Characterization of Triniti virus supports its reclassification in the family Peribunyaviridae

Triniti virus (TNTV) has been isolated in Trinidad and Tobago and in Brazil. To date little is known about this virus, which is classified as an ungrouped virus within the family Togaviridae. Here, three isolates of TNTV were characterized both genetically and antigenically. The genome was shown to contain three RNA segments: small (S), medium (M) and large (L). Genome organization, protein sizes and protein motifs were similar to those of viruses in the genus Orthobunyavirus, family Peribunyaviridae. Antigenic reactivity revealed the three TNTV isolates to be closely related, but no serologic cross-reaction with other orthobunyaviruses. Morphological observation by transmission electron microscopy indicated that virus size and symmetry were compatible with those of viruses in the family Peribunyaviridae. Our serological, morphological and molecular results support the taxonomic reclassification of TNTV as a member of the genus Orthobunyavirus, family Peribunyaviridae.


INTRODUCTION
The prototype Triniti virus (TNTV), TRVL 7994, was isolated from a pool of 37 Trichoprosopon mosquitoes collected in Port of Spain, Trinidad, in 1955 ( Fig. 1) [1]. The virus remained uncharacterized until 1964, when scientists at the Trinidad Regional Virus Laboratory (TRVL) demonstrated that TNTV was a new, enveloped and probably arthropodborne virus [2]. In 1981, a study characterized some ungrouped viruses based on their morphological and physicochemical features; it concluded that TNTV was an enveloped spherical RNA virus about 65 nm in diameter, compatible with members of the family Togaviridae, and morphologically undifferentiated from Sindbis virus [3]. Since then, TNTV has been classified as an ungrouped virus in the family Togaviridae [4,5].
The Brazilian TNTV prototype, BeAn 235467, was isolated from the blood of a rodent (Dasyprocta aguti) captured in Barcarena City, Par a State, in 1973 [6]. Subsequently, additional isolates of TNTV have been isolated in the Brazilian Amazon region from a variety of mosquito genera, including Aedes, Sabethes, Wyeomyia and Anopheles ( Fig. 1) [7]. The present study sought to determine the taxonomic identity of TNTV by applying morphological, serological and genomic characterization of the virus prototype and two Brazilian isolates.

Viral isolates
The TNTV prototype was provided by the World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch, Galveston, TX, USA. Two Brazilian TNTV isolates, BeAn 235 467 and BeAr 800584, were obtained from the virus collection of the Department of Arbovirology and Hemorrhagic Fevers, Evandro Chagas Institute, Ananindeua, Par a State, Brazil. BeAr 800 584 was isolated from Aedes (Och) fulvus mosquitoes collected in Bel em City, Par a State, in 2014 (Table 1).

Serological tests
To determine the antigenic relationship of TNTV with other New World orthobunyaviruses and phleboviruses, the TNTV antigen was used in complement fixation (CF) tests against hyperimmune mouse ascitic fluids prepared for each of the 66 viruses listed in Table S1 (available in the online version of this article). CF tests were carried out using a micro-technique, with two full units of complement [8].
The preparation of antigen and antisera was performed as described previously [8].

Transmission electron microscopy
For structural analysis, ultra-thin sections of baby hamster kidney (BHK-21) cells infected with TNTV (TRVL 7994) were fixed for 1 h in a 2.5 v/w formaldehyde mixture prepared from paraformaldehyde powder and 0.1 v/v glutaraldehyde in 0.05 M cacodylate buffer, pH 7.3, to which 0.01 v/v picric acid and 0.03 w/w CaCl 2 were added. The monolayer was washed in 0.1 M cacodylate buffer, cells were scraped off and the pellets were retained for further processing. The pellets were post-fixed in 1 % OsO 4 in 0.1 M cacodylate buffer, pH 7.3, for 1 h, washed with distilled water and en bloc stained with 2 v/v aqueous uranyl acetate for 20 min at 60 C. The pellets were dehydrated in ethanol,  Removal of contaminating host RNA and nucleic acid isolation Supernatant culture fluids from African green monkey kidney (Vero) or BHK-21 cells infected with the three TNTV isolates were collected between 3 and 5 days post-infection, when 80-90 % cytopathic effect was observed. They were centrifuged for 10 min at 2000 rpm (700 g) and then for 5 min at 4000 rpm (2800 g), followed by filtration through a 0.2 µm sterile filter and polyethylene glycol precipitation to enrich for virus particles. Subsequently, the virus pellets were resuspended in 250 µl phosphate-buffered saline, and 25 µl RNase A (final concentration 2 mg ml À1 ) was added to digest cellular RNAs. Next, viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions.

Nucleotide sequencing
Complementary DNA (cDNA) synthesis was performed using the cDNA Synthesis System Kit (Roche Diagnostics, Rotkreuz, Switzerland) and 400 µM random primers (Roche Diagnostics). The reaction solution was purified with the Agencourt AMPure XP Reagent (Beckman Coulter, LaBrea, CA, USA). The cDNA libraries of the Brazilian TNTV isolates were subjected to semiconductor sequencing [9] (Ion Torrent PGM; ThermoFisher Scientific, Waltham, MA, USA) at the genomic core facility of the Center for Technological Innovation, Evandro Chagas Institute. The cDNA library of the TNTV prototype was sequenced on a HiSeq 2500 System (Illumina, San Diego, CA, USA) at the genomic core facility of the University of Texas Medical Branch. The 5¢ and 3¢ termini of non-coding regions (NCR) from the Brazilian isolates were amplified by 5¢/3¢ RACE kits following the manufacturer's instructions (Roche, Basel, Switzerland) using Sanger sequencing method [10].

