CSF antibody tests targeting echo virus, coxsackie virus, EV71, EB virus, cytomegalovirus, herpes simplex virus, varicella-zoster virus and measles virus were all negative. The blood culture for putative bacterium was also negative. Under the appropriate treatment, the boy was cured and left the hospital two weeks later. To conduct the pathogen pursuing research, the informed consent was acquired from the parents of the child before the study. Subsequently, the deep sequencing method was applied to look for potential novel pathogens. The CSF of the boy suffering encephalitis was collected. Since the viral nucleic acids were enclosed in their capsid protein, a mixture of nuclease (Turbo DNase from Ambion, Carlsbad, USA; Baseline-ZERO from Epicentre, Madison, USA; Benzonase from Darmstadt, Germany; and RNase from Fermentas, Carlsbad, USA) was applied to digest unprotected nucleic acids in the filtrates. The nucleic acids were then extracted using QIAamp viral RNA extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s manual and stored at –80 °C. The produced nucleic acid (containing both DNA and RNA viral sequences) were subjected to the reverse transcription with N8 random primers (Sangon, Shanghai, China), and the second stand was generated using Klenow enzyme (NEB, Ipswich, USA). The sequencing library was then constructed by the employment of the Nextera XT DNA Sample Preparation Kit (Illumina, CA, USA) following the suggested protocol. Subsequently, the prepared library was subjected to sequencing in the Illumina MiSeq platform.
Figure 1. (A) Magnetic resonance imaging (MRI) on ad-missio.MRI scans were performed on the paWent using a 3T MRI scanner (750W GE) and a 12-channel head coil. Hyperintense lesions (indicated by the white arrows) in the right parietal cortex are clearly identified on T2WI (A-a), and a DWI (A-b). (B) The genome organization of TTMV-Zhenjiang. The genome structure diagram was generated using Vector NTI 10 software. (C) The phylogenetic tree based on ORF1 amino acid of TTMV-Zhenjiang constructed using the maximum-likelihood (ML) method. The amino acids were firstly aligned using Mafft and edited to retain the conserved domains. And then calculating the phylogenetic tree using PhyML3.0 software. The generated tree file was displayed and edited by the Figtree software
The raw sequencing data generated were processed according to the standard procedure as described previously, which included decarcoding, trimming and assembling (Deng et al., 2015). A total of 1,732,414 reads were generated in the Illumina MiSeq platform. Then the assembled contigs were analyzed using Blasx algorithm against the Genbank database using the BLAST + 2.5.0. The putative ORFs of the targeted query sequence was inferred by the ORF finder in the NCBI online service (https://www.ncbi.nlm.nih.gov/orffinder/), and the DNA or amino acid identities with other referential sequences were calculated using MegAlin algorithm implemented in the Lasergene software package v7.1 (DNAstar, Madison, WI). One potential virus was discovered exclusively in the assembled contigs, excluding host sequence and unparsed short reads.
Identity (%) with strain* 1 2 3 4 5 6 7 8 9 10 11 12 1. TTMV-Zhenjiang 37.3 35.9 36.2 45.4 42.8 41.7 38.6 42 41.2 29 28.8 2. TTMV1-CBD279 27.3 41.8 43.2 41.2 35.8 44.1 39.1 39.5 38.4 28.8 29.9 3. TTMV2-NLC023 26 36.5 47.6 40 34.5 39.7 37.3 37.1 36.5 28.9 32.1 4. TTMV3-NLC026 26.9 33.7 41.4 44 36.4 40.6 38.2 38.6 38.1 28 29.8 5. TTMV4-Pt-TTV8II 38.2 30.9 33 33.9 41.1 44.7 42.7 42.7 38 28.4 30.7 6. TTMV5-TGP96 33.2 26.7 25.5 26.8 32.7 39.4 37.2 40.3 38.6 30.9 28.8 7. TTMV6-CBD203 34.6 32 32.3 32.3 37.3 27.6 47 46.8 43.4 28.5 31.4 8. TTMV7-CLC156 28 29.6 29.9 29 34.7 24.7 37.5 47.2 40.2 26.4 29.8 9. TTMV8-PB4TL 33.2 31.1 29.3 30.1 36.9 29.5 37 37.3 50.3 29.5 32.1 10. TTMV9-NLC030 32.1 29.6 28.2 27.8 34.7 28.5 36.3 34.3 43.1 29.8 28.1 11. TTV 20.7 18.1 21.5 16.3 17.1 20.7 18 17.6 20.7 20.5 29.4 12. TTMDV 18.7 23.3 23.7 18 24 22.2 22.4 18.9 22.7 18.9 22.6 Note: * The numbers represent the identities of nucleotide (above the diagonal) and amino acid (below the diagonal).
