Iflavirus increases its infectivity and physical stability in association with baculovirus

Insects and viruses

A virus-free S. exigua colony (SUI) was obtained from Andermatt Biocontrol AG (Grossdietwil, Switzerland) and reared in continuous culture at a constant temperature (25 ± 1 °C), relative humidity (RH; 50% ± 5%), and photoperiod (16-h/8-h light-dark cycle) on artificial diet (Elvira et al., 2010) in the insectary facilities of the Universidad Pública de Navarra (UPNa, Pamplona, Spain). Insects from the UPNa colony were used to start a sister colony reared at Universitat de Valencia (UV, Valencia, Spain) using similar conditions. Insects were routinely tested for the presence of SeIV1 and SeMNPV, and confirmed to be virus-free ahead and during each experiment.

SeIV1 was detected and isolated from the laboratory colony of S. exigua (Millán-Leiva et al., 2012). The SeMNPV isolate used for the establishment of covert infections was originally isolated from spontaneous infections observed in the laboratory-reared offspring of field-caught S. exigua females (Cabodevilla et al., 2011). For this study, a fresh stock of SeMNPV OBs was amplified in vivo by droplet-feeding S. exigua fourth instar larvae. OBs were purified from cadavers by washing in 0.01% SDS twice, once in double-distilled water and finally diluted in sterile double-distilled water. OB suspension was stored at −20 °C until required. OBs were quantified by counting in triplicate with a Neubauer chamber.

A SeIV1-free isolate of SeMNPV was obtained after PCR screening of the UPNa baculovirus collection. The Spanish isolate SeMNPV-SP2 was found to be negative for the presence of SeIV1 and was selected for subsequent studies. This isolate was originally obtained from a group of cadavers collected during a baculovirus epizootic in greenhouse crops in southern Spain (Caballero et al., 1992; Muñoz et al., 1999). OBs had been maintained at −20 °C since 1996.

OBs from the SeMNPV-SP2 isolate associated with SeIV1 (SeMNPV-SeIV1) and SeIV-free OBs (SeMNPV) were produced for biological comparison. For this, groups of 25 newly molted fourth instars were allowed to drink from a suspension of 10⁴ OBs/ml in a solution of 10% (w/v) sucrose and 0.001% (w/v) food dye. A second batch of 25 larvae was fed with 20 µl of an identical OB concentration mixed with 80 µl of 1.34 × 10⁻¹ ng/µl SeIV1 particles (≈1.46 × 10⁷ SeIV1 genomes/µl). Larvae were reared individually until death or pupation. OBs were purified from cadavers and tested for SeIV1 by RT-qPCR as described below. The SeIV1 load present in OBs produced by this method was estimated at 18.9 ± 2.3 SeIV1 genomes per OB.

Infection with baculovirus

To infect S. exigua individuals with SeMNPV, larvae from a virus-free laboratory colony were challenged per os with an estimated 50% lethal concentration of OBs (Cabodevilla et al., 2011). Briefly, a batch of 30 pre-molt S. exigua third instars were starved overnight, allowed to molt to the fourth instar and then allowed to drink an OB suspension containing 9 × 10³ OBs/ml during a 10-min period (Hughes & Wood, 1981). Control insects (VF) consumed droplets that did not contain OBs. Inoculated larvae were individually placed in 25-ml plastic cups perforated for ventilation and provided with artificial diet. OB-challenged (VT) larvae that did not succumb to polyhedrosis disease were reared through to pupation at 25 ± 1 °C and 50% ± 5% RH. According to previous experiments (Cabodevilla et al., 2011), these larvae were expected to carry a persistent baculovirus infection as the experiment was originally planned to examine the expression of baculovirus and insect genes in persistently infected larvae. We were, however, unable to confirm establishment of the persistent infection as originally planned, which led us to investigate the relationship between SeIV1 and SeMNPV. Pupae were sexed and, once adults emerged, one male–female pair of adults was placed in a paper bag, and allowed to mate and oviposit to obtain the subsequent generation (F1). An identical procedure was applied to mock-inoculated insects that were defined as control samples. Egg masses were collected and placed in 300-ml plastic containers provided with artificial diet until larvae reached the second instar. From these containers 25 larvae were individualized and reared through to the fifth instar as described above. All procedures were performed in triplicate. Four F1 fifth-instar larvae per replicate were randomly selected and pooled for RNA extraction and microarray analysis. For each sample (VT and VF), three different pools were obtained and used for subsequent gene expression analysis.

