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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Virology Journal 1/2017

Complete genome sequence of T’Ho virus, a novel putative flavivirus from the Yucatan Peninsula of Mexico

Zeitschrift:
Virology Journal > Ausgabe 1/2017
Autoren:
Thomas Briese, Maria A. Loroño-Pino, Julian E. Garcia-Rejon, Jose A. Farfan-Ale, Carlos Machain-Williams, Karin S. Dorman, W. Ian Lipkin, Bradley J. Blitvich
Abbreviations
ALFV
Alfuy virus
BAGV
Bagaza virus
C
Capsid
cDNA
Complementary DNA
CPCV
Cacipacore virus
ds
Double stranded
E
Envelope
ILHV
Ilheus virus
ITV
Israel turkey meningoencephalitis virus
JE
Japanese encephalitis
JEV
Japanese encephalitis virus
KOUV
Koutango virus
KUNV
Kunjin virus
LB
Lysogeny broth
ML
Maximum likelihood
MP
Maximum parsimony
MVEV
Murray Valley encephalitis virus
NJ
Neighbor-joining
NS
Nonstructural
nt
Nucleotide
NTAV
Ntaya virus
ORF
Open reading frame
Poly (A)
Polyadenylate
prM/M
Premembrane/membrane
RACE
Rapid amplification of cDNA ends
ROCV
Rocio virus
RT-PCR
Reverse transcription-polymerase chain reaction
SLEV
St. Louis encephalitis virus
TMUV
Tembusu virus
USUV
Usutu virus
UTRs
Untranslated regions
WNV
West Nile virus
YAOV
Yaounde virus
ZIKV
Zika virus

Background

The genus Flavivirus (family Flaviviridae) contains more than 70 viruses, most of which are transmitted to vertebrates by arthropod vectors such as mosquitoes and ticks [23]. The genus is divided into at least 14 groups on the basis of nucleotide (nt) and deduced amino acid sequence data, antigenic relatedness and other characteristics. Two groups within the genus are the Ntaya and Japanese encephalitis (JE) groups. According to the Ninth Report of the International Committee on Taxonomy of Viruses, the Ntaya group consists of six viruses: Bagaza virus (BAGV), Ilheus virus (ILHV), Israel turkey meningoencephalitis virus (ITV), Ntaya virus (NTAV), Tembusu virus (TMUV) and Zika virus (ZIKV). Rocio virus (ROCV) is considered to represent a subtype of ILHV. The JE group consists of eight viruses: Cacipacore virus (CPCV), Japanese encephalitis virus (JEV), Koutango virus (KOUV), Murray Valley encephalitis virus (MVEV), St. Louis encephalitis virus (SLEV), Usutu virus (USUV), West Nile virus (WNV) and Yaounde virus (YAOV), in addition to Alfuy virus (ALFV) and Kunjin virus (KUNV) which are considered to represent subtypes of MVEV and WNV, respectively.
All flaviviruses possess a single-stranded, positive-sense RNA genome of approximately 11 kb [29]. The genome encodes a major open reading frame (ORF) that is flanked by 5′ and 3′ untranslated regions (UTRs) of ~100 and ~400–700 nt, respectively. The ORF encodes a polyprotein that is co- and post-translationally cleaved to generate three structural proteins, designated the capsid (C), premembrane/membrane (prM/M) and envelope (E) proteins, and at least seven nonstructural (NS) proteins in the gene order: 5′–C–prM (M)–E–NS1–NS2A–NS2B–NS3–NS4A–2K–NS4B–NS5–3′. Some viruses in the JE group utilize efficient −1 ribosomal frameshifting to produce a larger NS1-related protein (NS1’) [18, 33].
Previously, we provided evidence that a novel flavivirus (designated T’Ho virus) occurs in the Yucatan Peninsula of Mexico [16]. The putative virus was identified in a pool of Culex quinquefasciatus mosquitoes collected in 2007 at the Merida zoo, Yucatan State. A 1358-nt region of the NS5 gene was amplified and sequenced by reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing using flavivirus-specific primers. Application of BLASTn analysis revealed that the sequence is genetically equidistant to the corresponding regions of SLEV (72.6% identical), ILHV (72.2%), JEV (72.1%), USUV (71.8%), ROCV (71.4%), MVEV (71.3%), WNV (71.1%) and BAGV (70.1%). Although we successfully amplified T’Ho virus RNA, we were not able to obtain an isolate by virus isolation in African Green Monkey kidney (Vero) or Aedes albopictus (C6/36) mosquito cells or suckling mouse brain inoculation. In this study, the complete genome sequence of T’Ho virus was determined and its genetic relatedness to other flaviviruses was assessed.

