A new Aura virus isolate in Brazil shows segment duplication in the variable region of the nsP3 gene

A new Aura virus isolate in Brazil shows segment duplication in the variable region of the nsP3 gene Background: A new isolate of Aura virus serendipitously discovered as a cell culture contaminant is reported in this manuscript. Aura virus belongs to the family Togaviridae and is classified in the genus Alphavirus. There are only two reports of Aura virus isolation from mosquitoes in the scientific literature, and the existence of a vertebrate host is still unknown. The discovery of this new isolate was based on transmission electron microscopy and nucleic acid amplification through a non-specific RT-PCR amplification protocol followed by sequencing. Results: Genetic analysis has shown that the new virus shares a high degree of identity with the previously described isolate (GenBank: AF126284.1). A major difference was observed in the nsP3 gene in which a 234-nucleotide duplication has been identified. Furthermore, a pronounced difference was observed in cell cultures compared to the data available for the previously described isolate. Cell permissiveness and phenotypic characteristics in C6/36, Vero and BHK-21 cells were found to differ from previous reports. This may be due to the genetic differences that have been observed. Conclusions: The genetic and biological characteristics of thenew Aura virusisolate aresuggestiveofviral adaptation to the cell substrate. The development of a cDNA clone will lend a perspective and better understanding of these results as well as open avenues for its use as a biotechnological tool, as seen for other alphaviruses. Keywords: Aura virus, nsP3, Duplication Background The first isolations of AURAV were carried out in Aura virus (AURAV) is a member of the family Togaviridae, 1959, 1960 and 1961 by Causey et al. [3]frompools genus Alphavirus. Most alphaviruses are arthropod-borne of Culex sp. and Aedes serratus mosquitoes that were viruses (arboviruses) that are involved in the etiology of collected in the vicinity of the city of Belém (Pará, human viral diseases whose main symptoms are rash, fever Brazil). Some years later, this same virus was isolated and arthralgia (Chikungunya virus, Mayaro virus, Ross River from Aedes serratus collected in Misiones Province in virus, and O’nyong-nyong virus) or encephalitis (Western Argentina [4]. As there are no other reports in the equine encephalitis virus, Eastern equine encephalitis virus scientific literature of new isolations, the distribution and Venezuelan equine encephalitis virus) [1]. Their gen- is considered to be restricted to South America [5]. ome consists of a positive sense single-stranded RNA of Despite being a virus that seems to be restricted to approximately 11.7 kb presenting two open reading frames mosquitoes, it is not considered an insect-specific with a cap at its 5' end and a poly-A tail at its 3' end [2]. virus according to Bolling et al. [6]. It also does not possess a known vertebrate host; to date, it is considered non-pathogenic to humans [3]. Initial hemagglutination inhibition and complement fixation * Correspondence: clsantos@fiocruz.br studies indicate that this virus is more closely related Laboratory of Molecular Virology, Instituto Carlos Chagas, FIOCRUZ, Rua Prof. to Western equine encephalitis virus (WEEV) and Algacyr Munhoz Mader 3775, Cidade Industrial, Curitiba, PR 81350-010, Brazil Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This 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. Mosimann et al. Parasites & Vectors (2018) 11:321 Page 2 of 10 Sindbis virus (SINV). However, serum neutralization Results studies indicate that it is quite antigenically different To address this question, we performed transmission from these viruses [7, 8]. In later studies, the nucleo- electron microscopy (TEM) of C6/36 cells infected with tide sequencing of the prototype strain of AURAV BR/P05. As seen in Fig. 1, most of the identified viral (BeAR 10315) showed a higher genetic identity with particles were in close proximity to the cell, as if they SINV [9], and more recent phylogenetic studies of had just budded from the cell membrane. This result the genus Alphavirus have confirmed a closer genetic was not compatible with TEM of DENV infection [10]. relationship with SINV and WEEV [5]. Next, we used an adapted non-specific RT-PCR While working with a supernatant of the fifth amplification protocol that was designed to amplify passage (BR/P05) in an insect cell culture of a clin- any kind of viral nucleic acid. First, we carried out an ical sample in which dengue virus (DENV) type 3 enrichment step, which consisted of supernatant fil- had been previously identified, a phenotype that was tration through 0.22 μm, virion precipitation using not compatible with DENV infection was noticed. polyethylene glycol 8000 and NaCl and DNase diges- During infection kinetics (24, 48 and 72 h) in the tion to eliminate any cellular DNA. Next, total nu- Huh7.5 and C6/36 cells, the percentage of infected cleic acids were extracted and either directly used in cells could not clearly be distinguished from the degenerate-oligonucleotide primed polymerase chain mock-infected cells when measured through flow cy- reaction (DOP-PCR), as previously described by tometry using an anti-flavivirus monoclonal antibody Nanda et al. [11], or first subjected to reverse tran- (4G2). However, when the supernatants of these in- scription using random primers. The amplified DNA fection kinetics were titrated by plaque assay in C6/ was cloned into a TA vector and sequenced. The se- 36 cell cultures, the titer of the supernatants from quences presented homology with AURAV strain the C6/36 cell cultures increased over time, while al- BeAr10315 (NC_003900.1 or AF126284.1), which is most no virus could be detected in the supernatant the only complete sequence of AURAV available in of Huh7.5 cell cultures. These results raised suspi- the GenBank database (Additional file 1: Table S1 and cion of the presence of a different virus in the BR/ Additional file 1: Supplementary sequence data). In P05 sample. addition, the virions seen in the TEM presented an Fig. 1 TEM of mock (a) and BR/P05 (b-d) C6/36 infected cells at 48 h post-infection. Arrows point to some of the virus particles that are budding or have just budded from the cell membrane. b through d represent progressively higher magnification fields of infected cells. Scale-bars: a, b, 200 nm; c, d, 100 nm Mosimann et al. Parasites & Vectors (2018) 11:321 Page 3 of 10 average size of 55.0 nm (Fig. 1) in accordance with a or focus immunodetection assays, failed, suggesting previous report on AURAV [12]. an insect-host specificity. We have also tried to detect Once the virus had been identified, we proceeded with infection in Vero and BHK-21 cells through IFA. Only its full genetic characterization through genome sequen- when using very high MOIs (40 and 80 MOI for cing. For that purpose, specific primers (Additional file BHK-21 and 80 MOI for Vero) was it possible to 1: Table S2) were designed taking into account the gen- visualize a few scattered positive cells (Additional file 1: ome sequence available in GenBank (AF126284.1) and Figure S6). the results obtained through the sequencing of the non-specifically amplified cloned RT-PCR fragments Discussion during the identification of this new isolate. The genome Rümenapf et al. [14] had previously shown that BHK-21 amplification strategy consisted of RT-PCR fragments clone 15, Vero, primary chicken embryo fibroblasts covering the whole genome that possessed a minimal (CEF) and C6/36 cells were permissive to AURAV infec- overlap required for full genome assembly. A compari- tion. In their work, AURAV was titrated in BHK-21 cells son of the sequence of this new isolate (GenBank: through plaque assay [14]. On the other hand, Lascano MG761767) with the one previously described shows et al. [12] carried out a study on the morphogenesis of that they share significant nucleotide identity (95.4%) Aura virus particles in CEF and the brains of newborn and deduced-amino acidic sequence (ORF1: 92.9% and mice infected through intracerebral inoculation. New- ORF2: 96.6%). Detailed homology information for each born mice intracerebral inoculation was also used for gene is shown in Fig. 2. The specific polymorphic virus titration in this later case [12]. In addition to our nucleotide and amino acid residues are shown in results, no report of the CPE in mosquito cell lines after Additional file 1: Alignment, and its genetic relationship infection with AURAV has been found in the scientific with other alphaviruses is depicted in Fig. 3. The most literature. Notwithstanding, Garmashova et al. [15] have striking difference is seen in the sequence of the variable shown that nsP2 is associated with CPE development in region of the nsP3 gene, which shows a 234-nucleotide the SINV model. In accordance with this finding, follow- duplication (highlighted in light yellow and light green ing nsP3, the gene that presented the highest number of in Additional file 1: Alignment and Fig. 2). non-synonymous mutations in BR/P05 was nsP2 (Fig. 2 As mentioned, BR/P05 was first detected in the fifth and Additional file 1: Alignment). passage of a DENV clinical