Virus isolation and host-range determination

In April 2014, a total of 12 sediment samples were collected from soda lakes in Southern Nhecolândia, Pantanal, Brazil. In 2012, 8 ocean sediments samples were collected from 3000 meters below water line surface at Bacia de Campos, in Campos dos Goytacazes, Rio de Janeiro, Brazil. The collection was performed by a submarine robot, during petroleum prospection studies performed by the Petrobras Company, and kindly provided to our group. The samples were stored at 4 °C until the inoculation process. The samples were transferred to 15 mL flasks, and 5 mL of Page’s Amoebae Saline (PAS) was added. The solution was stored for 24 h to decant the sediment. The liquid was then subjected to a series of filtrations: first through paper filter and then through a 5 μm filter to remove large particles of sediment and to concentrate any giant viruses present. For co-culture, the cells used were A. castellanii (strain NEFF) and V. vermiformis (strain CDC 19), purchased from ATCC. These cell strains were stored in 75 cm2 cell culture flasks containing 30 mL of peptone yeast extract glucose medium (PYG) at 28 °C. After 24 h of growth, cells were harvested and pelleted by centrifugation. The supernatant was removed, and the amoebae were resuspended three times in sterile PAS. After the third washing, 500,000 A. castellanii or V. vermiformis were resuspended in PAS or TS solutions and seeded in 24-well plates. The amoebae suspensions were added to an antibiotic mix containing ciprofloxacin (20 μg/mL; Panpharma, Z.I., Clairay, France), vancomycin (10 μg/mL; Mylan, Saint-Priest, France), imipenem (10 μg/mL; Mylan, Saint-Priest, France), doxycycline (20 μg/mL; Mylan, Saint-Priest, France), and voriconazole (20 μg/mL; Mylan, Saint-Priest, France). Each 100 μL of sample was mixed and inoculated in the numbered (1–12) wells and incubated at 30 °C in a humid chamber. A negative control was used in each plate. The wells were observed daily under an optical microscope. After 3 days, new passages of the inoculated wells were performed in the same manner until the third passage. In this passage, the content of the wells presenting lysis and cytopathic effects were collected and stored for production and analysis of the possible isolates by haemacolour staining and electron microscopy using the negative stain technique. Of the twelve tested samples from Pantanal, we found Tupanvirus (soda lake) in three. In only one ocean sample we did isolate Tupanvirus (deep ocean). To evaluate the Tupanvirus soda lake host range, a panel of cell lines was subjected to virus infection at an multiplicity of infection (MOI) of 5: Acanthamoeba castellanii (ATCC30010), Acanthamoeba royreba (ATCC30884), Acanthamoeba griffin (ATCC50702), Acanthamoeba sp. E4 (IHU isolate), Acanthamoeba sp. Micheline (IHU isolate), Vermamoeba vermiformis (ATCC50237), Dictyostelium discoideum (ATCC44841), Willartia magna (ATCC50035), Tetrahymena hyperangularis (ATCC 50254), Trichomonas tenax (ATCC 30207), RAW264.7 (Mouse leukemic monocyte-macrophage) (ATCCTIB71) and THP-1 (human monocytic cell line) (ATCCTIB202). Cell lines were tested for mycoplasma. The assays were carried out in 24-well plates, and cells were incubated for 24 or 48 h. The tupanvirus titer was measured in A. castellanii by end-point and calculated by the Reed-Muench method34. In parallel, the samples were subjected to qPCR, targeting the tupanvirus tyrosyl RNA synthetase (5ʹ-CGCAATGTGTGGAGCCTTTC-3ʹ and 5ʹ-CCAAGAGATCCGGCGTAGTC-3ʹ) and aiming to verify viral genome replication (Biorad, CA, USA). Tupanvirus was propagated in 20 A. castellanii 175 cm2 cell culture flasks in 50 mL PYG medium. The particles were purified by centrifugation through a sucrose cushion (50%), suspended in PAS and stored at −80 °C. Purified particles were used for genome sequencing, proteomic analysis10, and microscopic and biological assays.