Methods

High-throughput sequencing

Trizol Reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from the pool of Cx. quinquefasciatus previously shown to contain T’Ho virus RNA. Protocols used for the collection, identification and homogenization of mosquitoes have been described elsewhere [16]. RNA extracts were reverse transcribed using SuperScript III (Thermo Fisher, Waltham, MA, USA) with random hexamers. The complementary DNA (cDNA) was RNase-H treated prior to second strand synthesis with Klenow Fragment (NEB, Ipswich, MA, USA). The generated double stranded (ds) DNA was sheared to an average fragment size of 200 bp using manufacturer’s standard settings (Covaris focused-ultrasonicator E210; Woburn, MA, USA). Sheared products were purified (Agencourt Ampure DNA purification beads, Beckman Coulter, Brea, CA, USA) and libraries constructed. Sheared nucleic acid was end-repaired, dA-tailed, ligated to sequencing adapters (NEBNext modules, NEB), PCR amplified (Phusion High-Fidelity DNA polymerase, NEB) and quantitated by Bioanalyzer (Agilent, Santa Clara, CA, USA) for sequencing. Sequencing on the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) resulted in an average of 180 million reads per lane. Samples were de-multiplexed using Illumina software and FastQ files generated. Data were quality filtered and trimmed (Slim-Filter) and de novo assembled using Dwight assembler at custom settings [20]. The generated contiguous sequences (contigs) and unique singleton reads were subjected to homology search using BLASTn and BLASTx against the GenBank database.

5′ and 3′ rapid amplification of cDNA ends

The extreme 5′ and 3′ ends of the T’Ho virus genome were determined by 5′ rapid amplification of cDNA ends (RACE) and 3′ RACE, respectively. In the 5′ RACE reactions, total RNA was reversed transcribed using a T’Ho virus-specific primer. Complementary DNAs were purified by ethanol precipitation and oligo (dC) tails were added to the 3′ ends using 15 units of terminal deoxynucleotidyl transferase (Invitrogen, Carlsbad, CA, USA). Tailing reactions were performed at 37 °C for 30 min and then terminated by heat-inactivation (65 °C for 10 min). Oligo dC-tailed cDNAs were purified by ethanol precipitation then PCR-amplified using a consensus forward primer specific to the C-tailed termini (5′-GACATCGAAAGGGGGGGGGGG-3′) and a reverse primer specific to the T’Ho virus cDNA sequence. In the 3′ RACE reactions, polyadenylate [poly (A)] tails were added to the 3′ ends of the T’Ho virus genomic RNA using 6 units of poly (A) polymerase (Ambion, Austin, TX, USA). Tailing reactions were performed at 37 °C for 1 h and then terminated by heat-inactivation (65 °C for 10 min). Poly (A)-tailed RNA was reverse transcribed using a poly (A) tail-specific primer (5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′). Complementary DNAs were PCR amplified using a forward primer specific to the T’Ho virus cDNA sequence and a reverse primer that matched the 5′ half of the poly (A)-specific reverse transcription primer (5′-GGCCACGCGTCGACTAGTAC-3′). PCR products generated from the 5′ and 3′ RACE reactions were inserted into the pCR4-TOPO cloning vector (Invitrogen, Carlsbad, CA, USA) and ligated plasmids were transformed into competent TOPO10 Escherichia coli cells (Invitrogen, Carlsbad, CA). Cells were grown on Lysogeny broth (LB) agar containing ampicillin (50 μg/ml) and kanamycin (50 μg/ml), and colonies were screened for inserts by PCR amplification. An aliquot of each PCR product was examined by 1% agarose gel electrophoresis and several PCR products were purified using a QIAquick spin column (Qiagen, Valencia, CA, USA) and sequenced using a 3730 × 1 DNA sequencer (Applied Biosystems, Foster City, CA, USA).