Table 1. The nucleotides and amino acids identities of TTMV-Zhenjiang with published TTMVs
The results showed that the potential virus was most likely a novel species of TTMV. The targeted contig of the putative TTMV (named TTMV-Zhenjiang) was of 3037 bp, and the assembly contained the overlapping sequences in both end of the genomes, suggesting a circular genomic organization. The exact genome sequence was further confirmed and re-edited based on the results of PCR combing Sanger sequencing. Finally, the confirmed genome of this virus was of 2943 nt length containing 3 major ORFs (Figure 1B). The ORF1 shared 45.4% (TTMV4) ~ 35.9% (TTMV2) identities with established TTMV species, and < 35% with the other TT viral species ( Table 1). The ORF1 encoded a 680 aa protein, sharing identities with other TTMV species ranging from 38.2% (TTMV4) to 26% (TTMV2). For the ORF2, based on the online BLASTN searching in GenBank, the TTMV-Zhenjiang shared the highest nucleotide homology (identity: 70%; query cover: 45%; E-value: 0.003) with TTMV-Emory1, while the ORF3 with TTMV8-PB4TL (identity: 75%; query cover: 50%; E-value: 1e-27). According to the species demarcation criteria in the Genus Betatorquevirus suggested by ICTV, the TTMV-Zhenjiang can be regarded as a new TTMV species. Like other TTMV species, the ORF3 of TTMV-Zhenjiang overlapped with ORF1, and the ORF2 partially overlapped with ORF1. The genome sequence of the TTMV-Zhenjiang had been deposited into GenBank (accession number: KY856742).
To get insight of the evolutionary relationship between the newly discovered TTMV-Zhejiang and other TTMVs, we collected all the established TTMV species as well as some other unclassified viruses in genus Betatorquevirus (Supplementary Table S2). The entire protein sequence of ORF1 was utilized to infer their phylogenetic relationship. The genome sequences were firstly aligned using E-INS-i algorithm in MAFFT version 7, and edited using TrimAl to remove ambiguously aligned regions. The phylogenetic tree was constructed using the maximum-likelihood (ML) method implemented in PhyML3.0 software, with the best-fit model (LG) determined using Prot-Test 3.4. In addition, we inferred the phylogenetic tree using Mrbayes3.2.6 to confirm the topology of the ML tree. The result showed, the TTMV-Zhenjiang was distantly related to the viruses TTMV4 and TTMV5 (Figure 1C). The tree generated by MrBayes showed a similar topology. Based on the sequence information of the TTMV-Zhenjiang, we tried to detect this virus in the blood of the patient using PCR, but the result was negative. The possible reason may be the virus is neurotropic and its replication efficiency in blood is too low to detect.
Since the first report about TTV in 1997, a large number of TT viruses have been discovered. It is reported that there was a ubiquitous presence of TT viruses in human population, even in the healthy individuals (Moustafa et al., 2017). The strain Emory1, with which TTMV-Zhenjiang shared highest identity, was detected in the human glioblastoma. And other closed related strains in the phylogenetic tree, like ALH8, TGP96 and LYs were also found in various human tissues, specially the LYs, was detected in parapneumonic empyema in children, deserving more clinical attention. Besides, many studies have demonstrated the possible relationship of the TTVs with various diseases. Although TT viruses were considered by someone to be absent of human pathologies due to the high prevalence, which indicating the virus-host coevolution for a long periods. Nevertheless, many epidemiological studies revealed the association of TT viruses with different pathological conditions. Since the definite involvement of the TT viruses in human disease deserve more data, the accumulation of the viral diversity and related pathology information is meaningful (Maggi and Bendinelli, 2010; García-Álvarez et al., 2013; Zhang et al., 2016). Viral agent is the major cause of human encephalitis (Kennedy, 2004). To date, several viruses were found to be associated with encephalitis, including Japanese B encephalitis virus, West Nile encephalitis virus, Adenoviruses, Herpes simplex encephalitis, enterovirus, etc. Like other infectious disease, the pathogen spectrum could be extended in the light of deep sequencing approaches (Chan et al., 2014). This report firstly described a novel TTMV from the cerebrospinal fluid in an encephalitis patient, which gives a new clue for the pathogen spectrum of encephalitis. Since the TT viruses were ubiquitous around human population, the infection caused by other viral or bacterial agents combining with TTMV-Zhenjiang could not be denied. Then, the potential disease association of TTMV with encephalitis requires further investigation.
