The genus Alphavirus of the Togaviridae family comprises enveloped, single-stranded positive-sense RNA viruses and currently includes over 30 species. Members of this genus have an ∼11.7 Kb genome with a capped 5′ end and a poly-A tail at the 3′ end and are divided into two open reading frames, the nonstructural and the structural domains . Most alphaviruses are mosquito-borne viruses and have a wide range of vertebrate hosts, including humans . Recently, with advances in viral detection methodologies using next-generation sequencing and bioinformatics tools, a growing number of novel viruses have been described in the Togaviridae family, including viruses with unidentified vertebrate hosts [3–5] and those with fishes as natural hosts . Humans infected by pathogenic alphaviruses exhibit febrile illnesses that may culminate either in encephalitis or arthritis, depending upon the viral etiology. Alphaviruses are broadly distributed on all continents and are primarily transmitted to humans by female mosquitoes during blood meal feeding. These viruses are classically divided into Old World and New World viruses , with Old World viral infections often causing clinical symptoms such as fever, rash, and arthritis, whereas New World viral infections are associated with encephalitis. However, the recent spread of the Chikungunya virus (CHIKV), originally from the Old World to the Americas [8–9], demonstrates the dissemination dynamics of arboviruses worldwide.
In the first half of 2017, the Reference Laboratory of Emerging Viruses from Fiocruz-PR received sixty-two serum samples from patients who presented to the health service with fever, myalgia, headache, backache, retroorbital pain, and arthralgia. Ten of the serum samples were collected during the acute phase of the disease (between 1 and 6 days after the onset of symptoms). These acute-phase serum samples were screened for infection by flavivirus and alphavirus with generic RT–PCR protocols, as well as for MAYV, WNV, and OROV using specific primers, and all rendered negative results. All of the patients were inhabitants of Marilena municipality (22° 44′ 09″ S, 53° 02′ 24″ W), Paraná State, Southern Brazil. As the etiological agent causing this outbreak was not identified, mosquito samplings were performed in the field and near the houses of the patients.
Marilena is a city with approximately 7,000 inhabitants located at the left margin (downstream) of the Paraná River in confluence with the Paranapanema River (Figure 1). This region is at a low altitude and has a humid subtropical climate zone according to the Köppen-Geiger climate classification map [updated in ref. 10]. In the past few years, this region has presented a high incidence of arbovirus circulation, primarily the dengue virus . Nevertheless, during the epidemiological period 2016/2017, approximately 88% of the patients seeking health care services with clinical symptoms of a dengue-like disease had no laboratory-confirmed diagnosis of dengue , thus constituting an interesting context to perform prospection of new viruses.
Figure 1. Sampling site of CAAV positive pools of mosquitoes (black dots) and the location of Marilena municipality, State of Paraná, Brazil. In the last frame, the county detail with the place of the collection of pool code MS364 is highlighted in red. Pool codes MS680 and MS681 were collected in the margin of Paraná River.
This study describes a novel alphavirus, tentatively named Caainguá virus (CAAV), that was isolated from pools of Culex mosquitoes collected at Marilena municipality, Brazil, during an outbreak of an undiagnosed arthritogenic disease. Here we present the phylogenetic analysis as well as a biological characterization of CAAV, including viral replication in the mosquito, vertebrate and primary human cells, and electron microscopy. So far, the results suggest the discovery of a new insect-specific alphavirus circulating during an undiagnosed human outbreak scenario. Nevertheless, we cannot rule out an epidemiological link between CAAV and human cases due to the intriguing property of CAAV to infect human primary cells.
Materials and methods
A sampling of mosquitoes and molecular screening
Mosquito collection was performed by the Epidemiological Surveillance group of the Paraná State Health Service, concomitant with the ongoing human outbreak of the unknown disease (May and December 2017). Adult mosquitoes were collected using insect aspirators and by active collection techniques between 9:00 am and 4:00 pm and were pooled according to morphological identification, sampling location, and date. Morphological identification was performed according to Lane and Frattini [13.14].