sample in cell culture. To The new AURAV isolate reported in this manuscript carry out the biological characterization experiments, was first identified in the fifth passage in cell culture of a different dilutions of the BR/P05 supernatant were DENV-3 isolate. This sample was not isolated in our la- incubated with the anti-flavivirus monoclonal antibody boratory and was initially received as a cell culture (mAb) 4G2, and this mixture was used to infect C6/36 supernatant (then named as P01). We have tested all cells. The goal was to neutralize any DENV particle that previous passages (P01-P04) through RT-PCR for DENV could still be present in the BR/P05 sample and hence and AURAV. Amplification was positive for both targets have a homogeneous preparation of AURAV. The super- in all of these passages (Additional file 1: Figure S7). natant of the infection corresponding to the highest Mock infected C6/36 cells used as control, showed no dilution of BR/P05 incubated with the anti-flavivirus amplification. This result indicates that the cell line used antibody was used to prepare an AURAV stock for the in our laboratory was not contaminated. Unfortunately, cell culture experiments (BR/P07) (Additional file 1: there is no information on the passage history of this Figure S1). It is important to emphasize that we could sample previous to its arrival in our laboratory. As this not detect any DENV in BR/P05 neither through virus is considered non-pathogenic to humans we can one-step RT-PCR (Additional file 1: Figure S2a) nor only hypothesize that the identification of AURAV in through indirect immunofluorescence using mAb 4G2 this sample was a result of cell culture contamination (Additional file 1: Figure S2b). previous to its arrival in our laboratory; however, this is AURAV stock titration was carried out in C6/36 cells very difficult to track at this point. (Additional file 1: Figure S3) through a plaque assay Therefore it can be hypothesized that the genetic and once this cell line exhibits a cytopathic effect (CPE) (Fig. 4 biological differences observed in the present work are and Additional file 1: Figure S4). Infection could be con- the result of a long period of interaction of this virus firmed through an indirect immunofluorescence assay with the same cell substrate (possibly C6/36 cells), which (IFA) using anti-alphavirus mAb 1A4B-6 [13](Fig. 4). The resulted in a high adaptation to it. In favor of this theory, Aedes pseudoscutellaris (AP-61) mosquito cell line has Weaver et al. [16] reported the substitution of the opal also been shown to be permissive to viral infection stop codon present in the C-terminus of the nsP3 gene (Additional file 1: Figure S5). Our attempts to titrate the by an arginine or cysteine after Eastern equine encephal- present AURAV isolate in BHK-21 cells, either by plaque itis virus (EEEV) adaptation to C6/36 cells. Furthermore, Mosimann et al. Parasites & Vectors (2018) 11:321 Page 4 of 10 Fig. 2 Comparison of the sequences of AF126284 and the new isolate of AURAV (BR/P05). A schematic representation of the Alphavirus genome is also shown. At the very top is an enhanced representation of the nsP3 gene that highlights the 234-nucleotide duplication that has been identified in BR/P05. The green and yellow boxes represent the duplicated sequence, and the black line in AF126284 represents the absence of the duplicated sequence in this genome EEEV adaptation to C6/36 also resulted in a fitness loss Results of this study suggest that EILV structural pro- for BHK-21 viral infection [16]. In AURAV BR/P05, the teins do not mediate efficient attachment and entry into substitution of the opal stop codon with an arginine was mammalian cells. Furthermore, EILV non-structural pro- also observed. teins are unable to sustain continued viral replication in According to Bolling et al. [6] the only insect-specific these cells. Thus, EILV host-restriction was considered alphavirus is Eilat virus (EILV). A recent study by Nasar to depend on multiple genes. As observed for the et al. [17] investigated the host restriction of EILV. AURAV new isolate (BR/P05), EILV can also be titrated Mosimann et al. Parasites & Vectors (2018) 11:321 Page 5 of 10 Fig. 3 Phylogenetic analysis based on the alignment of the nucleotide sequences of the alphavirus concatenated ORFs. Segments of the nsP3 and C were excluded for not presenting reliable alignments. The tree was inferred using the MrBayes (v.3.2.6) software and is based on the general time reversible model with gamma-distributed rate variation and a proportion of invariable sites (GTR+I+G). The numbers shown to the right of the nodes represent posterior probabilities. Representatives from all species of alphaviruses have been included, except for the WEEV complex. The tree was midpoint rooted, and the sequence of the new isolate is highlighted in the black box (MG761767/AURAV BR/P05). Strains were labeled according to GenBank accession number/abbreviation, and the bar indicates nucleotide substitutions per site. Further details on the dataset used for phylogenetic analysis can be accessed in Additional file 1: Table S3 Fig. 4 Indirect immunofluorescence assays of C6/36 cells infected or not (mock) with 1 MOI BR/P07. At 3 days post-infection, the cell monolayer was fixed and permeabilized with methanol:acetone (1:1) at -20 °C and then incubated with anti-alphavirus monoclonal antibody (mAb) clone 1A4B-6 followed by goat anti-mouse IgG (H+L) Alexa Fluor 488 conjugate. Pictures were taken using a Leica DMI 6000B inverted microscope attached to a Leica DFC365 FX camera, and the images were visualized and processed using the Leica Application Suite Advanced Fluorescence 3.1.0 software Mosimann et al. Parasites & Vectors (2018) 11:321 Page 6 of 10 through plaque assay in insect cells [18] suggesting this with SFV, SINV and CHIKV nsP3 has also been observed. may be an insect-specific alphavirus feature. As previ- The duplication also resulted in the presence of the DILV- ously reported by Nasar et al. [18] our analysis shows a QAEVH motif in triplicate (underlined in red in Additional close phylogenetic relationship between AURAV and file 1: Alignment), whose significance is unknown. In EILV (Fig. 3). In spite of this, they constitute different addition, a difference in the hydrophobicity plot can be species making it difficult to identify potential detected in the region of duplication, which presents insect-specific amino acid motifs. non-synonymous substitutions (Additional file 1:Figure S8). The role of non-structural protein 3 (nsP3) of alpha- This difference may influence nsP3 interaction with virus is still not fully elucidated. Studies using nsP3 mu- membranes and/or other hydrophobic residues of other tants showed it to be required in the viral RNA molecules [24]. synthesis [19]. It has also been shown to co-localize with Speculating about the origin of the observed dupli- other non-structural proteins to sites of viral RNA cation, it has been noticed that when the sequence of replication [20]. These data are also supported by AURAV AF126284 is aligned against itself using the co-immunoprecipitation experiments [20]. Although the BLAST 2 sequences algorithm, the expected results of N-terminal portion of the protein is conserved among 100% identity were observed. In addition, in the gen- different alphaviruses, its C-terminal portion is not [2]. omic region where the duplication has been identified Three domains have been identified in nsP3: the macro in the new isolate, there is also a segment (5238– domain, the alphavirus unique domain (AUD) and the 5417, in green) which presents high identity (82%) hypervariable domain (HVD) [19]. The macro domain is with another neighboring segment (5415–5594, in located in the N-terminal portion and is evolutionarily blue) (Additional file 1: Figure S9a). This could have conserved across different Phylos [19]. The AUD is also resulted in homologous recombination [25]orreplica- a conserved element, albeit only among alphaviruses, tion error, which may have originated this duplication. and is located downstream of the macro domain in the During the synthesis of the negative strand, when the central part of nsP3 [19]. On the other hand, the HVD, replication complex reaches position 5238, the two which is located in the C-terminal portion of nsP3, toler- strands of the replicative intermediate may temporar- ates significant changes in sequence [19]. Nevertheless, ily detach, or the replication complex may switch the detection of conserved elements among the isolates strands. Then, the recently copied upstream segment of the same alphavirus species points to the evolutionar- (5238–5417, in green) in the negative strand may ily advantageous characteristic of these sequences [19]. hybridize to the neighboring downstream segment HVD sequences have also been shown to have an impact (5415–5594, in blue) in the positive strand and re- on the formation of distinct virus-specific protein sume the synthesis of the negative strand, resulting in complexes [21]. the duplication of the upstream segment (dashed The scientific literature indicates that the segment du- green line, see Additional file 1: Figure S9b for fur- plication observed in the variable region of the nsP3 ther details). However, as this genomic