Microarray design, hybridization and analysis

A 44K Agilent oligonucleotide microarray was designed to study different aspects of the interaction of S. exigua larvae with viral (iflavirus and baculovirus) and bacterial pathogens (Jakubowska, Vogel & Herrero, 2013; Bel et al., 2013). In that sense, the array mainly comprised probes representing unigenes from the S. exigua transcriptome (Pascual et al., 2012; Jakubowska, Vogel & Herrero, 2013), but also included 60-mer non-overlapping tiling probes covering both strands of the SeIV1 and SeMNPV genomes. In total, the microarray contained 167 probes covering the positive strand of SeIV1 and 166 probes covering the negative strand. The SeMNPV genome was represented by 2,260 tiling probes covering both virus strands. The microarray was also used to determine the expression levels of 139 open reading frames (ORFs) predicted for the SeMNPV genome (Ijkel et al., 1999). Two different 60-mer probes were included for each of the predicted ORFs. The probes were designed using the eArray application from Agilent.

Synchronized F1-larvae from the VT and VF groups were collected and total RNA was extracted using RNAzol reagent (Molecular Research Center, Inc., Cincinnati, OH), according to the manufacturer’s protocol. To further purify RNA, an RNAeasy Kit (Qiagen, Hilden) was used following the protocol provided by the manufacturer. The quality of RNA was assessed by Agilent 2100 Bioanalyzer using the EukaryoteTotal RNA Nano protocol.

Agilent One-Color Spike-in Mix was added and 600 ng of total RNA was used for cRNA (complimentary RNA) synthesis. The obtained cRNA (1.65 µg) was fluorescently labeled with cyanine-3-CTP, fragmented and hybridized to S. exigua microarray slides following the One-Color Microarray-Based Gene Expression Analysis (Quick-Amp labelling) protocol. Microarrays were scanned using G2505B Agilent scanner and data were extracted using Agilent Feature Extraction 9.5.1 software. Spike-in transcripts are a mix of unique 55-mer probes that specifically anneal to complementary control probes on the Agilent’s microarrays and were used for linear normalization performed by the Agilent Extraction 9.5.1 software. Before data analysis, hybridization quality control reports were verified as correct. RNA labelling and hybridization, as well as array scanning and data extraction were performed by the Microarray Analysis Service of Principe Felipe Research Centre (CIPF, Valencia, Spain) following standard protocols.

Data analysis was performed using Babelomics 4.3 software (http://babelomics.bioinfo.cipf.es/) (Medina et al., 2010). First, between-arrays normalization was performed using the quartile normalization method in Babelomics (Bolstad et al., 2003). Normalized arrays of the VT samples were compared to VF controls and expressed as a fold-change in gene expression or abundance. For those probes having low signal levels for one of the samples (as indicative of absence in one of the samples), changes in gene expression were estimated by comparison with the overall background intensity.