Cycle and virion characterization

All biological tests were performed with Tupanvirus soda lake only. To investigate the viral replication cycle by TEM, 25 cm2 cell culture flasks were filled with 10 × 106 A. castellanii per flask, infected by Tupanvirus at a multiplicity of infection of 10 and incubated at 30 °C for 0, 2, 4, 6, 8, 12, 15, 18, and 24 h. One hour after virus-cell incubation, the amoeba monolayer was washed three times with PAS buffer to eliminate non-internalized viruses. A total of 10 mL of the infected cultures was distributed into new culture flasks. A culture flask containing only amoeba was used as the negative control. The infected cells and control sample were fixed and prepared for electron microscopy14. For immunofluorescence, A. castellanii cells were grown, infected by Tupanvirus at a multiplicity of infection of 1 as described and added to coverslips for 0, 2, 4, 6, 8, 12, 15, 18, and 24 h. After infection, the cells were rinsed in cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) in PAS for 10 mins. After fixation, cells were permeabilized with 0.2% Triton X-100 in 3% bovine serum albumin (BSA)–PAS for 5 min, followed by rinsing with 3% BSA–PBS three times. Cells were then stained for 1 h at room temperature with an anti-tupanvirus antibody produced in mice (According Aix Marseille University ethics committee rules). After incubation with an anti-mouse secondary antibody, fluorescently labeled cells were viewed using a Leica DMI600b microscope. For Tupanvirus virion characterization, we also used scanning electron microscopy (SEM)35. Chemical treatment with proteases and sonication was performed as described elsewhere35 to investigate fiber composition and the attachment between capsid and tail. For tomography videos, tilt series were acquired on a Tecnai G2 transmission electron microscope (FEI) operated at 200 keV and equipped with a 4096 × 4096 pixel resolution Eagle camera (FEI) and Explore 3D (FEI) software. The tilt range was 100°, scanned in 1° increments. The magnification ranged between 6,500 and 25,000, corresponding to pixel sizes between 1.64 and 0.45 nm, respectively. The image size was 4,096 × 4,096 pixels. The average thickness of the obtained tomograms was 298 ± 131 nm (n = 11). The tilt series were aligned using ETomo from the IMOD software package (University of Colorado, USA) by cross-correlation (http://bio3d.colorado.edu/imod/). The tomograms were reconstructed using the weighted-back projection algorithm in ETomo from IMOD. ImageJ software was used for image processing.

Genomes sequencing and analyses

The tupanviruses genomes were sequenced using an Illumina MiSeq instrument (Illumina Inc., San Diego, CA, USA) with the paired end application. The sequence reads were assembled de novo using ABYSS software and SPADES, and the resulting contigs were ordered by the Python-based CONTIGuator.py software. The obtained draft genomes were mapped back to verify the read assembly and close gaps. The best assembled genome was retained, and the few remaining gaps (three) were closed by Sanger sequencing. The gene predictions were performed using the RAST (Rapid Annotation using Subsystem Technology) and GeneMarkS tools. Transfer RNA (tRNA) sequences were identified using the ARAGORN tool. The functional annotations were inferred by BLAST searches against the GenBank NCBI non-redundant protein sequence database (nr) (e-value < 1 × 10−3) and by searching specialized databases through the Blast2GO platform. Finally, the genome annotation was manually revised and curated. The predicted ORFs that were smaller than 50 amino acids and had no hits in any database were ruled out. Tupanvirus codon and aa usages were compared with those of A. castellanii and other lineages of mimiviruses. Sequences were obtained from NCBI GenBank and subjected to CGUA (General Codon Usage Analysis). The global distribution of Tupanvirus tRNAs was analyzed and compared manually with viral aa usage considering the corresponding canonical codons related to each aa. Phylogenetic analyses were carried out based on the separate alignments of several genes, including family B DNA polymerase, 18 S rRNA intronic regions (copies 1/2) and 20 aminoacyl-tRNA synthetases (aaRS). The predicted aa sequences were obtained from NCBI GenBank and aligned using Clustal W in the Mega 7.0 software program. Trees were constructed using the maximum likelihood evolution method and 1000 replicates. The analysis of aaRS domains was carried out using NCBI Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). A search for promoter sequences was performed in intergenic regions based on a search for the mimivirus canonical AAAATTGA promoter sequence, as previously described20. Single-nucleotide polymorphisms (SNP) in the AAAATTGA promoter sequence were also considered for each base, considering all possibilities. Gene sets available for members of the family Mimiviridae and those of Tupanvirus soda lake and Tupanvirus deep ocean were used for analyses of the mimivirus pangenome. Groups of orthologues were determined using the Proteinortho tool V51 with 1e−3 and 50% as the e-value and coverage thresholds, respectively. Concurrently, BLAST searches were performed using ORFs of all mimivirus genomes available in the NCBI GenBank nucleotide sequence database against the set of clusters of orthologous groups previously delineated for mimiviruses (mimiCOGs) (n = 898), with 1e−3 and 50% as the e-value and coverage thresholds, respectively. For rhizome preparations, all coding sequences were blasted against the NR database, and results were filtered to retain the best hits. Taxonomic affiliation was retrieved from NCBI. For the construction of a translation-associated elements network, the different classes of translation elements of each organism included in the analysis were obtained by searching for each component within their genome, according to protein function annotation using Blastp searches against the GenBank NCBI non-redundant protein sequence database. The tRNA components were obtained using the ARAGORN tool. Different tRNA molecules were included in the analysis, considering the anti-codon sequence. Repeated elements were eliminated to avoid analysis of duplicate events. The layout of the network was generated by a force-directed algorithm—followed by local rearrangement for visual clarity, leaving the network’s overall layout unperturbed—using the program Gephi (https://gephi.org).