segment, gene could play a role in the adaptation of AURAV to which presents partial identity, has a smaller size (181 different hosts. Foy et al. [22] demonstrated that the nucleotides) than the observed duplication (234 nu- phosphorylation of the HVD of nsP3 is more critical for cleotides), other additional events would have been the growth of Venezuelan equine encephalitis virus in necessary to account for all the observed differences. insect cells (C7/10) than in vertebrate cell lines (BHK-21 and NIH 3T3). They have also shown that the permis- siveness to different cell lines is selectively affected by Conclusions the substitution of the nsP3 HVD by a heterologous In summary, we report the finding of a new isolate of protein-encoding sequence [22]. In addition, Neuvonen AURAV. It is difficult to track its origin, but its genetic et al. [23] have identified SH3-binding motifs in the and biological characteristics are suggestive of viral C-terminal portion of SFV, SINV and Chikungunya virus adaptation to the cell substrate. To better understand (CHIKV) nsP3. These SH3-binding motifs specifically the impact of the observed genetic differences on the interact with the host cell proteins amphiphysin 1 and biological phenotype, an infectious cDNA clone is amphiphysin 2, and this interaction has been shown to needed. In addition to contributing to unraveling some play a role in viral replication. The 234-nucleotide of the questions raised in this manuscript (i.e. What is duplication observed in AURAV BR/P05 also results in the role of the nsP3 segment duplication in the virus the duplication of an SH3-binding motif (PVPPPR). As adaptation to the insect cell line? Are the nsP2 muta- discussed by Neuvonen et al. [23], the insect amphiphy- tions related to CPE development?), it can become a sin gene is very similar to the mammalian genes and valuable molecular tool as already described for other encodes a homologous SH3-binding motif whose interaction alphaviruses [26, 27]. Mosimann et al. Parasites & Vectors (2018) 11:321 Page 7 of 10 Methods culture (mock) supernatant was subjected to the same Isolate identification procedure as a negative control. Transmission electron microscopy The total nucleic acid extracted as described above For transmission electron microscopy, C6/36 cells were was used as a template in reverse transcription, using infected or not (mock) with BR/P05 for 48 h and fixed the ImProm-II Reverse Transcriptase (Promega, Madison, with 2.5% glutaraldehyde in 0.1 M sodium cacodylate WI, USA) with random primers (Invitrogen, Carlsbad, buffer for 1 h. Cells were washed twice with 0.1 M caco- CA, USA), followed by PCR or directly used as a template dylate buffer, pH 7.2, and subsequently fixed in 1% in the PCR reaction. Reverse transcription was carried out OsO , 0.8% KFe (CN) and 5 mM CaCl diluted in 0.1 according to the manufacturer’s instructions. The PCR re- 4 6 2 M cacodylate buffer for 1 h. After fixation, the cells were action was performed in 20 mM Tris-HCl (pH 8.4), 50 washed, dehydrated in increasing concentrations of mM KCl, 1.5 mM MgCl ,200 μMdNTPs,0.06U/μlTaq acetone and embedded in Poly/Bed 812 resin for 72 h at DNA polymerase (IBMP, Curitiba, Brazil) and 1.2 μM 60 °C. Ultrathin sections were stained for 30 min with DOP Primer (Additional file 1: Table S2), as previously uranyl acetate and for 2 min with lead citrate before described by Nanda et al. [11]. The following cycling con- analysis in a JEOL JEM-1400 transmission electron ditions were applied: one cycle of 95 °C for 5 min, 5 cycles microscope (JEOL, Tokyo, Japan) at 80 kV [28]. For of 94 °C for 1 min, 30 °C for 1.5 min, ramping to 72 °C at average size estimation, 152 virions were manually mea- 0.2°C/s, and72°Cfor 3 min,followedby 35cyclesof94°C sured based on the size scale for the mean calculation. for 1 min, 55 °C for 1 min, and 72 °C for 2 min, with the addition of 14 s/cycle to the extension step [11]. The amplified DNA was purified using the High Pure PCR Non-specific RT-PCR nucleic acid amplification Product Purification Kit (Roche, Mannheim, Germany) fol- The supernatant (10 ml) of a three-day post-infection in- lowing the protocol for the purification of PCR products in sect cell culture was centrifuged at 3220× g for 30 min solution after amplification, cloned in the pGEM-T-easy at 4 °C to pull-down any cell debris. The supernatant of vector (Promega) and used to transform Escherichia coli this centrifugation was then filtered through a 0.22 μm Top10F’ cells. Plasmid DNA was purified from twenty se- sterile filter [29]. The filtered supernatant (10 ml) was lected white colonies through miniprep using the Wizard precipitated with 1.4 g of polyethylene glycol 8000 and Plus SV Minipreps DNA Purification System (Promega), 0.47 g of NaCl and incubated overnight at 4 °C under and the presence of an insert was confirmed through NotI gentle agitation. Then, it was centrifuged at 3200× g (New England Biolabs, Ipswich, MA, USA) digestion. The for 30 min at 4 °C. The supernatant was discarded, concentration of the purified plasmid DNAs was measured the pellet was resuspended with 0.5 ml of DPBS using a Nanodrop ND-1000 (Thermo Fisher Scientific, ++ ++ containing Ca and Mg (Lonza, Walkersville, MD, Wilmington, DE, USA), and then sent to Macrogen (Seoul, USA) [30] and incubated with 100 U of Turbo Korea), where they were sequenced using an Applied DNAse I (Ambion, Austin, TX, USA) at 37 °C for 2 h. The Biosystems 3730xl DNA Analyzer (Applied Biosystems, viral particle lysis was carried out through incubation Foster City, IA, USA). with 10% (v/v) 10% SDS and 1% (v/v) 14.3 M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, Genetic characterization USA) at 72 °C for 3 min. Total nucleic acids were ex- RNA was extracted from the supernatant of BR/P05 tracted through the addition of an equal volume of using the QIAamp Viral RNA Mini Kit (Qiagen, phenol:chlorophorm:isoamyl alcohol (25:24:1), mixing Valencia, CA, USA) according to the manufacturer’s and centrifugation at 20238× g for 2 min at room instructions. This RNA was amplified through reverse temperature (RT). The aqueous phase was then mixed transcription using the ImProm-II Reverse Transcriptase with an equal volume of chloprophorm:isoamyl alcohol and random primers (Invitrogen) followed by PCR using (24:1) and centrifuged at 20238× g for 2 min at RT. The the Qiagen LongRange PCR System (Qiagen) according aqueous phase was mixed with 2.5 volumes of ethanol to the manufacturer’s instructions. Specific primers and 0.8 M LiCl (Ambion) and incubated at -20 °C for (Additional file 1: Table S2) were designed taking into approximately 64 h. Subsequently, this solution was account only the full-length genome sequence available centrifuged at 20817× g for 15 min at 4 °C, the super- in GenBank (NC_003900.1 or AF126284.1) and the results natant was discarded and 0.3 ml of 70% ethanol was obtained through the sequencing of the non-specifically added to the pellet. Finally, this mixture was vortexed cloned and amplified RT-PCR fragments during the and centrifuged at 20817× g for 7 min at 4 °C, the identification of this new isolate. The RT-PCR amplified supernatant discarded, and the pellet dried at RT and fragments were purified using either the High Pure PCR resuspended with 50 μlofH O. All reagents cited in Product Purification Kit or the QIAquick Gel Extraction this section were nuclease free. A non-infected cell Kit (Qiagen). The concentration of the purified DNA Mosimann et al. Parasites & Vectors (2018) 11:321 Page 8 of 10 fragments was measured using a Nanodrop ND-1000, and (Gibco) at 28 °C. AP61 cells were maintained in Leibo- then sent to Macrogen, where they were sequenced using vitz’s L-15 medium supplemented with 10% FBS, 0.56% an Applied Biosystems 3730xl DNA Analyzer. tryptose and 25 μg/ml of gentamicin at 28 °C. Vero To sequence the 5' and 3' ends of the AURAV that (ATCC, CCL-81) and BHK-21 (ATCC, CCL10) cells were identified in BR/P05, RNA extracted from the were maintained in DMEM-F12 (Gibco) supplemented supernatant of the infected cell cultures was first with 10% FBS, 100 U/ml of penicillin and 100 μg/ml of decapped through incubation with tobacco acid pyro- streptomycin (Sigma-Aldrich). All cells used in this work phosphatase (Epicentre, Madison, WI, USA) at 37 °C for tested negative for mycoplasma contamination. 