SeIV1 detection by RT-PCR

RT-qPCR was used to detect SeIV1 genomic RNA in the purified preparations of SeMNPV and SeIV1. SeMNPV occlusion bodies (OBs) as well as SeIV were purified on discontinuous sucrose gradients as described below. RNA was extracted from the samples using Tripure reagent (Roche), according to the manufacturer’s protocol. RNase-free glycogen (5 µg/µl) was added as a carrier during the RNA precipitation step. Purified RNA was used for cDNA synthesis using PrimeScript RT reagent kit from Takara Bio Inc. (Otsu Shiga, Japan) following the manufacturer’s protocol. RT-qPCR was carried out in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). All reactions were performed using HOT FIREPOL EvaGreen qPCR mix Plus (ROX) from Solis BioDyne (Tartu, Estonia), in a total reaction volume of 25 µl. Forward and reverse primers, designed using Primer Express software (Applied Biosystems, Foster City, CA), were added to a final concentration of 0.3 µM. These specific primers were designed to amplify a 97-bp fragment in the RNA-dependent RNA polymerase (RdRp) region from 9743-9840 nt on the genome (Forward: 5′-TGTGAAGTTAGACACGCATGGAA-3′ and Reverse: 5′-CGACTTGTGCTACTCTCTTCATCAA-3′). For relative quantification of virus genomes, Ct values from the RT-qPCR were compared to the standard curves obtained for known number of copies of virus genome fragment cloned in the pGEMTeasy vector. A fragment of SeIV1 genome was cloned into pGEMTeasy vector and the standard curve prepared from the serial dilutions of known copies of the vector DNA.

Semiquantitative RT-PCR was used to detect the negative RNA strand of SeIV1 in larvae as well as in SeMNPV OBs. Tagged primer was used for the specific synthesis of cDNA due to the occurrence of self-priming, often observed for RNA viruses. RNA was extracted as described above. For this, 0.5 µg of RNA were used for cDNA synthesis using tagged specific primer (5′ -ggatgcaggctacgtgaagatacgGTGTCAACAACAGACCCTAGCG-3′, tag in lowercase, SeIV1 specific sequence in uppercase). cDNA synthesis was performed using PrimeScript RT reagent kit from Takara Bio Inc (Otsu Shiga, Japan) following the manufacturer’s protocol, at 42 °C for 30 min. 2 µl were used for subsequent PCR reaction using the following primers: forward 5′ -ggatgcaggctacgtgaagatacg-3′ and reverse 5′ -gcagccatgttcaacctc-3′ , and the following conditions: 94 °C for 5 min, annealing at 55 °C and elongation at 72 °C for 30 cycles. The resulting PCR product had a size of 1,495 bp.

Virus purification by gradient centrifugation

For electron microscopy, OBs from SeMNPV and SeIV1-associated SeMNPV samples were additionally purified through sucrose gradients (King & Possee, 1992). Briefly, 3 ml of ∼10⁸ OB/ml suspensions were loaded onto a 30–60% (w/w) continuous sucrose gradient and then centrifuged at 40,000 × g for 1 h at 4 °C. The OB band was harvested by puncturing the tube with a needle and collecting the sucrose fraction in a syringe. OBs in sucrose were diluted in 2 vol. of 1× TE buffer and centrifuged at 40,000 × g for 1 h. OBs collected in the pellet were suspended in sterile double-distilled water.

SeIV1 particles were purified from S. exigua larvae as follows. Approximately one hundred fourth and fifth instar larvae were freeze-killed and lyophilized. The lyophilized larvae were homogenized in 0.01 M potassium phosphate pH 7.4 containing 0.45% diethyldethiocarbamic acid (DIECA) and 0.2% β-mercaptoethanol (2–5 ml of buffer per gram of larvae). The homogenate was then sonicated for 20 s and subsequently filtered through a double layer of cheesecloth and then centrifuged for 15 min at 5,000 × g to remove large debris. The supernatant was centrifuged for 2.5 h at 82,000 × g (Beckman centrifuge, S28 rotor). The pellet was resuspended in 2 ml TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) buffer at pH 7.3 and left overnight at 4 °C. The suspension was then applied onto 20% sucrose in TAE buffer and centrifuged 2.5 h at 100,000 × g at 4 °C. The pellet was again resuspended in a small volume of TAE buffer and left overnight at 4 °C. Next, 10–40% discontinuous sucrose gradients were prepared and the sample was centrifuged for 2.5 h at 100,000 × g. SeIV1 purified particles were collected from the white virus fraction.