Nucleotide and amino acid sequence alignments

The genomic and predicted amino acid sequences of T’Ho virus were aligned to all other sequences in the Genbank database by application of BLASTn and BLASTp, respectively [1]. Sequence alignments using Clustal Omega (available at http://​www.​ebi.​ac.​uk/​Tools/​msa/​clustalo/​) were performed to calculate percent nucleotide and amino acid identities between select sequences.

Phylogenetic analysis

The complete ORF sequences of T’Ho virus and other flaviviruses were aligned using Muscle v3.8.31 [14]. Phylogenetic trees were constructed by MrBayes v3.2.6 (evolutionary model GTR with gamma-distributed rates in six categories plus invariant sites, two chains run for 3 × 106 generations with 25% burn-in, resulting in maximum PSRF 1.005 among all continuous parameters) [38], neighbor-joining (NJ, Mega v7.0.20 using Maximum Composite Likelihood distances with Gamma-distributed rates, alpha set to 0.58, pairwise gap deletion and 500 bootstraps), maximum parsimony (MP, Mega v7.0.20 using complete gap deletion and 500 bootstraps) [26] and maximum likelihood (ML, RaxML v8.2.8, using GTRGAMMAIX model and bootstopping criterion autoMRE) [42]. The GTR + G + I model was selected by sample size corrected AICc in jModelTest v2.1.6, and this finding motivated the models chosen for the NJ and ML analyses [36].

Results

Genomic organization and BLAST analysis

The T’Ho virus genome consists of 10,937 nt (Genbank Accession No. EU879061.2) and contains a single 10,284-nt ORF flanked by 5′ and 3′ UTRs of 97 and 556-nt, respectively. The ORF encodes the three structural and seven nonstructural proteins common to all known flaviviruses. The position and length of each gene and untranslated region are shown in Table 1. The complete genome and deduced polyprotein amino acid sequences of T’Ho virus were aligned to all other flaviviruses sequences for which complete genome data are available. The nucleotide sequence alignments indicated that T’Ho virus is most closely related to ROCV (67.4% identity) and ILHV (65.9%) followed by other Ntaya group viruses (58.8–63.3%) and JE group viruses (62.0–63.7%) (Table 2). The polyprotein of T’Ho virus has greatest amino acid identity to ROCV (72.2%) and ILHV (70.6%) followed by other Ntaya group viruses (57.1–66.3%) and JE group viruses (63.2–66.3%). The genome sequence of T’Ho virus was inspected for potential overlapping genes but none were identified.
Table 1
Genomic organization of T’Ho virus
Gene/genetic region
Size (nt)
Genome
position
Protein size (aa)
Polyprotein position
5′ UTR
97
1–97
-
-
C
357
98–454
119
1–119
VirC
306
98–403
102
1–102
AnchC
51
404–454
17
103–199
prM
501
455–955
167
120–286
pr
276
455–730
92
120–211
M
225
731–955
75
212–286
E
1503
956–2458
501
287–787
NS1
1059
2459–3517
353
788–1141
NS2A
678
3518–4195
226
1142–1367
NS2B
393
4196–4588
131
1368–1498
NS3
1857
4589–6445
619
1499–2117
NS4A
378
6446–6823
126
2118–2243
2 K
69
6824–6892
23
2244–2266
NS4B
771
6893–7663
257
2267–2523
NS5
2718
7664–10,381
905
2524–3428
3′ UTR
556
10,382–10,937
-
-
Table 2
Genetic relatedness between T’Ho virus and select other flaviviruses
Virus
% nucleotide identity
(genome)
% nucleotide identity
(ORF)
% amino acid identity
(polyprotein)
Ntaya group viruses
 Bagaza virus
62.6
63.5
66.0
 Ilheus virus
65.9
65.8
70.6
   1Rocio virus
67.4
67.3
72.2
 Israel turkey meningoencephalitis virus
63.3
64.0
66.3
 Ntaya virus
62.9
63.6
65.2
 Tembusu virus
63.0
63.4
65.3
 Zika virus
58.8
59.4
57.1
Japanese encephalitis group viruses
 Cacipacore virus
n/a
62.5
63.2
 Japanese encephalitis virus
63.5
63.8
64.7
 Koutango virus
n/a
63.4
64.0
 Murray Valley encephalitis virus
62.8
63.3
64.4
   2Alfuy virus
62.3
62.6
64.1
 St. Louis encephalitis virus
63.7
64.1
66.3
 Usutu virus
63.1
63.4
64.3
 West Nile virus
62.8
63.4
64.5
   3Kunjin virus
62.0
62.8
64.5
 Yaounde virus
n/a
63.8
65.5
Other flaviviruses
 Cell fusing agent virus
42.1
42.0
29.2
 Dengue virus type-2
56.4
56.9
51.7
 Modoc virus
50.2
48.1
38.4
 Tick-borne encephalitis virus
48.5
49.1
41.9
 Yellow fever virus
53.4
53.7
45.8
1, 2, 3Subtype of ILHV, MVEV and WNV, respectively
n/a not available (genome has not been fully sequenced)