Patient data Normal range Routine blood test White blood cell count (WBC) 8.21 × 109/L 4~10 × 109/L Basophil count (BASO#) 0.07 × 109/L 0~0.1 × 109/L Basophil ratio (BASO%) 0.80% 0~1% Neutrophil count (NEUT#) 6.26 × 109/L 2.0~7.5 × 109/L Neutrophil ratio (NEUT%) 76.20% 50%~70% Eosinophil count (EO#) 0.16 × 109/L 0~0.7 × 109/L Eosinophil ratio (EO%) 2.00% 0.5%~5% Lymphocyte count (LYMPH#) 1.34 × 109/L 0.8~4.0 × 109/L Lymphocyte ratio (LYMPH%) 16.30% 17%~50% Monocyte count (MONO#) 0.38 × 109/L 0.3~0.8 × 109/L Monocyte ratio (MONO%) 4.70% 3%~10% Red blood cell count (RBC) 4.07 × 1012/L 4.0~5.50 × 1012/L Hemoglobin (HGB) 125.00 g/L 120~160 g/L Mean corpuscular volume (MCV) 100.70 FL 80~100 FL Mean corpuscular hemoglobin (MCH) 30.70 pg 26~38 pg Mean corpuscular hemoglobin concentration (MCHC) 305.00 g/L 300~360 g/L Platelet (PLT) 235.00 × 109/L 100~300 × 109/L CSF analysis WBC 9 × 106/L 0~15 × 106/L Glucose 3.6 mmol/L 2.8~4.4 mmol/L Chloride 118 mmol/L 111~123 mmol/L Protein 0.40 g/L 0.15~0.45 g/L
Table S1. Laboratory data for the patient with encephalitis
Virus Abbreviation Accession no. Host Source Torque teno mini virus 1 TTMV1-CBD279 AB026931 human plasma Torque teno mini virus 2 TTMV2-NLC023 AB038629 human plasma Torque teno mini virus 3 TTMV3-NLC026 AB038630 human plasma Torque teno mini virus 4 TTMV4-Pt-TTV8-II AB041963 chimpanzees sera Torque teno mini virus 5 TTMV5-TGP96 AB041962 human sera Torque teno mini virus 6 TTMV6-CBD203 AB026929 human serum Torque teno mini virus 7 TTMV7-CLC156 AB038627 human plasma Torque teno mini virus 8 TTMV8-PB4TL AF291073 human Peripheral blood
Torque teno mini virus 9 TTMV9-NLC030 AB038631 human plasma Torque teno mini virus -LIL-y1 TTMV-LIL-y1 EF538880 human plasma Torque teno mini virus -LIL-y2 TTMV-LIL-y2 EF538881 human plasma Torque teno mini virus -LIL-y3 TTMV-LIL-y3 EF538882 human plasma TTV-like mini virus- LIL-y4 TTMV-LIL-y4 EF538883 human plasma TTV-like mini virus CLC062 TLMV-CLC062 AB038625 human Plasma TTV-like mini virus CLC138 TLMV-CLC138 AB038626 human plasma TTV-like mini virus Emory1 TTMV-Emory1 KX810063 human glioblastoma TTV-like mini virus Emory2 TTMV-Emory2 KX810064 human glioblastoma Torque teno mini virus ALA22 TTMV-ALA22 KM259873 human oral mucosa Torque teno mini virus ALH8 TTMV-ALH8 KM259874 human oral mucosa TTV-like mini virus LY2 TTMV-LY2 JX134045 human pleural effusion TTV-like mini virus LY3 TTMV-LY3 JX134046 human pleural effusion TTV-like mini virus LY1 TTMV-LY1 JX134044 human pleural effusion
Table S2. The basic information of accepted TTMV members and some representative unclassified species in genus Betatorquevirus using in this study.