Determination of SeIV1 infectivity in larvae

In order to compare the ability of SeIV1 alone and associated with SeMNPV OBs to enter and replicate in host cells, virus-free S. exigua fourth instar larvae were starved overnight and then orally inoculated by allowing them to drink from an OB suspension of SeMNPV-SeIV1 containing 1 × 10⁶ OBs/ml by the droplet-feeding method (Hughes & Wood, 1981). Another batch of larvae was orally inoculated with the same amount of SeIV1 particles that were estimated to be present in the SeMNPV-SeIV1 OBs (estimated by RT-qPCR). Both SeMNPV-SeIV1 OBs and SeIV1 particles used in the experiment originated from two separate preparations. At 72 hpi larval midguts were dissected and SeIV1 loads measured by RT-qPCR as described above. Ten larvae were infected with each virus preparation and ten larvae served as controls, to confirm that the SUI colony was virus-free, and that the insects were not contaminated during the experiment. Larvae were processed individually. Ct values of SeIV1 were normalized to Cts for the ATP synthase reference gene (Herrero et al., 2005), and −Δ Ct values compared between larvae infected with SeIV1 alone or associated with OBs. The resulting –Δ Ct values were compared by Mann–Whitney test, due to the presence of outliers.

Determination of dose-mortality response

The pathogenicity of SeMNPV OBs and SeMNPV-SeIV1 OBs was determined by the droplet-feeding method (Hughes & Wood, 1981). Briefly, groups of 30 newly molted second instars were starved overnight and orally inoculated with one of the following OB concentrations: 2.54 × 10⁵, 8.18 × 10⁴, 2.72 × 10⁴, 9.09 × 10³ and 3.03 × 10³ OBs/ml. This range of concentrations was estimated to kill between 95% and 5% of the experimental insects. Larvae that ingested droplets within 10 min were individually transferred to a 24-well tissue culture plate. A cohort of 24 larvae was allowed to drink from an OB-free suspension as controls. Larvae were reared on a semisynthetic diet at 25 ± 2 °C and mortality was recorded daily for 7 days post-inoculation. The entire bioassay was performed three times. Data were subjected to Probit regression analysis using the Polo-PC program (Le Ora Software, 2002). Lethal concentration (LC₅₀) values and relative potencies were estimated when a parallelism test confirmed that the regressions for each treatment could be fitted with a common slope (Robertson & Preisler, 1992).

Determination of OB production and virulence

Groups of 30 fourth instar larvae were starved overnight and allowed to drink for 10 min from an OB suspension containing 5 × 10⁷ OBs/ml of either SeMNPV/SeIV+ or SeMNPV/SeIV1-. Individual weight measurements were first taken immediately before inoculation. Inoculated larvae were individualized in 24-well plates containing diet and checked daily for virus-induced mortality. Larvae were monitored every eight hours for mortality and weighed daily during a six day post-inoculation period. Virus-killed larvae were frozen at −20 °C to avoid liquefaction. Cadavers were individually homogenized in 1 ml sterile distilled water. Each homogenate was filtered through cheesecloth to remove debris and the resulting suspension was counted in triplicate in a Neubauer chamber at 400× magnification using a phase contrast microscope. The experiment was performed six times. OB production and weight gain data were not normally distributed, and were compared by Kruskall–Wallis or Mann–Whitney test (SPSS statistics V.21, 2012).

Effect of UV and temperature treatment on the stability of SeIV1

The effect of environmental factors such as UV radiation and high temperature on the stability of SeIV1, alone or in association with OBs, was indirectly estimated by genome integrity measured by RT-qPCR. To this aim, two preparations of SeIV1 (SeMNPV OBs containing SeIV1 or SeIV1 alone) were purified by sucrose gradient centrifugation and then exposed to different intensities of UV-C radiation or temperature for different periods of time. The amounts of SeIV1 that remained viable for amplification were estimated by RT-qPCR.