Organization of the polyprotein

Cleavage sites in the T’Ho virus polyprotein are shown in Table 3. These sites were predicted by aligning the polyprotein sequence of T’Ho virus to those of select other flaviviruses for which cleavage sites have previously been predicted and in some instances experimentally verified. The predicted cleavage sites for T’Ho virus are in agreement with the rules previously established for flaviviruses. The C/prM, prM/E, E/NS1 and 2K/NS4B polyprotein junctions of most known flaviviruses conform to predicted signalase cleavage sites [6] and similar sites were identified at these locations for T’Ho virus (Table 3). The NS1/NS2A junctions of most known flaviviruses occur after a Val-X-Ala site that fulfills the ‘-1, −3’ rule for a signalase site but lacks an upstream hydrophobic domain [6]. The predicted NS1/NS2A junction of T’Ho virus fulfills these requirements. For most flaviviruses, the predicted pr/M junction occurs after an Arg-X-Lys/Arg-Arg or Arg-X-X-Arg motif. T’Ho virus adheres to this rule. The flavivirus virion C/anchor, NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4A/2K and NS4B/NS5 junctions are commonly cleaved after two basic amino acid residues (KR, RR or RK) [57] and sites in T’Ho virus are consistent with that rule.
Table 3
Predicted cleavage sites in the polyproteins of T’Ho virus and select other flaviviruses
Virus
Junction
 
1VirC/Anch
2C/prM
3pr/M
2prM/E
2E/NS1
4NS1/NS2A
T’Ho virus
KNNKR↓GMTTT
ATTMA↓ARLSS
RRGRR↓SINIA
VPAYS↓LNCLG
MNVHA↓DTGCV
SMVVA↓GHGSS
ROCV
RKAKR↓GNGSV
TGSMA↓LRLGT
RRGRR↓SVNIP
APAYS↓INCLG
MNVHA↓DTGCA
SKVTA↓GTGND
ILHV
KEKKK↓SFSTA
TAVAG↓LKISS
RRGRR↓AINIP
APAYS↓LNCLG
VNVHA↓DTGCA
SKVSA↓GNGQT
BAGV
GKKKR↓GGTTV
GVAQA↓IKIGS
RRSRR↓SITVH
APAYS↓FNCLG
TNVHA↓DTGCA
SRVTA↓YDGAG
WNV
KQKKR↓GGTAG
ACAGAVTLSN
RRSRR↓SLTVQ
APAYS↓FNCLG
VNVHA↓DTGCA
SRVNA↓YNADM
 