1 h. The decapped RNA was purified by phenol extrac- The RNA extracted from BR/P05 tested negative for tion as described in the “Nonspecific RT-PCR nucleic dengue in a one-step RT-PCR protocol used for dengue acid amplification” section, but instead of 0.8 M of LiCl, virus serotyping [38]. However, to be sure that the 10% (v/v) 3 M sodium acetate, pH 5.3, was used in the supernatant that was going to be used in the biological precipitation step. This decapped RNA was ligated characterization experiments was free from dengue virus -1 -6 through incubation with T4 RNA ligase (New England serotype 3, different dilutions (10 –10 ) of the Biolabs, Ipswich, MA, USA) at 37 °C for 30 min followed supernatant of BR/P05 were incubated at 37 °C with by 16 h at 16 °C. The ligated product was purified by anti-flavivirus monoclonal antibody (4G2) for 2 h. This phenol extraction as described above and used as the mixture was incubated with C6/36 cells (3.5 × 10 cells/ template for an RT-PCR reaction with the AURAV23F well, seeded the day before in a 6-well plate) for 1 h at and AURAV2R primers. The PCR product was purified 28 °C. Then, the inoculum was discarded, the cell with a High Pure PCR Product Purification Kit and either monolayer was washed once with a sterile PBS solution directly sequenced or inserted into the pGEM-T-easy and 3 ml/well of medium added. Two days post-infection, vector for nucleotide sequencing. the supernatants were collected, aliquoted and stored at The nucleotide sequences were assembled using the -80 °C. The supernatant of the infection that was carried phred (version 0.020425.c), phrap (version 1.080812) and out with the least amount of virus was titrated and used consed (version 17.0) software packages [31–34], and to infect C6/36 cells at a multiplicity of infection (MOI) of whenever needed, pairwise alignment was carried out 0.01 to produce the viral stock (BR/P07) for the biological using the Basic Local Alignment Search Tool (BLAST) characterization experiments (Additional file 1:Figure S1). [35]. The complete genome consensus sequence was When needed, titration was carried out in C6/36 submitted to the GenBank database under the accession monolayers as follows. Twenty-four-well plates were number MG761767. seeded the day before with 1 × 10 cells/well and in- The dataset used in the phylogenetic analysis fected with tenfold dilutions (in duplicate) of viral super- (Additional file 1: Table S3) was based on the dataset natants. Dilutions were made in the medium without used by Nasar et al. [18]; however, we have excluded FBS supplementation, and incubation was carried out at WEEV and WEEV-like and concatenated the two ORFs 28 °C for 1 h. After the incubation period, the inoculum as done by Forrester et al. [36] when undertaking was discarded, and the cells were overlaid with 500 μlof full-genome analysis. The sequences were aligned using a 1:1 mixture of CMC 3,2% and Leibovitz’s L-15 medium the muscle algorithm [37] as implemented in MEGA supplemented with 10% FBS, 0.52% tryptose and 50 μg/ml (version 7.0.14), and the segments of the nsP3 and C of gentamicin. Plates were then sealed with tape and that did not present reliable alignments, were excluded. incubated for 7 days at 28 °C. At this point, the overlay A consensus tree was inferred using MrBayes (version was discarded, and cell monolayers were washed thrice 3.2.6 ×86). MrBayes analyses were carried under the with PBS, fixed with 3% paraformaldehyde in PBS at RT general time reversible model with gamma-distributed for 20 min and stained with a solution of 0.8% crystal rate variation and a proportion of invariable sites (GTR violet (w/v), 0.5% NaCl (w/v) and 10% formamide (v/v) in +I+G) using three hot chains and one cold chain and ethanol. Plaques were counted in duplicate wells, and the was run for 2 million generations with a 25% burn-in. mean was calculated. This value was divided by the For the production of the Additional file 1: Alignment volume of inoculum and multiplied by the dilution factor and Fig. 2, the alignment in the nsP3 genomic region to obtain the result in PFU/ml. was manually edited to clearly represent the duplication. The permissiveness of AP61, BHK-21 and Vero cells was tested. For this purpose, 48-well plates were seeded Cell culture, viral stock and biological characterization the day before with either 1 × 10 cells/well (Vero and C6/36 (ATCC, CRL-1660) cells were maintained in BHK-21) or 5 × 10 cells/well (AP61). Cells were then Leibovitz’s L-15 medium (Gibco, Grand Island, NY, infected (duplicate wells) with MOIs of 1, 10 and 40 in USA) supplemented with 5% fetal bovine serum (FBS; the case of AP-61 or 10, 40 and 80 in the case of Vero Gibco), 0.26% tryptose and 25 μg/ml of gentamicin and BHK-21. Infection conditions were incubation at 28 °C Mosimann et al. Parasites & Vectors (2018) 11:321 Page 9 of 10 for 1 h for AP61 and at 37 °C for 1 h for Vero and BHK-21 Funding FIOCRUZ, Fundação Araucária and Secretaria de Ciência, Tecnologia e Ensino cells. After this incubation period, the cell monolayer was Superior do Estado do Paraná. washed twice with a sterile PBS solution, and 500 μl/well of medium was added. Three days post-infection, the su- Availability of data and materials All data generated or analyzed during this study are included in this published pernatants were collected, aliquoted, and stored at -80 °C, article and its additional files. The sequence generated in this study was and the cells were fixed and permeabilized with a mixture submitted to the GenBank database under the accession number MG761767. of methanol:acetone (1:1) at -20 °C for at least 1 h. This Supplementary sequence data are included in the additional files. time point was chosen based on the results of Rümenapf Authors’ contributions et al. [14]. Afterward, the cells were incubated at 37 °C for ALPM: genetic characterization, phylogenetic analysis, infection experiments 1 h with anti-alphavirus monoclonal antibody 1A4B-6 and manuscript preparation. MKS: genetic characterization and infection (EMD Millipore, Temecula, CA, USA) diluted 1:300, experiments. LFC: transmission electron microscopy and virion size estimation. CNDS: study design and manuscript preparation. All authors read washed three times with PBS, incubated at 37 °C for 1 h and approved the final manuscript. with goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa fluor 488 (Life Technologies, Eugene, OR, Competing interests The authors declare that they have no competing interests. USA) diluted 1:100 and washed again three times with PBS. Finally, the cells were overlaid with 100 μl/well of PBS containing 10% glycerol and observed in a Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published Leica DMI 6000B inverted microscope (Leica, Mannheim, maps and institutional affiliations. Germany). Pictures were taken using this equipment, which is attached to a Leica DFC365 FX camera, and the Author details Laboratory of Molecular Virology, Instituto Carlos Chagas, FIOCRUZ, Rua Prof. images were visualized and processed using the Leica Algacyr Munhoz Mader 3775, Cidade Industrial, Curitiba, PR 81350-010, Brazil. Application Suite Advanced Fluorescence 3.1.0 software. Present Address: Department of Genetics, Evolution and Bioagents, Institute of Biology, University of Campinas, Campinas, SP 13083-970, Brazil. Laboratory of Cell Biology, Instituto Carlos Chagas, FIOCRUZ, Rua Prof. Additional file Algacyr Munhoz Mader 3775, Cidade Industrial, Curitiba, PR 81350-010, Brazil. Additional file 1: Alignment. Nucleotide and amino acid alignment of Received: 17 January 2018 Accepted: 20 May 2018 AF126284 and BR/P05. Figure S1. Workflow depicting the protocol used for DENV neutralization and production of AURAV viral stock (BR/P07) from BR/P05. Figure S2. DENV detection in BR/P05. Figure S3. Result of References BR/P07 titration by plaque assay in C6/36 cells. Figure S4. Brightfield 1. Griffin DE. Alphaviruses. In: Knipe DM, Howley PM, editors. Fields virology. images of C6/36 cells infected or not (mock) with 0.1 MOI BR/P07 at 3 4th ed. Philadelphia: Lippincott Williams & Wilkins; 2001. p. 917–62. days post-infection. Figure S5. IFA of AP-61 cells infected or not (mock) 2. Strauss JH, Strauss EG. The alphaviruses: gene expression, replication, and with 1 MOI BR/P07. Figure S6. IFA of Vero and BHK-21 cells infected or evolution. Microbiol Rev. 1994;58:491–562. not (mock) with 80 MOI BR/P07. Figure S7. RT-PCR testing of P01-P04 for 3. Causey OR, Casals J, Shope RE, Udomsakdi S. Aura and Una, two new group a AURAV and b DENV. Figure S8. nsP3 hydrophobicity plot of AF126284 A arthropod-borne viruses. Am J Trop Med Hyg. 1963;12:777–81. and BR/P05. Figure S9. Mechanism of duplication hypothesis. Table S1. 4. Oro JGB, Sabattini M, LFG F. Aura, nuevo arbovirus del grupo A para la BLAST analyses of DOP-PCR sequencing results. Table S2. Primers used in República Argentina. Cienc Invest. 1967;23:180–3. RT-PCR and sequencing. Table S3. Dataset used in phylogenetic analysis. 5. Weaver SC, Winegar R, Manger ID, Forrester NL. Alphaviruses: population Supplementary sequence data. (PDF 2561 kb) genetics and determinants of emergence. Antiviral Res. 2012;94:242–57. 6. Bolling B, Weaver S, Tesh R, Vasilakis N. Insect-specific virus discovery: significance for the arbovirus community. Viruses. 2015;7:4911–28. Abbreviations 7. Karabatsos N. Antigenic relationships of group A arboviruses by plaque AUD: Alphavirus unique domain; AURAV: Aura virus; CHIKV: Chikungunya reduction neutralization testing. Am J Trop Med Hyg. 1975;24:527–32. virus; CPE: Cytopathic effect; DENV: Dengue virus; DOP-PCR: Degenerate- 8. Calisher CH, Karabatsos N, Lazuick JS, Monath TP, Wolff JS. Reevaluation of oligonucleotide primed polymerase chain reaction; EEEV: Eastern equine the western equine encephalitis antigenic complex of alphaviruses (family encephalitis virus; EILV: Eilat virus; FBS: Fetal bovine serum; Togaviridae) as determined by neutralization tests. Am J Trop Med Hyg. HVD: Hypervariable domain; IFA: Indirect immunofluorescence assay; 1988;38:447–52. mAb: Monoclonal antibody; MOI: Multiplicity of infection; PFU: Plaque 9. Rümenapf T, Strauss EG, Strauss JH. Aura virus is a New World forming units; RT: Room temperature; RT-PCR: Reverse transcription representative of Sindbis-like viruses. Virology. 