Genome assembly and phylogenetic analysis
Sequence assembly was carried out using the de novo assembler within the MIRA 4.0 program [11]. Inspection and annotations of putative open reading frames (ORFs) were performed using Geneious 9.1.6 software (Biomatters,   Multiple sequence alignment was performed using the PROMALS3D (http://prodata.swmed.edu/promals3d/pro-mals3d.php) open source program [12]. The dataset used to reconstruct the phylogenetic trees consisted of amino acid sequences obtained for each genome segment of representative members of the genus Orthobunyavirus. A maximum likelihood tree was used to reconstruct the TNTV phylogenetic relationship [13] using RAxML 8.2.9 software [14]. Bootstrap analysis was conducted over 1000 replicates [15].
Confidence values used as criteria for group inclusion or exclusion were calculated based on the mean of amino acid sequence divergence within and between groups, in line with classical taxonomic classification within the genus Orthobunyavirus. A paired t-test was applied and group clustering was considered statistically significant at P-values <0.05. Evaluation of genetic reassortment Natural genetic reassortment was evaluated at nucleotide (nt) level using concatenated ORFs for all three RNA segments (SRNA, MRNA, LRNA). To evaluate genomic shifting, since this is a common process within the Peribunyaviridae family, multiple sequence alignment was carried out first with MAFFT 7 [16] and then with SimPlot 3.5.1 software [17]. Values of permutation trees were assigned as percentages. Genetic reassortment was considered when the percentage of permutation trees was higher than 90 % across the entire genome segment.

RESULTS
Antigenic relationships CF tests confirmed that, based on antigenic reactivity, the three TNTV isolates were closely related to each other ( Table 2). In contrast, no serologic cross-reactions were observed between the TNTV antigen and antibodies prepared against any of the 66 New World orthobunyaviruses and phleboviruses tested ( Table 2).  Fig. 3. The genomes consisted of singlestranded, tri-segmented, negative-sense RNA molecules.

Ultrastructural studies
he Brazilian strains of Triniti virus showed high identity between each other to all segments in comparison with the Trinidad strain (Table 3). Furthermore, BlastX analysis identified the S segment to be most closely related to Lukuni    1212). Additionally, within region III, it was possible to detect the conserved pre-motif A (951 to 980) and motif E (1213 to 1223) described previously [18]. Finally, region IV, located at positions 1236 to 1251 and 1288 to 1304, was also conserved in TNTV.

Phylogenetic analysis and group definition
Regardless of RNA segment, phylogenetic analysis of nucleotide and amino acid sequences showed similar topologies for the N gene, M polyprotein and RdRp. This suggested that the three TNTV isolates belonged to a monophyletic group (bootstrap 1000, P-value <0.05 based on a paired t-test). TNTV isolates were most closely related to Tacaiuma virus, Anopheles A group and Tataguine virus (Fig. 4a-c).

Reassortment analysis
SimPlot analysis failed to provide evidence of any natural reassortment events between the genomic segments of TNTV and other closely related orthobunyaviruses.

DISCUSSION
Despite its isolation more than 60 years ago, relatively little information is available about TNTV. The first report on the virus, in 1964, described some of its physical and biological properties and concluded that TNTV was an enveloped RNA virus, probably arthropod-borne [2]. Results of a second morphological and physicochemical study, published in 1981, concluded that TNTV was an enveloped spherical RNA virus compatible with the Togaviridae family [3]. At the eighth International Committee on Taxonomy of Viruses Report on Virus Taxonomy, TNTV was listed as an unassigned virus among Togaviridae [4]. TNTV was not included in the ninth Report on Virus Taxonomy [19], and the Arbovirus Catalog still lists TNTV as an ungrouped togavirus [5].
Our analysis of the genome organization of TNTV revealed three RNA segments (SRNA, MRNA and LRNA), encoding the N, Gn-NSm-Gc and L proteins, respectively. This genome organization pattern, as well as protein sizes and motifs, is typically found in members of the recently established order Bunyavirales, family Peribunyaviridae [20]. The size of the three RNA segments, the presence of highly conserved nucleotides at the 3¢ and 5¢ complementary terminal sequences, as well as the Gn-NSm-Gc protein organization of the MRNA ORF are compatible with viruses in the genus Orthobunyavirus [20]. Interestingly, the SRNA ORF of TNTV does not encode a NS protein, as observed in other orthobunyaviruses, especially Tataguine, Tacaiuma and Anopheles A and B viruses. The NS protein is an important virulence factor because it acts as an interferon antagonist and thus helps viral particles escape the host immune system. However, it is not essential for viral replication [21][22][23]. TNTV appears to possess various conserved features previously identified in other orthobunyaviruses. These include the Cys residues on the M polyprotein; five potential N-linked glycosylation sites on Gn, including conserved sites at positions N 60 and N 1169 ; and functional motifs, such as zinc-finger and fusion peptide on Gc, as well as RdRp on the L protein [22].  (Fig. 4).
Regardless of the RNA segment, the paired t-test revealed a statistically significant P-value (P<0.05), suggesting that To date, all TNTV isolates have been recovered from mosquitoes and wild rodents. In the initial description of the virus, Spence and co-workers examined 95 human sera from two communities in eastern Trinidad and from TRVL staff. Using a newborn mouse neutralization test, they found that seven individuals (7.4 %) had protective antibodies against the virus. Interestingly, one individual (a TRVL employee) demonstrated seroconversion to the virus in two baseline sera collected six years apart. These data demonstrate the potential of TNTV to infect humans; however, only additional serological studies will confirm the pathogenesis of the virus and the actual threat that it may pose to public and veterinary health.
In summary, our analyses support the reclassification of TNTV into the genus Orthobunyavirus, family Peribunyaviridae and the possibility that it constitutes a new species in this genus. Additional molecular, serological and pathological studies will clarify the potential role of TNTV in human infections.