For the UV irradiation treatment, each of the SeIV1 preparations was exposed to 0, 3, 6, 9 and 12 J/cm² UV-C light using a crosslinker CL-1 (Herolab), at a wavelength of 254 nm. Samples of 200 µl of each preparation were placed in 24-well plates, to achieve a suspension depth of ∼1 mm, and were then exposed to continuous UV-C light 30 cm below the lamp, which allowed an exact dose to be administered to each sample. At each sample time, 300 µl of Tripure was immediately added to 150 µl of the sample, and the samples were frozen for further RNA purification. RNA was extracted and cDNA synthesized as described above. RT-qPCR was used to detect the presence and to estimate the number of SeIV1 genomes in the samples. The experiment was performed twice and the data were analyzed by two-way Anova.

For the heat treatment, each SeIV1 preparation was incubated at 72 °C for 0, 30, 60, 360 and 1440 min in an Eppendorf thermomixer. As before, at each sample time 300 µl of Tripure was immediately added to 150 µl of the sample, and then frozen until RNA purification. RNA was extracted and cDNA synthesized as described above. RT-qPCR was used to detect the presence and estimate the SeIV1 load in each sample. The experiment was repeated twice and the data were analyzed by two-way Anova. Starting quantities of SeIV1 genomes in all samples were normalized to 100%, and the decrease in estimated quantities at each sample time calculated accordingly.

Electron microscopy

Scanning electron microscopy (SEM) was used to examine the presence of SeIV1 particles on the surface of OBs. For this, OBs in suspension were fixed overnight by mixing with an equal volume of fixative (4% formaldehyde and 1% glutaraldehyde in 0.1M phosphate buffer, pH 7.4) and then washed twice with 0.1M phosphate buffer. Samples were then partially dehydrated with 70% ethanol, dried, placed on aluminum mounts using carbon tags, sputter-coated with gold-palladium and photographed at magnifications of 6,000× and 25,000× using a scanning electron microscope (Philips SEM 550).

Transmission electron microscopy (TEM) was used to examine the polyhedrin matrix within OBs. For this, OBs in suspension were fixed for 2 h at 4 °C with 1.5% glutaraldehyde. The samples were then concentrated in 0.4% agar and washed with phosphate buffer (0.2 M, pH 7.3). Samples were post-fixed with 2% osmium tetroxide (OsO₄) for 2 h, dehydrated and stained for 1 h with 2% uranyl acetate. The samples were then embedded in epoxy resin and polymerized for 48 h at 60 °C. After polymerization, samples were sectioned using an ultramicrotome (Leica UC6), transferred to TEM grids and stained with lead acetate. The resulting grids were observed under an electron transmission microscope of 100 kV (JEOL JEM 1010). Different fields of each sample were photographed at a magnification of 40,000× and visualized with image acquisition software.

Detection of iflavirus expression in S. exigua larvae treated with baculovirus

Gene expression comparison between S. exigua larvae derived from insects previously inoculated with SeMNPV (VT), or virus-free larvae (VF), was performed using a custom-made DNA-microarray containing S. exigua unigene probes; about 3,000 probes covering the complete genomes of SeMNPV and SeIV1 (tiling probes), and the predicted ORFs from SeMNPV. Microarray comparison showed high differential expression of the probes representing the positive (average log2 value of 11.1) as well as the intermediate (negative) strand (average log2 values of 7.2) of the SeIV1 genome (Fig. 1A), indicating the presence and active replication of SeIV1 in the offspring of insects that survived oral inoculation with SeMNPV OBs. In contrast, the presence of SeMNPV transcripts was not detected (ratio VT/VF equal to 1) in the VT insects (Figs. 1B and 1C) which also indicates that the iflavirus was capable of autonomous replication in the absence of baculovirus transcription.

Influence of the virus association on iflavirus infectivity and baculovirus pathogenicity and virulence