1995;208:621–33. associated with polymerase chain reaction; SFV: Semliki Forest virus; 10. Ko KK, Igarashi A, Fukai K. Electron microscopic observations on Aedes SINV: Sindbis virus; TEM: Transmission electron microscopy; WEEV: Western albopictus cells infected with dengue viruses. Arch Virol. 1979;62:41–52. equine encephalitis virus 11. Nanda S, Jayan G, Voulgaropoulou F, Sierra-Honigmann AM, Uhlenhaut C, McWatters BJP, et al. Universal virus detection by degenerate- Acknowledgments oligonucleotide primed polymerase chain reaction of purified viral nucleic The authors thank the Fiocruz Network of Technology Platforms for the use acids. J Virol Methods. 2008;152:18–24. of their core facilities for microscopy at Instituto Carlos Chagas/Fiocruz-PR, 12. Lascano EF, Berría MI, Oro JGB. Morphogenesis of Aura virus. J Virol. Brazil, and Bruna Hilzendeger Marcon for technical support. The authors also 1969;4:271–82. thank Adriana Delfraro Vázquez and Juliano Bordignon for critical reading 13. Powers AM, Roehrig JT. Alphaviruses. Methods Mol Biol. 2011;665:17–38. and Wagner Nagib de Souza Birbeire for the graphic in Additional file 1: 14. Rümenapf T, Strauss EG, Strauss JH. Subgenomic mRNA of Aura alphavirus is Figure S1 and for his help with the editing of Additional file 1: Figure S3. packaged into virions. 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A new Aura virus isolate in Brazil shows segment duplication in the variable region of the nsP3 gene

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Abstract

Background: A new isolate of Aura virus serendipitously discovered as a cell culture contaminant is reported in this manuscript. Aura virus belongs to the family Togaviridae and is classified in the genus Alphavirus. There are only two reports of Aura virus isolation from mosquitoes in the scientific literature, and the existence of a vertebrate host is still unknown. The discovery of this new isolate was based on transmission electron microscopy and nucleic acid amplification through a non-specific RT-PCR amplification protocol followed by sequencing. Results: Genetic analysis has shown that the new virus shares a high degree of identity with the previously described isolate (GenBank: AF126284.1). A major difference was observed in the nsP3 gene in which a 234-nucleotide duplication has been identified. Furthermore, a pronounced difference was observed in cell cultures compared to the data available for the previously described isolate. Cell permissiveness and phenotypic characteristics in C6/36, Vero and BHK-21 cells were found to differ from previous reports. This may be due to the genetic differences that have been observed. Conclusions: The genetic and biological characteristics of thenew Aura virusisolate aresuggestiveofviral adaptation to the cell substrate. The development of a cDNA clone will lend a perspective and better understanding of these results as well as open avenues for its use as a biotechnological tool, as seen for other alphaviruses. Keywords: Aura virus, nsP3, Duplication Background The first isolations of AURAV were carried out in Aura virus (AURAV) is a member of the family Togaviridae, 1959, 1960 and 1961 by Causey et al. [3]frompools genus Alphavirus. Most alphaviruses are arthropod-borne of Culex sp. and Aedes serratus mosquitoes that were viruses (arboviruses) that are involved in the etiology of collected in the vicinity of the city of Belém (Pará, human viral diseases whose main symptoms are rash, fever Brazil). Some years later, this same virus was isolated and arthralgia (Chikungunya virus, Mayaro virus, Ross River from Aedes serratus collected in Misiones Province in virus, and O’nyong-nyong virus) or encephalitis (Western Argentina [4]. As there are no other reports in the equine encephalitis virus, Eastern equine encephalitis virus scientific literature of new isolations, the distribution and Venezuelan equine encephalitis virus) [1]. Their gen- is considered to be restricted to South America [5]. ome consists of a positive sense single-stranded RNA of Despite being a virus that seems to be restricted to approximately 11.7 kb presenting two open reading frames mosquitoes, it is not considered an insect-specific with a cap at its 5' end and a poly-A tail at its 3' end [2]. virus according to Bolling et al. [6]. It also does not possess a known vertebrate host; to date, it is considered non-pathogenic to humans [3]. Initial hemagglutination inhibition and complement fixation * Correspondence: clsantos@fiocruz.br studies indicate that this virus is more closely related Laboratory of Molecular Virology, Instituto Carlos Chagas, FIOCRUZ, Rua Prof. to Western equine encephalitis virus (WEEV) and Algacyr Munhoz Mader 3775, Cidade Industrial, Curitiba, PR 81350-010, Brazil Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This 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. Mosimann et al. Parasites & Vectors (2018) 11:321 Page 2 of 10 Sindbis virus (SINV). However, serum neutralization Results studies indicate that it is quite antigenically different To address this question, we performed transmission from these viruses [7, 8]. In later studies, the nucleo- electron microscopy (TEM) of C6/36 cells infected with tide sequencing of the prototype strain of AURAV BR/P05. As seen in Fig. 1, most of the identified viral (BeAR 10315) showed a higher genetic identity with particles were in close proximity to the cell, as if they SINV [9], and more recent phylogenetic studies of had just budded from the cell membrane. This result the genus Alphavirus have confirmed a closer genetic was not compatible with TEM of DENV infection [10]. relationship with SINV and WEEV [5]. Next, we used an adapted non-specific RT-PCR While working with a supernatant of the fifth amplification protocol that was designed to amplify passage (BR/P05) in an insect cell culture of a clin- any kind of viral nucleic acid. First, we carried out an ical sample in which dengue virus (DENV) type 3 enrichment step, which consisted of supernatant fil- had been previously identified, a phenotype that was tration through 0.22 μm, virion precipitation using not compatible with DENV infection was noticed. polyethylene glycol 8000 and NaCl and DNase diges- During infection kinetics (24, 48 and 72 h) in the tion to eliminate any cellular DNA. Next, total nu- Huh7.5 and C6/36 cells, the percentage of infected cleic acids were extracted and either directly used in cells could not clearly be distinguished from the degenerate-oligonucleotide primed polymerase chain mock-infected cells when measured through flow cy- reaction (DOP-PCR), as previously described by tometry using an anti-flavivirus monoclonal antibody Nanda et al. [11], or first subjected to reverse tran- (4G2). However, when the supernatants of these in- scription using random primers. The amplified DNA fection kinetics were titrated by plaque assay in C6/ was cloned into a TA vector and sequenced. The se- 36 cell cultures, the titer of the supernatants from quences presented homology with AURAV strain the C6/36 cell cultures increased over time, while al- BeAr10315 (NC_003900.1 or AF126284.1), which is most no virus could be detected in the supernatant the only complete sequence of AURAV available in of Huh7.5 cell cultures. These results raised suspi- the GenBank database (Additional file 1: Table S1 and cion of the presence of a different virus in the BR/ Additional file 1: Supplementary sequence data). In P05 sample. addition, the virions seen in the TEM presented an Fig. 1 TEM of mock (a) and BR/P05 (b-d) C6/36 infected cells at 48 h post-infection. Arrows point to some of the virus particles that are budding or have just budded from the cell membrane. b through d represent progressively higher magnification fields of infected cells. Scale-bars: a, b, 200 nm; c, d, 100 nm Mosimann et al. Parasites & Vectors (2018) 11:321 Page 3 of 10 average size of 55.0 nm (Fig. 1) in accordance with a or focus immunodetection assays, failed, suggesting previous report on AURAV [12]. an insect-host specificity. We have also tried to detect Once the virus had been identified, we proceeded with infection in Vero and BHK-21 cells through IFA. Only its full genetic characterization through genome sequen- when using very high MOIs (40 and 80 MOI for cing. For that purpose, specific primers (Additional file BHK-21 and 80 MOI for Vero) was it possible to 1: Table S2) were designed taking into account the gen- visualize a few scattered positive cells (Additional file 1: ome sequence available in GenBank (AF126284.1) and Figure S6). the results obtained through the sequencing of the non-specifically amplified cloned RT-PCR fragments Discussion during the identification of this new isolate. The genome Rümenapf et al. [14] had previously shown that BHK-21 amplification strategy consisted of RT-PCR fragments clone 15, Vero, primary chicken embryo fibroblasts covering the whole genome that possessed a minimal (CEF) and C6/36 cells were permissive to AURAV infec- overlap required for full genome assembly. A compari- tion. In their work, AURAV was titrated in BHK-21 cells son of the sequence of this new isolate (GenBank: through plaque assay [14]. On the other hand, Lascano