Briefly, mosquitoes were mechanically lysed in RNase-free phosphate-buffered saline (PBS) using a PrecellysTM homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). RNA was extracted using a QIAamp Viral RNA Mini KitTM (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, and the remaining homogenates were stored at −80°C. A volume of 5.6 µL of the extracted RNA was used in the cDNA synthesis step. Alphavirus prospection was performed using generic primers described by Sánchez-Seco et al. . Nested PCR was performed in a final volume of 25 µL. The first-round PCR was performed following an initial denaturation of 2 min at 94°C followed by 40 cycles: 30 s at 94°C, 60 s at 52°C, and 60 s at 72°C, with a final extension at 72°C for 5 min. The PCR reaction contained 2.5 U of Platinum Taq DNA polymerase (Thermo Fisher Scientific, Waltham, USA), 1× buffer, 2.5 mM MgCl2, 0.1 mM of each dNTP, 20 pmol of each primer, and 5 µL of cDNA. The second-round PCR was performed following an initial denaturation of 2 min at 94°C followed by forty cycles: 30 s at 94°C, 30 s at 52°C, and 30 s at 72°C, with a final extension at 72°C for 5 min. The PCR reaction contained 2.5 U of Platinum Taq DNA polymerase, 1× buffer, 2.5 mM MgCl2, 0.1 mM of each dNTP, 20 pmol of each primer, and 1 µL of the first-round PCR product. PCR products were visualized on a 1.5% agarose gel stained with ethidium bromide. Mosquito pools were also screened for flavivirus infection according to the protocol described by Sánchez-Seco et al. .
CAAV isolation and titration
Viral isolation from the Culex mosquito MS681 pool lysate was performed in C6/36 cells (derived from Aedes albopictus larvae) (ATCC® CRL-1660; Manassas, USA). Cells were grown in Leibovitz’s L-15 medium (Gibco, Waltham, USA) supplemented with 5% fetal bovine serum (FBS) (Gibco), 0.26% tryptose (Sigma-Aldrich, St. Louis, USA) and 25 µg/mL gentamicin (Gibco) at 28°C. No cytopathic effect was observed until 14 d.p.i. A subsequent passage was performed (passage 1), in which viral isolation was confirmed by RT–PCR and immunofluorescence assay (IFA).
A foci-forming immunodetection assay in C6/36 cells was used for viral titration . Briefly, tenfold dilutions of viral supernatants were added to cell monolayers followed by a 1-hour incubation at 28°C. After absorption, the inoculum was removed and 500 µL of the overlay (composed of 1,6% carboxymethyl cellulose and L-15 medium supplemented with 5% FBS, 0.26% tryptose and 25 μg/mL of gentamicin) was added to each well. The plates were incubated at 28°C for 7 days. After discarding the overlay, the cells were fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100. Anti-Alphavirus E1 protein (clone 1A4B.6, cat. MAB8754, Merck, Temecula, USA) and anti-mouse IgG AP conjugate (Promega, Madison, USA) were used as the primary and secondary antibodies, respectively. Finally, foci were revealed by the addition of the NBT-BCIP substrate (Promega, Madison, USA), and the titer was expressed in the focus-forming unit (pfu/mL).
Viral genome characterization and phylogenetic analyses
Genomic RNA derived from the isolated virus was amplified and sequenced using a combination of the generic primers Alpha1+ from the protocol of Sánchez-Seco et al.  and Pan-Alpha-R1 from Hermanns et al.  to increase the fragment length. The PCR products were purified using a High Pure PCR Product Purification Kit (Roche, Mannheim, Germany) before sequencing.
Whole-genome sequencing of the isolated virus was performed to confirm the identity of the novel virus. RNA purification was performed using a QIAamp Viral RNA Mini Kit (Qiagen) following the manufacturer’s instructions, excluding the use of the RNA carrier reagent to facilitate sequencing procedures. Purified RNA was used for library construction using a TruSeq RNA kit (Illumina, San Diego, USA) and was sequenced on an Illumina MiSeq platform (2 × 75 bp). The obtained reads were uploaded to CLC Genomics Workbench v.10.5 (Qiagen) and assembled using the De novo assembly pipeline in the default configuration.