1NS2A/NS2B
1NS2B/NS3
1NS3/NS4A
1NS4A/2K
22K/NS4B
1NS4B/NS5
T’Ho virus
GMTRR↓GWPAS
RLQKR↓GGVLW
AAGKR↓SASAI
PEKQR↓SQTDN
SAVSA↓NEMGW
PKVKR↓GGGTG
ROCV
CATKR↓GWPAS
KIHKR↓GGVLW
AAGKR↓SAGSM
PEKQR↓SQTDN
SAVSA↓NEMGW
PKVKR↓GGIAA
ILHV
SLGKR↓GWPAS
KIHKR↓GGVMW
AAGKR↓SAGSV
PEKQR↓SQTDN
GAVSA↓NEMGW
PKLKR↓GGGSA
BAGV
PSNRR↓GWPVS
HSPKR↓SGAIW
ACGKR↓SAIGV
PEKQR↓SQTDS
GTVAS↓NEMGW
GSMRR↓GGGKG
WNV
PNRKR↓GWPAT
QYTKR↓GGVLW
ASGKR↓SQIGL
PEKQR↓SQTDN
GAVAA↓NEMGW
PGLKR↓GGAKG
Predicted cleavage sites for ROCV, WNV and BAGV were identified in earlier studies ([8]; [6]; [27]; [32]). Most of the predicted cleavage sites for ILHV are described in the corresponding Genbank entry (accession no. AAV34155) and the remainder determined here. Underlined sequences have been experimentally verified. Cleavage events are mediated by 1the viral NS2B/NS3 serine protease ([6]; [7]; [5]), 2a host signal peptidase [6], 3the cellular furin protease ([6]; [35]; [41]) and 4a membrane-bound host protease in the endoplasmic reticulum [15].
Potential N-linked glycosylation sites were identified using the NetNGlyc 1.0 Server (available at http://​www.​cbs.​dtu.​dk/​services/​NetNGlyc/​) with the consensus sequence defined as Asn-X-Ser/Thr (where X is not Pro) in the context of specific surrounding sequences. Fourteen Asn-X-Ser/Thr motifs were identified in the T’Ho virus polyprotein sequence. Eight motifs are predicted to be utilized by N-linked glycans: these are located at prM15, prM148, E154, NS1130, NS1207, NS2A122, NS5234 and NS5655.

Phylogenetic analysis

A ML phylogenetic tree was constructed by RAxML v8.2.8 using the complete ORF sequences of T’Ho virus and all 30 mosquito-borne flaviviruses (species and subtypes) listed in the Ninth Report of the International Committee on Taxonomy of Viruses (Fig. 1). Cell fusing agent virus (an insect-specific flavivirus) was used as the outgroup. Phylogenetic trees were also constructed using Bayesian, NJ and MP methods (data not shown). In all trees, T’Ho virus was most closely related to ROCV and ILHV. The Bayesian posterior support for this grouping was strong (100%), in agreement with similarly strong bootstrap support (100% for ML, 100% for MP, 94% for NJ). These viruses cluster within a larger clade that contains the Ntaya group viruses with the exception of ZIKV, although in phylogenies based on short conserved polymerase motifs used in taxonomic classification ZIKV appears more closely related to the Ntaya group viruses [24]. The Bayesian posterior support for this topological arrangement is 100%, but bootstrap support is weaker ranging from 49% for MP to 79% for NJ. Trees generated by all methods agree on the composition of all clades shown in Fig. 1 with 98% or more bootstrap support, but the relative placement of these clades is uncertain (data not shown).