MG761767) with the one previously described shows et al. [12] carried out a study on the morphogenesis of that they share significant nucleotide identity (95.4%) Aura virus particles in CEF and the brains of newborn and deduced-amino acidic sequence (ORF1: 92.9% and mice infected through intracerebral inoculation. New- ORF2: 96.6%). Detailed homology information for each born mice intracerebral inoculation was also used for gene is shown in Fig. 2. The specific polymorphic virus titration in this later case [12]. In addition to our nucleotide and amino acid residues are shown in results, no report of the CPE in mosquito cell lines after Additional file 1: Alignment, and its genetic relationship infection with AURAV has been found in the scientific with other alphaviruses is depicted in Fig. 3. The most literature. Notwithstanding, Garmashova et al. [15] have striking difference is seen in the sequence of the variable shown that nsP2 is associated with CPE development in region of the nsP3 gene, which shows a 234-nucleotide the SINV model. In accordance with this finding, follow- duplication (highlighted in light yellow and light green ing nsP3, the gene that presented the highest number of in Additional file 1: Alignment and Fig. 2). non-synonymous mutations in BR/P05 was nsP2 (Fig. 2 As mentioned, BR/P05 was first detected in the fifth and Additional file 1: Alignment). passage of a DENV clinical sample in cell culture. To The new AURAV isolate reported in this manuscript carry out the biological characterization experiments, was first identified in the fifth passage in cell culture of a different dilutions of the BR/P05 supernatant were DENV-3 isolate. This sample was not isolated in our la- incubated with the anti-flavivirus monoclonal antibody boratory and was initially received as a cell culture (mAb) 4G2, and this mixture was used to infect C6/36 supernatant (then named as P01). We have tested all cells. The goal was to neutralize any DENV particle that previous passages (P01-P04) through RT-PCR for DENV could still be present in the BR/P05 sample and hence and AURAV. Amplification was positive for both targets have a homogeneous preparation of AURAV. The super- in all of these passages (Additional file 1: Figure S7). natant of the infection corresponding to the highest Mock infected C6/36 cells used as control, showed no dilution of BR/P05 incubated with the anti-flavivirus amplification. This result indicates that the cell line used antibody was used to prepare an AURAV stock for the in our laboratory was not contaminated. Unfortunately, cell culture experiments (BR/P07) (Additional file 1: there is no information on the passage history of this Figure S1). It is important to emphasize that we could sample previous to its arrival in our laboratory. As this not detect any DENV in BR/P05 neither through virus is considered non-pathogenic to humans we can one-step RT-PCR (Additional file 1: Figure S2a) nor only hypothesize that the identification of AURAV in through indirect immunofluorescence using mAb 4G2 this sample was a result of cell culture contamination (Additional file 1: Figure S2b). previous to its arrival in our laboratory; however, this is AURAV stock titration was carried out in C6/36 cells very difficult to track at this point. (Additional file 1: Figure S3) through a plaque assay Therefore it can be hypothesized that the genetic and once this cell line exhibits a cytopathic effect (CPE) (Fig. 4 biological differences observed in the present work are and Additional file 1: Figure S4). Infection could be con- the result of a long period of interaction of this virus firmed through an indirect immunofluorescence assay with the same cell substrate (possibly C6/36 cells), which (IFA) using anti-alphavirus mAb 1A4B-6 [13](Fig. 4). The resulted in a high adaptation to it. In favor of this theory, Aedes pseudoscutellaris (AP-61) mosquito cell line has Weaver et al. [16] reported the substitution of the opal also been shown to be permissive to viral infection stop codon present in the C-terminus of the nsP3 gene (Additional file 1: Figure S5). Our attempts to titrate the by an arginine or cysteine after Eastern equine encephal- present AURAV isolate in BHK-21 cells, either by plaque itis virus (EEEV) adaptation to C6/36 cells. Furthermore, Mosimann et al. Parasites & Vectors (2018) 11:321 Page 4 of 10 Fig. 2 Comparison of the sequences of AF126284 and the new isolate of AURAV (BR/P05). A schematic representation of the Alphavirus genome is also shown. At the very top is an enhanced representation of the nsP3 gene that highlights the 234-nucleotide duplication that has been identified in BR/P05. The green and yellow boxes represent the duplicated sequence, and the black line in AF126284 represents the absence of the duplicated sequence in this genome EEEV adaptation to C6/36 also resulted in a fitness loss Results of this study suggest that EILV structural pro- for BHK-21 viral infection [16]. In AURAV BR/P05, the teins do not mediate efficient attachment and entry into substitution of the opal stop codon with an arginine was mammalian cells. Furthermore, EILV non-structural pro- also observed. teins are unable to sustain continued viral replication in According to Bolling et al. [6] the only insect-specific these cells. Thus, EILV host-restriction was considered alphavirus is Eilat virus (EILV). A recent study by Nasar to depend on multiple genes. As observed for the et al. [17] investigated the host restriction of EILV. AURAV new isolate (BR/P05), EILV can also be titrated Mosimann et al. Parasites & Vectors (2018) 11:321 Page 5 of 10 Fig. 3 Phylogenetic analysis based on the alignment of the nucleotide sequences of the alphavirus concatenated ORFs. Segments of the nsP3 and C were excluded for not presenting reliable alignments. The tree was inferred using the MrBayes (v.3.2.6) software and is based on the general time reversible model with gamma-distributed rate variation and a proportion of invariable sites (GTR+I+G). The numbers shown to the right of the nodes represent posterior probabilities. Representatives from all species of alphaviruses have been included, except for the WEEV complex. The tree was midpoint rooted, and the sequence of the new isolate is highlighted in the black box (MG761767/AURAV BR/P05). Strains were labeled according to GenBank accession number/abbreviation, and the bar indicates nucleotide substitutions per site. Further details on the dataset used for phylogenetic analysis can be accessed in Additional file 1: Table S3 Fig. 4 Indirect immunofluorescence assays of C6/36 cells infected or not (mock) with 1 MOI BR/P07. At 3 days post-infection, the cell monolayer was fixed and permeabilized with methanol:acetone (1:1) at -20 °C and then incubated with anti-alphavirus monoclonal antibody (mAb) clone 1A4B-6 followed by goat anti-mouse IgG (H+L) Alexa Fluor 488 conjugate. Pictures were taken using a Leica DMI 6000B inverted microscope attached to a Leica DFC365 FX camera, and the images were visualized and processed using the Leica Application Suite Advanced Fluorescence 3.1.0 software Mosimann et al. Parasites & Vectors (2018) 11:321 Page 6 of 10 through plaque assay in insect cells [18] suggesting this with SFV, SINV and CHIKV nsP3 has also been observed. may be an insect-specific alphavirus feature. As previ- The duplication also resulted in the presence of the DILV- ously reported by Nasar et al. [18] our analysis shows a QAEVH motif in triplicate (underlined in red in Additional close phylogenetic relationship between AURAV and file 1: Alignment), whose significance is unknown. In EILV (Fig. 3). In spite of this, they constitute different addition, a difference in the hydrophobicity plot can be species making it difficult to identify potential detected in the region of duplication, which presents insect-specific amino acid motifs. non-synonymous substitutions (Additional file 1:Figure S8). The role of non-structural protein 3 (nsP3) of alpha- This difference may influence nsP3 interaction with virus is still not fully elucidated. Studies using nsP3 mu- membranes and/or other hydrophobic residues of other tants showed it to be required in the viral RNA molecules [24]. synthesis [19]. It has also been shown to co-localize with Speculating about the origin of the observed dupli- other non-structural proteins to sites of viral RNA cation, it has been noticed that when the sequence of replication [20]. These data are also supported by AURAV AF126284 is aligned against itself using the co-immunoprecipitation experiments [20]. Although the BLAST 2 sequences algorithm, the expected results of N-terminal portion of the protein is conserved among 100% identity were observed. In addition, in the gen- different alphaviruses, its C-terminal portion is not [2]. omic region where the duplication has been identified Three domains have been identified in nsP3: the macro in the new isolate, there is also a segment (5238– domain, the alphavirus unique domain (AUD) and the 5417, in green) which presents high identity (82%) hypervariable domain (HVD) [19]. The macro domain is with another neighboring segment (5415–5594, in located in the N-terminal portion and is evolutionarily blue) (Additional file 1: Figure S9a). This could have conserved across different