Ribosomal RNA shutdown and toxicity assays

To investigate the toxicity of Tupanvirus particles, 1 million A. castellanii cells were infected with Tupanvirus or mimivirus at a multiplicity of infection of 1, 10, 50, or 100 and incubated at 32 °C. At 0 and 24 h post-infection, the cell suspensions were collected and titered as previously described. A fraction of this suspension (200 µL) was subjected to RNA extraction (Qiagen RNA extraction Kit, Hilden, Germany). The RNA was subjected to reverse transcription by using Vilo enzymes (Invitrogen, CA, USA) and then used as a template in qPCR targeting A. castellanii 18 S rRNA (5ʹ-TCCAATTTTCTGCCACCGAA-3ʹ and 5ʹ-ATCATTACCCTAGTCCTCGCGC-3ʹ). The values were expressed as arbitrary units (delta-Ct). Normalized amounts of the original RNA extracted from each sample were electrophoresed in 1% agarose gel with TBE buffer and run at 150 V. TEM over the entire testing period was performed to evaluate the presence of ribosome-containing vesicles and other cytological alterations. To investigate the nature of virion toxicity, purified Tupanvirus was inactivated by UV light (1 h of exposure, 60 W/m2) or heating (80 °C, 1 h)—inactivation was confirmed by inoculation on Acanthamoeba castellanii, CPE was observed for 5 days and lack of replication was confirmed by qPCR—and inoculated onto A. castellanii containing 500,000 cells at multiplicities of infection of 0.1, 1, 5, 10, 50, and 100. The assays were performed in PAS solution. The cytopathic effect was documented and quantified in a counting-cells chamber. Inactivated mimivirus was used for comparison. To determine whether Tupanvirus-induced shutdown of amoebal 18 S rRNA even after inactivation, 500,000 cells were infected (at a multiplicity of infection of 100) and collected at 3 and 9 h post-infection, and amoebal 18 S rRNA levels were measured by qPCR. APMV was used as the control. The sensitivity of Tupanvirus and mimivirus to the translation-inhibiting drugs geneticin and cycloheximide was tested. A total of 500,000 A. castellanii cells were pre-treated with different concentrations of the drugs (0–50 and 0–15 µg/ml, respectively) for 8 h and then infected at a multiplicity of infection of 10. Twenty-four hour post-infection, cells were collected, and the viral titers were measured. To investigate the toxicity effect of Tupanvirus particles in the non-host Tetrahymena sp., 1 million fresh cells were infected at a multiplicity of infection of 10 in a medium composed of 50% PYG and 50% PAS. The cytopathic effect was monitored for 4 days post-infection, given the reduction of cell movement and vacuolization (lysis or viral replication was not observed). Each day post-infection, 100 µL of infected cell suspension was collected and subjected to cytospin and haemacolour staining to observe vacuolization and other cytological alterations induced by the virus. Other 100 µL aliquots were used to investigate the occurrence of rRNA shutdown induced by Tupanvirus. To this end, the samples were subjected to RNA extraction and electrophoresis. Viral infection in Tetrahymena sp. was also observed by TEM at a multiplicity of infection of 10. To determine whether Tupanvirus particles affect the rate of Tetrahymena sp. phagocytosis because of toxicity, the rate of viral particle incorporation per cell was calculated during the period of infection. The ratio of TCID50 (infectious entities) and total particles was first calculated by counting the number of viral particles in a counting chamber (approximately 1 TCID 50 to 63 total particles). One million Tetrahymena cells were infected by Tupanvirus or mimivirus at an MOI of 10 TCID 50 . Twelve hour post-infection, the number of viral particles in the medium was estimated by counting the remaining (non-phagocytized) particles. An input of 10 TCID 50 per cell was added each day post-infection (in separate flasks, one for each day), and the rate of particles phagocytosis was calculated 12 h post-input. For the calculation, the remaining particles from the day before were considered (counted immediately before the input). Considering the toxicity caused by Tupanvirus, but not APMV, in tetrahymena, we conducted an in vitro experiment aiming to investigate the ability to maintain Tupanvirus or APMV in a system containing both Acanthamoeba (host) and Tetrahymena (non-host, predator of particles). Thus, A. castellanii (900,000 cells) and Tetrahymena (100,000 cells) were added simultaneously to the same flask, then infected by Tupanvirus or mimivirus at an MOI of 10 and observed for 12 days. One flask per observation day was prepared. At days 4 and 8, we added 500 µL of fresh medium (50% PYG and 50% PAS) and 100,000 A. castellanii, the permissive host. Each day post-infection, the corresponding flask was collected and subjected to titration as previously described. The same experiment was carried out by pre-treating Tetrahymena (8 h before infection) with 20 µg/ml of geneticin as negative controls.