To understand the evolutionary relationship of the virus being described in this study, phylogenetic and recombination analyses were performed. After genome sequencing, the generated consensus sequence was aligned with representative sequences for all alphavirus species, its variants and subtypes described so far. Up to three sequences per virus species were downloaded from GenBank (http://ncbi.nlm.nih.gov/genbank/) depending upon availability. Nucleotide sequences were codon aligned using MACSE v2.03 , and ambiguously aligned portions were cleaned using Gblocks v. 0.91b . In addition to the full genome sequences, with fragments of two open reading frames concatenated, alignments encompassing the alphavirus is and the complete sP regions were generated and analyzed separately (results in supplementary material).
Phylogenetic analyses were performed using the Maximum-likelihood (ML) and Bayesian approaches. ML trees were inferred using IQ-TREE, and branch support were calculated using ultrafast bootstrap approximation (UFBoot) with 1000 replicates . Prior to the tree reconstruction, the ModelFinder application , as implemented in IQ-TREE, was used to select the best-fitted nucleotide substitution model. Bayesian trees were estimated using MrBayes v3.2.7 , where the nucleotide substitution model was set according to the ModelFinder results. For each dataset, two runs of four chains each (one cold and three heated) were run for 3.8–10 × 106 generations, with a burn-in of 25%. All parameters estimated for each run showed ESS values above 200, and a final Bayesian majority-rule consensus tree was summarized for each dataset. Finally, the full-length genome alignment was screened for the presence of recombination breakpoints using the GARD algorithm from the HyPhy package v2.3.13  and boot scanning analysis, as implemented in Simplot 3.5.1 . A window size of 1000 bp with a step of 50 bp was used for boot scanning, and sequences were grouped into their respective complexes when appropriate.
DNA barcoding of CAAV hosts
To confirm the mosquito species identity in alphavirus-positive pools, fragments of the cytochrome C oxidase subunit I (COI) mitochondrial gene were amplified using two sets of genetic markers: the primer pair LCO1490 and HCO2198 described by Folmer et al.  and the primer pair F-COI50 and R-COI650 described by Hemmeter et al. . Initially, insect genetic material was obtained from mosquito homogenates using a Genomic DNA Extraction Kit (RBC Real GenomicsTM, Banqiao City, Taiwan). PCR was performed using an initial denaturation of 3 min at 95°C followed by thirty-five cycles: 30 s at 95°C, 30 s at 48°C, and 45 s at 72°C, with a final extension at 72°C for 5 min. PCR reactions contained 2.5 U of Taq DNA polymerase, 1 × PCR buffer, 2 mM MgCl2, 0.4 mM dNTP, 1 µM of each of the forward and reverse primers and 1 µL of template DNA in a final volume of 25 µL. PCR product was purified using a High Pure PCR Product Purification Kit before conventional Sanger sequencing.
Transmission electron microscopy
C6/36 cells were infected with CAAV or CHIKV BR/2015/15010 (isolated from a Brazilian human case in 2015, used as positive control) at an MOI of 1 for 72 h. Mock or infected C6/36 cells were fixed (2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2) at room temperature for one hour. After washing twice with 0.1 M cacodylate buffer, cells were fixed in 1% OsO4, 0.8% Fe(CN)6 and 5 mM CaCl2 diluted in 0.1 M cacodylate buffer at room temperature for 45 min. Cells were washed twice with 0.1 M cacodylate buffer, dehydrated in increasing concentrations of acetone, and embedded in Poly/Bed 812 resin for 72 h at 60°C. Ultrathin sections (60 nm) were collected in copper grids, stained for 45 min with uranyl acetate and for 1 min with lead citrate. Subsequently, the samples were observed in a JEOL JEM-1400 transmission electron microscope operating at 90 keV.