Phylos [19]. The AUD is also resulted in homologous recombination [25]orreplica- a conserved element, albeit only among alphaviruses, tion error, which may have originated this duplication. and is located downstream of the macro domain in the During the synthesis of the negative strand, when the central part of nsP3 [19]. On the other hand, the HVD, replication complex reaches position 5238, the two which is located in the C-terminal portion of nsP3, toler- strands of the replicative intermediate may temporar- ates significant changes in sequence [19]. Nevertheless, ily detach, or the replication complex may switch the detection of conserved elements among the isolates strands. Then, the recently copied upstream segment of the same alphavirus species points to the evolutionar- (5238–5417, in green) in the negative strand may ily advantageous characteristic of these sequences [19]. hybridize to the neighboring downstream segment HVD sequences have also been shown to have an impact (5415–5594, in blue) in the positive strand and re- on the formation of distinct virus-specific protein sume the synthesis of the negative strand, resulting in complexes [21]. the duplication of the upstream segment (dashed The scientific literature indicates that the segment du- green line, see Additional file 1: Figure S9b for fur- plication observed in the variable region of the nsP3 ther details). However, as this genomic segment, gene could play a role in the adaptation of AURAV to which presents partial identity, has a smaller size (181 different hosts. Foy et al. [22] demonstrated that the nucleotides) than the observed duplication (234 nu- phosphorylation of the HVD of nsP3 is more critical for cleotides), other additional events would have been the growth of Venezuelan equine encephalitis virus in necessary to account for all the observed differences. insect cells (C7/10) than in vertebrate cell lines (BHK-21 and NIH 3T3). They have also shown that the permis- siveness to different cell lines is selectively affected by Conclusions the substitution of the nsP3 HVD by a heterologous In summary, we report the finding of a new isolate of protein-encoding sequence [22]. In addition, Neuvonen AURAV. It is difficult to track its origin, but its genetic et al. [23] have identified SH3-binding motifs in the and biological characteristics are suggestive of viral C-terminal portion of SFV, SINV and Chikungunya virus adaptation to the cell substrate. To better understand (CHIKV) nsP3. These SH3-binding motifs specifically the impact of the observed genetic differences on the interact with the host cell proteins amphiphysin 1 and biological phenotype, an infectious cDNA clone is amphiphysin 2, and this interaction has been shown to needed. In addition to contributing to unraveling some play a role in viral replication. The 234-nucleotide of the questions raised in this manuscript (i.e. What is duplication observed in AURAV BR/P05 also results in the role of the nsP3 segment duplication in the virus the duplication of an SH3-binding motif (PVPPPR). As adaptation to the insect cell line? Are the nsP2 muta- discussed by Neuvonen et al. [23], the insect amphiphy- tions related to CPE development?), it can become a sin gene is very similar to the mammalian genes and valuable molecular tool as already described for other encodes a homologous SH3-binding motif whose interaction alphaviruses [26, 27]. Mosimann et al. Parasites & Vectors (2018) 11:321 Page 7 of 10 Methods culture (mock) supernatant was subjected to the same Isolate identification procedure as a negative control. Transmission electron microscopy The total nucleic acid extracted as described above For transmission electron microscopy, C6/36 cells were was used as a template in reverse transcription, using infected or not (mock) with BR/P05 for 48 h and fixed the ImProm-II Reverse Transcriptase (Promega, Madison, with 2.5% glutaraldehyde in 0.1 M sodium cacodylate WI, USA) with random primers (Invitrogen, Carlsbad, buffer for 1 h. Cells were washed twice with 0.1 M caco- CA, USA), followed by PCR or directly used as a template dylate buffer, pH 7.2, and subsequently fixed in 1% in the PCR reaction. Reverse transcription was carried out OsO , 0.8% KFe (CN) and 5 mM CaCl diluted in 0.1 according to the manufacturer’s instructions. The PCR re- 4 6 2 M cacodylate buffer for 1 h. After fixation, the cells were action was performed in 20 mM Tris-HCl (pH 8.4), 50 washed, dehydrated in increasing concentrations of mM KCl, 1.5 mM MgCl ,200 μMdNTPs,0.06U/μlTaq acetone and embedded in Poly/Bed 812 resin for 72 h at DNA polymerase (IBMP, Curitiba, Brazil) and 1.2 μM 60 °C. Ultrathin sections were stained for 30 min with DOP Primer (Additional file 1: Table S2), as previously uranyl acetate and for 2 min with lead citrate before described by Nanda et al. [11]. The following cycling con- analysis in a JEOL JEM-1400 transmission electron ditions were applied: one cycle of 95 °C for 5 min, 5 cycles microscope (JEOL, Tokyo, Japan) at 80 kV [28]. For of 94 °C for 1 min, 30 °C for 1.5 min, ramping to 72 °C at average size estimation, 152 virions were manually mea- 0.2°C/s, and72°Cfor 3 min,followedby 35cyclesof94°C sured based on the size scale for the mean calculation. for 1 min, 55 °C for 1 min, and 72 °C for 2 min, with the addition of 14 s/cycle to the extension step [11]. The amplified DNA was purified using the High Pure PCR Non-specific RT-PCR nucleic acid amplification Product Purification Kit (Roche, Mannheim, Germany) fol- The supernatant (10 ml) of a three-day post-infection in- lowing the protocol for the purification of PCR products in sect cell culture was centrifuged at 3220× g for 30 min solution after amplification, cloned in the pGEM-T-easy at 4 °C to pull-down any cell debris. The supernatant of vector (Promega) and used to transform Escherichia coli this centrifugation was then filtered through a 0.22 μm Top10F’ cells. Plasmid DNA was purified from twenty se- sterile filter [29]. The filtered supernatant (10 ml) was lected white colonies through miniprep using the Wizard precipitated with 1.4 g of polyethylene glycol 8000 and Plus SV Minipreps DNA Purification System (Promega), 0.47 g of NaCl and incubated overnight at 4 °C under and the presence of an insert was confirmed through NotI gentle agitation. Then, it was centrifuged at 3200× g (New England Biolabs, Ipswich, MA, USA) digestion. The for 30 min at 4 °C. The supernatant was discarded, concentration of the purified plasmid DNAs was measured the pellet was resuspended with 0.5 ml of DPBS using a Nanodrop ND-1000 (Thermo Fisher Scientific, ++ ++ containing Ca and Mg (Lonza, Walkersville, MD, Wilmington, DE, USA), and then sent to Macrogen (Seoul, USA) [30] and incubated with 100 U of Turbo Korea), where they were sequenced using an Applied DNAse I (Ambion, Austin, TX, USA) at 37 °C for 2 h. The Biosystems 3730xl DNA Analyzer (Applied Biosystems, viral particle lysis was carried out through incubation Foster City, IA, USA). with 10% (v/v) 10% SDS and 1% (v/v) 14.3 M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, Genetic characterization USA) at 72 °C for 3 min. Total nucleic acids were ex- RNA was extracted from the supernatant of BR/P05 tracted through the addition of an equal volume of using the QIAamp Viral RNA Mini Kit (Qiagen, phenol:chlorophorm:isoamyl alcohol (25:24:1), mixing Valencia, CA, USA) according to the manufacturer’s and centrifugation at 20238× g for 2 min at room instructions. This RNA was amplified through reverse temperature (RT). The aqueous phase was then mixed transcription using the ImProm-II Reverse Transcriptase with an equal volume of chloprophorm:isoamyl alcohol and random primers (Invitrogen) followed by PCR using (24:1) and centrifuged at 20238× g for 2 min at RT. The the Qiagen LongRange PCR System (Qiagen) according aqueous phase was mixed with 2.5 volumes of ethanol to the manufacturer’s instructions. Specific primers and 0.8 M LiCl (Ambion) and incubated at -20 °C for (Additional file 1: Table S2) were designed taking into approximately 64 h. Subsequently, this solution was account only the full-length genome sequence available centrifuged at 20817× g for 15 min at 4 °C, the super- in GenBank (NC_003900.1 or AF126284.1) and the results natant was discarded and 0.3 ml of 70% ethanol was obtained through the sequencing of the non-specifically added to the pellet. Finally, this mixture was vortexed cloned and amplified RT-PCR fragments during the and centrifuged at 20817× g for 7 min at 4 °C, the identification of this new isolate. The RT-PCR amplified supernatant discarded, and the pellet dried at RT and fragments were purified using either the High Pure PCR resuspended with 50 μlofH O. All reagents cited in Product Purification Kit or the QIAquick Gel Extraction this section were nuclease free. A non-infected cell Kit (Qiagen). The concentration of the purified DNA Mosimann et al. Parasites & Vectors (2018) 11:321 Page 8 of 10 fragments was measured using a Nanodrop ND-1000, and (Gibco) at 28 °C. AP61 cells were maintained in Leibo- then sent to Macrogen, where they were sequenced using vitz’s L-15 medium supplemented with 10% FBS, 0.56% an Applied Biosystems 3730xl DNA Analyzer. tryptose and 25 μg/ml of gentamicin at 28 °C. Vero To sequence the 5' and 3' ends of the AURAV that (ATCC, CCL-81) and BHK-21 (ATCC, CCL10) cells were identified in BR/P05, RNA extracted from the were maintained in DMEM-F12 (Gibco) supplemented supernatant of the infected cell cultures was first with 10% FBS, 100 U/ml of penicillin and 100 μg/ml of decapped through incubation with tobacco acid pyro- streptomycin (Sigma-Aldrich). All cells used in this work phosphatase (Epicentre, Madison, WI, USA) at 37 °C for tested negative for mycoplasma contamination. 