Semi-quantitative PCR as well as reverse transcription quantitative PCR (RT-qPCR) revealed the presence of SeIV1 genomes in the SeMNPV OB preparation that had been purified after several centrifugation steps prior to being used to inoculate VT insects. Both positive and negative strand of the virus were detected, nevertheless the negative strand was present only in trace amounts when compared with its abundance in the larvae. This finding was indicative of a possible association between both viruses. We decided to explore the influence of such association on the insect–virus relationship of both types of virus. To determine whether iflavirus association with baculovirus favors iflavirus infectivity, the ability to establish an infection in host insects was estimated using iflavirus inoculum alone or associated with SeMNPV OBs. For this, SeIV1-free insects were orally inoculated with a preparation of SeMNPV OBs containing SeIV1 particles (quantified by RT-qPCR). In a side-by-side approach, a second batch of virus-free insects was inoculated with the same amount of SeIV1 particles, quantified by RT-qPCR method described above, but in the absence of SeMNPV OBs. Three days after inoculation, insects were dissected and the abundance of SeIV1 genomes in the midgut of the larvae, as an estimation of the SeIV1 ability to establish a viral infection, was determined by RT-qPCR. Results revealed that the iflavirus was present in all inoculated insects, but a 2.8-fold difference in SeIV1 load, indicated by a 1.5-fold difference in qPCR Ct values, was detected in insects inoculated with iflavirus associated with OBs than when inoculated alone (Fig. 2A). Control insects were confirmed to be negative for both viruses.

We also determined the effect of the viral association on the virulence and pathogenicity of SeMNPV OBs. In contrast to the results for iflavirus, the pathogenicity of SeMNPV OBs, estimated by peroral bioassay, was reduced by ∼40% when iflavirus was associated with OBs compared to equal OB inoculum free from iflavirus (Fig. 2B). However, the co-transmission of iflavirus and SeMNPV did not significantly affect the mean speed of kill of SeMNPV that varied between 98 and 102 h post-inoculation (Fig. 2C). Similarly, the growth of infected host insects (Fig. 2D-left), or the total OB production (Fig. 2D-right), in insects inoculated by baculovirus OBs with iflavirus, did not differ significantly from that of insects inoculated with iflavirus-free OBs.

Influence of the virus association on iflavirus persistence in the environment

Occlusion of baculovirus ODVs greatly improves the persistence of these virions in the environment following death of the insect host (Granados & Federici, 1987). If SeIV1 is detected in association with the baculovirus occlusion body, it may be expected that such an association could contribute to increasing its stability in the environment. In agreement with such a hypothesis, experiments involving exposure to an ultraviolet light source (UV-C, 254 nm wavelength) or high temperature (72 °C) revealed that SeIV1 particles associated with OBs maintained physical stability significantly better than naked particles. Following exposure to 3–12 J/cm² of UV-C radiation, the stability of occluded iflavirus particles, measured as the relative viral load, was approximately two orders of magnitude greater at each time point than that of naked particles (Fig. 2E). Similarly, exposure of iflavirus particles to high temperature resulted in ∼100-fold greater stability of OB-associated iflaviruses, following 1 or 6 h exposure, compared to naked particles (Fig. 2F).

Detection of the iflavirus in baculovirus OBs

RT-qPCR revealed the presence of SeIV1 genomes in the SeMNPV OB preparation that had been purified by several centrifugation steps prior to being used to inoculate larvae. Moreover, the iflavirus particles present in the preparation were able to establish a persistent infection. This finding suggested that a physical association may exist between both viruses. The physical association between the viruses is likely to involve the localization of iflavirus particles inside or outside of OBs. To determine this, purified OBs from insects that died of SeMNPV infection in the presence of iflavirus (SeIV1+), or the absence of iflavirus (SeIV1−), were observed by scanning electron microscopy (SEM) (Figs. 3A–3H). No differences were observed in the external appearance of each type of OB. In contrast, transmission electron microscopy (TEM) of the OBs revealed the presence of ODVs of baculovirus, each comprising 1–4 nucleocapsids (Figs. 4A–4C), as well as dark spots dispersed in the matrix resembling in size and form (Figs. 4D and 4E), the icosahedral particles of iflavirus embedded in the polyhedrin matrix (Fig. 4F). To confirm this, the TEM procedure was repeated in an independent laboratory (Laboratory of Virology of Wageningen University, the Netherlands) to exclude the possibility of generating artifacts during sample preparation. Similarly, samples processed in the Netherlands laboratory showed the presence of dark spots resembling iflavirus particles in a certain number of OBs (Figs. 4G and 4H).