Infection of mosquito and vertebrate cells lines
The arthropod-derived cell lines Aag-2 (ATCC® CCL-125™, derived from Aedes aegypti larvae), AP-61 (derived from Aedes pseudoscutellaris larvae), and C6/36 (ATCC® CRL-1660; derived from Aedes albopictus larvae) were either cultured in Schneider’s Drosophila Medium (Aag-2) or Leibovitz’s L-15 (AP-61 and C6/36) and were maintained at 28°C. The vertebrate cell lines used in this study included Vero E6 (Sigma-Aldrich, 85020206, epithelial cells derived from Cercopithecus aethiops kidney), UMNSAH/DF-1 (ATCC® CRL-12203™, Gallus gallus embryo fibroblasts lineage), BHK-21 (ATCC® CCL-10™, baby Mesocricetus auratus kidney fibroblasts line), ZEM-2S (ATCC® CRL-2147™, Danio rerio embryo fibroblast line), Huh7.5 (ATCC® PTA-8561™, human hepatocellular carcinoma cell line), C6 (ATCC® CCL-107™, Rattus norvegicus glial cells) and A549 cells (ATCC® CCL-185, human lung epithelial cells), all of which were cultivated in the appropriate growth medium at 28°C (ZEM-2S) or 37°C with 5% CO2. Cells were infected with CAAV at MOIs of 1 or 10 for one hour. Venezuelan Equine Encephalitis Virus (VEEV) TC83 was used as a positive control for UMNSAH/DF-1 and ZEM-2S cells, and CHIKV BR/2015/15010 was the positive control for the other cell lines. Subsequently, to avoid any interference of the remaining virus in the inoculum, the cell monolayers were washed three times with PBS before the addition of the appropriate media. Viral replication was evaluated 0, 24 and 72 h.p.i by IFA using a commercial anti-alphavirus antibody (cat. MAB8754, Merck). Images were obtained with a Leica AF6000 Modular System with a 40× objective. Images were acquired with different exposition times to evidence the infected cells, without interfering with the percentage of positive cells.
Primary human cell infection
Peripheral blood samples from six healthy adult donors, with ages ranging between 22 and 40 years and with no history of arbovirus infection, were collected after obtaining written consent (Human Research Ethics Committee from Fiocruz under the number CAAE: 60643816.6.0000.5248). Peripheral blood mononuclear cells (PBMCs) were obtained by density gradient separation with Ficoll-Paque PLUS (density 1.077 g/mL) (GE Life Science). Cells (5 × 105/well, 96 well plates) were infected with CAAV or CHIKV BR/2015/15010 (positive control) at MOIs of 1 or 10 for one hour. After removing the viral inoculum, the cells were washed twice with PBS and PBMCs were maintained at 37°C with 5% CO2 in RPMI 1640 medium (Lonza) supplemented with 100 IU/ml penicillin (Gibco), 100 g/ml streptomycin (Gibco), and 10% FBS. Uninfected C6/36 cell supernatants were used as a mock control. Supernatants were recovered at 24, 48 and 72 h.p.i. and stored at −80°C for viral titration, and the cells were used for flow cytometry analysis. Briefly, the cells were stained with a mix of anti-human monoclonal antibodies (mAb) for surface markers, including anti-CD3-APC (Immunotools), anti-CD4-APC-H7 (BD Biosciences), anti-CD8-PE-Cy5 (BD Biosciences), anti-CD19-PE-Cy7 (BD Biosciences), and anti-CD14-BV450 (BD Biosciences), or their respective isotype controls and were incubated at 4°C for 20 min. After permeabilization with Cytofix/Cytoperm (BD Biosciences), the cells were stained with 100 µL of an anti-alphavirus (cat. MAB8754, Merck) at 1:100 (vol/vol) diluted in Perm/Wash (BD Biosciences). After incubation, the cells were washed and stained with 100 µL of Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) at 1:400 (v/v) in Perm/Wash solution. Finally, the cells were washed and recovered in 200 µL of PBS/1.5% paraformaldehyde. The cell suspensions were analyzed by flow cytometry on a FACSCanto II instrument (BD Biosciences). Data were expressed as means ± SEM. Statistical analyses were performed using PRISM (version 7.0; GraphPad, San Diego, USA). Significance was determined using a paired nonparametric test (Wilcoxon) among different stimulations in the same group. Values of p ≤ .05 were considered significant.