1 h. The decapped RNA was purified by phenol extrac- The RNA extracted from BR/P05 tested negative for tion as described in the “Nonspecific RT-PCR nucleic dengue in a one-step RT-PCR protocol used for dengue acid amplification” section, but instead of 0.8 M of LiCl, virus serotyping [38]. However, to be sure that the 10% (v/v) 3 M sodium acetate, pH 5.3, was used in the supernatant that was going to be used in the biological precipitation step. This decapped RNA was ligated characterization experiments was free from dengue virus -1 -6 through incubation with T4 RNA ligase (New England serotype 3, different dilutions (10 –10 ) of the Biolabs, Ipswich, MA, USA) at 37 °C for 30 min followed supernatant of BR/P05 were incubated at 37 °C with by 16 h at 16 °C. The ligated product was purified by anti-flavivirus monoclonal antibody (4G2) for 2 h. This phenol extraction as described above and used as the mixture was incubated with C6/36 cells (3.5 × 10 cells/ template for an RT-PCR reaction with the AURAV23F well, seeded the day before in a 6-well plate) for 1 h at and AURAV2R primers. The PCR product was purified 28 °C. Then, the inoculum was discarded, the cell with a High Pure PCR Product Purification Kit and either monolayer was washed once with a sterile PBS solution directly sequenced or inserted into the pGEM-T-easy and 3 ml/well of medium added. Two days post-infection, vector for nucleotide sequencing. the supernatants were collected, aliquoted and stored at The nucleotide sequences were assembled using the -80 °C. The supernatant of the infection that was carried phred (version 0.020425.c), phrap (version 1.080812) and out with the least amount of virus was titrated and used consed (version 17.0) software packages [31–34], and to infect C6/36 cells at a multiplicity of infection (MOI) of whenever needed, pairwise alignment was carried out 0.01 to produce the viral stock (BR/P07) for the biological using the Basic Local Alignment Search Tool (BLAST) characterization experiments (Additional file 1:Figure S1). [35]. The complete genome consensus sequence was When needed, titration was carried out in C6/36 submitted to the GenBank database under the accession monolayers as follows. Twenty-four-well plates were number MG761767. seeded the day before with 1 × 10 cells/well and in- The dataset used in the phylogenetic analysis fected with tenfold dilutions (in duplicate) of viral super- (Additional file 1: Table S3) was based on the dataset natants. Dilutions were made in the medium without used by Nasar et al. [18]; however, we have excluded FBS supplementation, and incubation was carried out at WEEV and WEEV-like and concatenated the two ORFs 28 °C for 1 h. After the incubation period, the inoculum as done by Forrester et al. [36] when undertaking was discarded, and the cells were overlaid with 500 μlof full-genome analysis. The sequences were aligned using a 1:1 mixture of CMC 3,2% and Leibovitz’s L-15 medium the muscle algorithm [37] as implemented in MEGA supplemented with 10% FBS, 0.52% tryptose and 50 μg/ml (version 7.0.14), and the segments of the nsP3 and C of gentamicin. Plates were then sealed with tape and that did not present reliable alignments, were excluded. incubated for 7 days at 28 °C. At this point, the overlay A consensus tree was inferred using MrBayes (version was discarded, and cell monolayers were washed thrice 3.2.6 ×86). MrBayes analyses were carried under the with PBS, fixed with 3% paraformaldehyde in PBS at RT general time reversible model with gamma-distributed for 20 min and stained with a solution of 0.8% crystal rate variation and a proportion of invariable sites (GTR violet (w/v), 0.5% NaCl (w/v) and 10% formamide (v/v) in +I+G) using three hot chains and one cold chain and ethanol. Plaques were counted in duplicate wells, and the was run for 2 million generations with a 25% burn-in. mean was calculated. This value was divided by the For the production of the Additional file 1: Alignment volume of inoculum and multiplied by the dilution factor and Fig. 2, the alignment in the nsP3 genomic region to obtain the result in PFU/ml. was manually edited to clearly represent the duplication. The permissiveness of AP61, BHK-21 and Vero cells was tested. For this purpose, 48-well plates were seeded Cell culture, viral stock and biological characterization the day before with either 1 × 10 cells/well (Vero and C6/36 (ATCC, CRL-1660) cells were maintained in BHK-21) or 5 × 10 cells/well (AP61). Cells were then Leibovitz’s L-15 medium (Gibco, Grand Island, NY, infected (duplicate wells) with MOIs of 1, 10 and 40 in USA) supplemented with 5% fetal bovine serum (FBS; the case of AP-61 or 10, 40 and 80 in the case of Vero Gibco), 0.26% tryptose and 25 μg/ml of gentamicin and BHK-21. Infection conditions were incubation at 28 °C Mosimann et al. Parasites & Vectors (2018) 11:321 Page 9 of 10 for 1 h for AP61 and at 37 °C for 1 h for Vero and BHK-21 Funding FIOCRUZ, Fundação Araucária and Secretaria de Ciência, Tecnologia e Ensino cells. After this incubation period, the cell monolayer was Superior do Estado do Paraná. washed twice with a sterile PBS solution, and 500 μl/well of medium was added. Three days post-infection, the su- Availability of data and materials All data generated or analyzed during this study are included in this published pernatants were collected, aliquoted, and stored at -80 °C, article and its additional files. The sequence generated in this study was and the cells were fixed and permeabilized with a mixture submitted to the GenBank database under the accession number MG761767. of methanol:acetone (1:1) at -20 °C for at least 1 h. This Supplementary sequence data are included in the additional files. time point was chosen based on the results of Rümenapf Authors’ contributions et al. [14]. Afterward, the cells were incubated at 37 °C for ALPM: genetic characterization, phylogenetic analysis, infection experiments 1 h with anti-alphavirus monoclonal antibody 1A4B-6 and manuscript preparation. MKS: genetic characterization and infection (EMD Millipore, Temecula, CA, USA) diluted 1:300, experiments. LFC: transmission electron microscopy and virion size estimation. CNDS: study design and manuscript preparation. All authors read washed three times with PBS, incubated at 37 °C for 1 h and approved the final manuscript. with goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa fluor 488 (Life Technologies, Eugene, OR, Competing interests The authors declare that they have no competing interests. USA) diluted 1:100 and washed again three times with PBS. Finally, the cells were overlaid with 100 μl/well of PBS containing 10% glycerol and observed in a Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published Leica DMI 6000B inverted microscope (Leica, Mannheim, maps and institutional affiliations. Germany). Pictures were taken using this equipment, which is attached to a Leica DFC365 FX camera, and the Author details Laboratory of Molecular Virology, Instituto Carlos Chagas, FIOCRUZ, Rua Prof. images were visualized and processed using the Leica Algacyr Munhoz Mader 3775, Cidade Industrial, Curitiba, PR 81350-010, Brazil. Application Suite Advanced Fluorescence 3.1.0 software. Present Address: Department of Genetics, Evolution and Bioagents, Institute of Biology, University of Campinas, Campinas, SP 13083-970, Brazil. Laboratory of Cell Biology, Instituto Carlos Chagas, FIOCRUZ, Rua Prof. Additional file Algacyr Munhoz Mader 3775, Cidade Industrial, Curitiba, PR 81350-010, Brazil. Additional file 1: Alignment. Nucleotide and amino acid alignment of Received: 17 January 2018 Accepted: 20 May 2018 AF126284 and BR/P05. Figure S1. Workflow depicting the protocol used for DENV neutralization and production of AURAV viral stock (BR/P07) from BR/P05. Figure S2. DENV detection in BR/P05. Figure S3. Result of References BR/P07 titration by plaque assay in C6/36 cells. Figure S4. Brightfield 1. Griffin DE. Alphaviruses. In: Knipe DM, Howley PM, editors. Fields virology. images of C6/36 cells infected or not (mock) with 0.1 MOI BR/P07 at 3 4th ed. Philadelphia: Lippincott Williams & Wilkins; 2001. p. 917–62. days post-infection. Figure S5. IFA of AP-61 cells infected or not (mock) 2. Strauss JH, Strauss EG. The alphaviruses: gene expression, replication, and with 1 MOI BR/P07. Figure S6. IFA of Vero and BHK-21 cells infected or evolution. Microbiol Rev. 1994;58:491–562. not (mock) with 80 MOI BR/P07. Figure S7. RT-PCR testing of P01-P04 for 3. Causey OR, Casals J, Shope RE, Udomsakdi S. Aura and Una, two new group a AURAV and b DENV. Figure S8. nsP3 hydrophobicity plot of AF126284 A arthropod-borne viruses. Am J Trop Med Hyg. 1963;12:777–81. and BR/P05. Figure S9. Mechanism of duplication hypothesis. Table S1. 4. Oro JGB, Sabattini M, LFG F. 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