Analysis involving Tupanvirus 18 S rRNA intronic region

All analyses involving the genomic environment of copies 1 and 2 were conducted based on the annotation of Tupanvirus. In the best-hits evaluation, the core sequences of copies 1 and 2 were used for nucleotide BLAST analysis using blastn. The resulting 100 best hits were tabulated, and the information was used to construct diagrams. For the phylogenetic analysis, the sequences of these best hits were also aligned, using Clustal W in the Mega 7.0 software, and constructed using the maximum likelihood evolution method of 1,000 replicates. To analyze subjacent regions of the core sequence of 18 S rRNA intronic regions in the Mimiviridae family, one member of the lineages A (HQ336222.2), B (JX962719.1) and C (JX885207.1) was chosen and analyzed. The expression of both copies was checked using fluorescence in situ hybridization (FISH) and qPCR. For this, A. castellanii cells were infected with Tupanvirus with a multiplicity of infection of 5 and collected at 30 min and at 6 and 12 h post-infection. As a control, A. castellanii cells were also incubated with PAS alone and collected. At the indicated times, cells and the supernatant were collected and centrifuged at 800 × g for 10 min. For FISH, the pellet was resuspended in 200 µL of PAS, submitted to cytospin and the cells were fixed in cold methanol for 5 min. Specific probes targeting the 18 S rRNA of A. castellanii (5ʹ-TTCACGGTAAACGATCTGGGCC-3ʹ-fluorophore Alexa 488), copy 1 RNA (5ʹ-AGTGGAACTCGGGTATGGTAAAA-3ʹ-fluorophore Alexa 555) and copy 2 RNA (5ʹ-GGCCAAGCTAATCACTTGGG-3ʹ-fluorophore Alexa 555) were diluted and applied at 2 µM in hybridization buffer (900 mM NaCl, 20 mM Tris/HCL, 5 mM EDTA, 0.01% SDS, 10–25% deionized-formamide in distilled-H 2 O). The hybridization buffer containing the probes was added to the slides and the hybridization was carried out at 46 °C overnight in a programmable temperature-controlled slide-processing system (ThermoBrite StatSpin, IL, USA). Post-hybridization washes consisted of 0.45–0.15 M NaCl, 20 mM Tris/HCL, 5 mM EDTA, and 0.01% SDS at 48 °C for 10 min. Slides were analyzed using a DMI6000B inverted research microscope (Leica, Wetzlar, Germany). To qPCR the pellet of infected cells was also washed with PAS and then used for total RNA extraction using the RNeasy mini kit (Qiagen, Venlo, Netherlands). The extracted RNA was treated with the Turbo DNA-free kit (Invitrogen, CA, USA) and then used as a template in reverse transcription (RT) reactions carried out using SuperScript Vilo (Invitrogen, CA, USA). The resultant cDNA was used as a template for quantitative real-time PCR assays using the QuantiTect SYBr Green PCR Kit (Qiagen RNA extraction Kit, Hilden, Germany) and targeting copies 1 (primers 5ʹ-GCATCAAGTGCCAACCCATC-3ʹ and 5ʹ-CTGAAATGGGCAATCCGCAG-3ʹ) and 2 (primers 5ʹ-CCAAGTGATTAGCTTGGCCATAA-3ʹ and 5ʹ-CGGGAAGTCCCTAAAGCTCC-3ʹ) of the intergenic18S rRNA region in TPV. To normalize the results, primers targeting the GAPDH housekeeping gene of Acanthamoeba (primers 5ʹ-GTCTCCGTCGTCGATCTCAC-3ʹ and 5ʹ-GCGGCCTTAATCTCGTCGTA-3ʹ) were also used. qPCR assays were performed in a BioRad Real-Time PCR Detection System (BioRad) using the following thermal conditions for all genes: 15 min of pre-incubation at 95 °C followed by 40 amplification cycles of 30 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. The results were analyzed using the relative quantification methodology of 2(-ΔΔct).