Discussion

We report the complete genome sequence of T’Ho virus, a novel putative flavivirus discovered in Cx. quinquefasciatus from the Yucatan Peninsula of Mexico. The closest known relatives of T’Ho virus are ROCV and ILHV, that both belong to the Ntaya group of mosquito-borne flaviviruses, with 67.4% and 65.9% nt. identity, respectively. Based on genetic criteria, we propose that T’Ho virus is classified as a new species within the genus Flavivirus. It has been suggested that flaviviruses with >84% nucleotide sequence identity should be classified within the same species [28]. The genome of T’Ho virus has only 58.8–67.4% nt. identity (mean: 63.4%) to the seven viral species and subtypes currently assigned to the Ntaya group, and this amount of genetic relatedness is not dissimilar to that observed between group members (≥57.9% identity; mean: 65.9%). Phylogenetic analysis of full polyprotein sequences revealed that T’Ho virus forms a distinct clade with Ntaya group viruses, except for ZIKV. Based on previous sequence analysis of a 1358-nt region of the NS5 gene of T’Ho virus that indicated it to be genetically equidistant to ILHV, SLEV, WNV, ROCV and JEV, we speculated that T’Ho virus could also serologically resembled WNV [16]; however, the NS5 gene is one of the most conserved regions of the flavivirus genome [6] and sequence alignments and phylogenetic studies performed using relatively short, conserved sequences are not as robust as those performed with complete genomes.
The two closest known relatives of T’Ho virus are recognized human pathogens. ROCV was responsible for several epidemics of severe encephalitis in Brazil in the 1970s [10, 11, 17, 44]. A case fatality rate of 10% and long-term sequelae in 20% of the surviving patients were reported. ILHV has been sporadically isolated from humans in Central America, South America and the Caribbean, and symptomatic infections are usually characterized by fever, headache, myalgia and arthralgia although central nervous system manifestations have also been reported [25, 37, 39, 40, 45]. Because T’Ho virus is most closely related to known human pathogens, it too could be a cause of human disease. Most other Ntaya group viruses are also recognized pathogens; ZIKV is a cause of febrile illness, neonatal microcephaly and linked to Guillain-Barré syndrome [47]. BAGV, ITV and TMUV are known avian pathogens [4, 9, 30]. Most JE group viruses are also serious pathogens of humans and other vertebrates [21].
An isolate of T’Ho virus is not available; thus, experimental infection studies cannot currently be performed to assess the vector and reservoir competence of mosquito and vertebrate species likely to be involved in its amplification. However, probable vectors and reservoir hosts of T’Ho virus can be inferred from the information available for its closest known relatives. The amplification cycles of ROCV and ILHV are not well defined but most isolations have been made from Aedes and Psorophora spp. mosquitoes (particularly Ps. ferox) with birds implicated as principal reservoir hosts [2, 12, 17, 19, 31, 34]. Most other Ntaya group viruses are primarily maintained in transmission cycles between Culex spp. mosquitoes and birds [13, 22, 43], the notable exception being ZIKV which cycles between Aedes spp. mosquitoes and primates [47]. JE group viruses are primarily maintained in transmission cycles between Culex spp. mosquitoes and birds [21]. It is therefore likely that the principal amplification vectors of T’Ho virus are Culex, Aedes and/or Psorophora spp. mosquitoes and the principal reservoir hosts are birds.

Conclusion

In conclusion, we describe a novel species, T’Ho virus, of the genus Flavivirus, whose closest known relatives are human pathogens; thus, it is feasible to suggest that T’Ho virus may be an unrecognized cause of human disease in the Yucatan Peninsula of Mexico. Our report enables directly the creation of specific PCR diagnostic assays. However, serological cross-reactivity is common with flaviviruses [3], as exemplified in the ongoing ZIKV pandemic where the differential diagnosis of ZIKV and dengue virus infections is difficult [46]. Accordingly, virus isolates or a recombinant virus will be needed for serosurveillance studies in Latin America where several other flaviviruses, including dengue virus, yellow fever virus, ZIKV, WNV, ROCV and ILHV, may co-circulate in overlapping geographic areas.

Acknowledgments

This study was supported by the National Institutes of Health awards 5R21AI067281, U19-AI057158 (Northeast Biodefense Center), U19-AI109761 (Center for Research in Diagnostics and Discovery) and AI088647; the United States Department of Defense and an intramural grant from Iowa State University.

Availability of data and materials

The genome sequence of T’Ho virus has been deposited into the Genbank database.

Authors’ contributions

TB and WIL coordinated the high-throughput sequencing experiments, BJB performed the 5′ and 3′ RACE experiments and prepared the first draft of the manuscript, KSD constructed the phylogenetic trees and MAL, JEG, JAF and CMW coordinated the mosquito collections. All authors reviewed and approved the manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not Applicable.

Ethics approval and consent to participate

Not Applicable.

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