Molecular prospecting and phylogenetic analyses
A total of 1,923 mosquitoes grouped into one hundred twenty-one pools were collected at Marilena municipality, Brazil, where an unknown arthritogenic disease outbreak was taking place (Table S1). Nine genera of mosquitoes were identified by morphological identification: Aedes, Aedeomyia, Anopheles, Culex, Limatus, Mansonia, Psorophora, Sabethes, and Weyomyia. Three out of 121 pools of mosquitoes analyzed yielded positive results for the alphavirus by RT-PCR. Two positive-pools (codes MS680 and MS681) had exclusively female mosquitoes (n = 21 and 9 mosquitoes per pool, respectively), while the third positive-pool (code MS364) contained 7 mosquitoes and was not characterized by gender. Forty-four out of the mosquito pools tested positive for flavivirus in screening by a generic RT–PCR protocol . Amplicons varying from 98 bp to 1167 bp were sequenced. These amplicons were identified as insect-specific flaviviruses (ISFs) using the BLAST algorithm (available in https://blast.ncbi.nlm.nih.gov/Blast.cgi). The alphavirus cDNA fragments, amplified from the mosquito pools, were identified via genomic sequencing of a 455-bp amplicon from the nonstructural protein 4 (nsP4) gene. Nucleotide alignment with sequences retrieved from GenBank showed by the BLAST algorithm a 74% nucleotide identity with a group of encephalitogenic viruses, such as VEEV, Western Equine Encephalitis (WEEV), and Eastern Equine Encephalitis (EEEV). The use of the combined protocols of Sánchez-Seco et al.  and Hermanns et al.  for the same mosquito pools resulted in the amplification of a larger fragment of 1,028 bp corresponding to the nsP4 region that displayed a 76% of similarity to the equine encephalitis complexes. This low score raised the possibility that the identified alphavirus was novel since it met the criteria of the International Committee on Taxonomy of Viruses for a distinct species within an antigenic complex (minimum of a 21% divergence at the nucleotide level).
The virus was successfully isolated from the MS680, MS681 and MS364 pools in C6/36 cells, which presented a mild cytopathic effect. The isolation was confirmed by RT–PCR and by indirect IFA and the identity of the three isolates was confirmed by sequencing of the RT–PCR amplicons. An insect-specific flavivirus was co-isolated with CAAV from the MS680 and MS364 pools. For this reason, the subsequent viral characterization was performed with the virus isolated from the MS681 pool. Viral titers in C6/36 cells ranged from 105 pfu/mL on passage 1 to 107 of/mL on passages 2 and 3.
Next-generation sequencing (NGS) of the third passage of the isolated virus yielded the viral genome in a single contig containing 12,096 bp with an average coverage of 1,846×. Comparison of the complete genome sequence with the GenBank nucleotide database using BLASTn showed a 73% identity with equine encephalitis complexes, corroborating the previous results. The nucleotide sequence was deposited in the GenBank database under the accession number MK353339.
ML and Bayesian methods were used to infer the phylogenetic relationship of the newly isolated virus, hereafter referred to as CAAV, within the genus Alphavirus. Three sequence alignments were analyzed – (i) complete genomes; (ii) nonstructural protein (SCP) region; (iii) structural protein (sP) region – and both Bayesian and ML methods generated the same tree topologies when comparing the same alignment. Using the full genome analysis (Figure 2), CAAV was placed basal (bootstrap support of 77 and posterior probability of 99) to the encephalitogenic alphaviruses belonging to the New World group which comprises VEEV, EEEV, and part of the WEEV complexes.
Figure 2. Maximum likelihood analysis of Caainguá virus incorporated with previously described alphaviruses and its variants based on nucleotide sequences of complete genomes. Bootstrap values over 95% were suppressed, those values lower than 95% are shown above the branches, and the related posterior probability values from Bayesian analysis are shown below the branches. The scale bar indicates the number of substitutions per site