Investigation of the nature of ribosomal RNA shutdown

To investigate the shutting down of the host rRNA and verify whether this phenomenon was related to the canonical ribophagy/autophagy process, tests using two acidification and lysosome-vesicle fusion inhibitors (chloroquine and bafilomycin A) were performed. The pH of infected cells and the effect of Atg8-2 silencing on shutdown were also tested. For the inhibitor assays, 5 × 105 A. castellanii cells cultured in PYG medium were infected with Tupanvirus or mimivirus at a multiplicity of infection of 100 and incubated at 32 °C. At 1 h post-infection, chloroquine (Sigma-Aldrich, MO, USA) at a final concentration of 100 µM or bafilomycin A (Sigma-Aldrich, MO, USA) at a final concentration of 10 nM was added to the infected cell suspensions. As a control, A. castellanii cells not infected were also treated with these inhibitors under the same conditions. After 3 and 9 h post-infection, cells and the supernatant were collected and centrifuged at 800 × g for 10 min. The supernatant was discarded, and the pellet was submitted to RNA extraction (Qiagen RNA extraction Kit, Hilden, Germany). From the extracted RNA, 10 µL of each sample was electrophoresed in 1.5% agarose gel with TBE buffer and run at 135 V, and 14 µL was submitted to reverse transcription to measure the amoebal 18 S rRNA levels by qPCR as previously described. To investigate the acidification caused by Tupanvirus or mimivirus infection, A. castellanii cells were also submitted to the same pattern of infection and treatment with bafilomycin A, as previously described. In addition, 1 h before the collection time, the cells were incubated with LysoTracker Red DND-99 (Thermo Fisher Scientific, Massachusetts, United States) at a final concentration of 75 nM. After 9 h post-infection, cells and the supernatant were collected and centrifuged at 800 × g for 10 min. The supernatant was discarded, and the pellet was resuspended in 1 mL of PAS medium containing only bafilomycin A (10 nM). A total of 20 µL of this suspension was added to glass slides and cover slipped. Analyses were performed using a confocal microscope (Zeiss, Jena, Germany). For gene silencing, small interfering RNA (siRNA) targeting the Atg8-2 gene of A. castellanii was synthesized by Eurogentec (Liège, Belgium) based on the cDNA sequence of the gene. The siRNA duplex with sense (5ʹ-GAACUCAUGUCGCACAUCUTT-3ʹ) and anti-sense (5ʹ-AGAUGUGCGACAUGAGUUCTT-3ʹ) sequences was used. The siRNA tagged with a fluorescence dye was transfected onto A. castellanii trophozoites at a density of 1 × 106 cells. The control of transfection was performed using fluorescence microscopy. The biological effect of siRNA was check by qPCR and by the observation of the inhibition of acanthamoebal encystment, which is dependent on Atg8-2. Finally, modifications of A. castellanii nucleus/nucleolus structure after infection with Tupanvirus and mimivirus were investigated. A total of 106 cells were infected with Tupanvirus or mimivirus at an MOI of 10, stained by haemacolour and treated with SYTO RNASelect Green Fluorescent cell stain (Invitrogen, USA) following the manufacturer’s instructions. After 9 h.p.i., cells were observed under an immunofluorescence microscope to observe modifications to the nucleus /nucleolus of infected and control cells. In parallel, this preparation was submitted to electron microscopy.

Data availability

The Tupanvirus genome sequences have been deposited in GenBank under accession codes KY523104 (soda lake) and MF405918 (deep ocean). Proteomic data have been deposited in PRIDE archive under accession code PXD007583. All other data supporting the findings of this study are available within the article and its Supplementary Information, or from the corresponding author upon reasonable request.