Confirmation of Oryctes rhinoceros nudivirus infections in G-haplotype coconut rhinoceros beetles (Oryctes rhinoceros) from Palauan PCR-positive populations
Insects and virusOryctes rhinoceros was collected from Amami, Kagoshima, Japan in 2017 and Ishigaki, Okinawa, Japan in 2018. The insects were brought back to the lab in Tokyo and maintained in a moisture mushroom mat substrate (Mushroom Mat, Tsukiyono Kinokoen, Japan) which was also served as food for larvae. The temperature was held at 25–30 °C with a 16-h light / 8-h dark photoperiod. To collect eggs, 2 or 3 female adults were put in a plastic case containing a moisture mushroom mat substrate with a male adult beetle. The insect jelly (Dorcus Jelly, Fujikon, Japan) was provided ad libitum as food for adults. After 2 weeks, we collected eggs, and about 10 eggs were placed in a plastic cup with a moisture mushroom mat substrate until hatched larvae developed to the second instar. This strain was used in all bioassays in this study. All Japanese O. rhinoceros were confirmed as CRB-G.The OrNV-X2B isolate used in this study was originally isolated from Philippine CRB and obtained from AgResearch in New Zealand.Cell culturesFRI-AnCu-35 (AnCu35) cells were obtained from Genebank of NARO (Tsukuba, Japan)27. This continuous cell line was developed from embryos of the cupreous chafer, Anomala cuprea (Coleptera: Scarabaeidae). The cells were maintained as adherent cultures in 25 cm2 tissue culture flasks (Falcon, Corning, USA) at 25 °C in 5 ml of 10% Fetal Bovine Serum (Gibco, Thermo Fisher Scientific, USA) supplemented Grace’s insect medium (Gibco). Cells were passaged in the above culture medium until the cell monolayer reached 70% confluence.DNA extraction and identification of haplotypes in Palauan populationCRB specimens were collected in Palau using pheromone traps containing ethyl 4-methyloctanoate (ChemTica Internacional, Costa Rica). Adults were dissected to collect midgut and gut tissues to avoid cross contamination between dissection of individuals, which were immediately soaked into 0.1 μg/ml gentamicin solution to prevent bacterial contamination during transportation at room temperature. Specimens were stored at − 30 °C after arrival to Tokyo. The tissues were homogenized in cell lysis solution (10 mM Tris–HCl, 100 mM EDTA, 1% SDS, pH 8.0) using pestles in 1.5 ml microcentrifuge tubes. Homogenates were centrifuged at 12,000× g for 5 min at 4 °C. Proteinase K (200 µg/ml final concentration) (Nippon Gene Co. Ltd., Japan) was added to the supernatant and incubated at 50 °C for 5 h. To remove contaminating RNA, RNase A solution (100 µg/ml final concentration) (Nippon Gene Co. Ltd.) was added. After a 30 min incubation at 37 °C, the mixture was placed on ice and supplemented with 200 μl of Protein Precipitation Solution (Qiagen, Germany), and then centrifuged at 17,000× g for 15 min at 4 °C. The supernatant was isopropanol-precipitated, pelleted by centrifugation, and washed with 70% ethanol. Finally, precipitated DNA was dissolved in distilled MilliQ water. The concentrations of each DNA solution were measured by using NanoVue Plus (GE Healthcare, Buckinghamshire, England, UK). The sample DNA was diluted to 10 ng/μl and used for PCR. The following primer pair was used to amplify a 523 bp fragment of the COI gene: C1-J-1718Oryctes (5′-GGAGGTTTCGGAAATTGACTTGTTCC-3′) and C1-N-2191Oryctes (5′-CCAGGTAGAATTAAAATRTATACCTC-3′)9. Each 10 μl PCR reaction contained: 5 μl Emerald Amp (Takara, Japan), 0.3 μl forward primer (10 μM), 0.3 μl reverse primer (10 μM), 3.4 μl Milli-Q water (Merck Millipore, USA), and 1 µl template DNA. PCR amplifications were performed in a Life ECO thermocycler (Bioer Technology, China) with a cycling profile of 35 cycles of 94 °C denaturation (30 s), 50 °C annealing (45 s), 72 °C extension (1 min) with an initial denaturation of 3 min at 94 °C and a final extension of 5 min at 72 °C. A 5 μl aliquot of each PCR amplicon was checked by agarose gel electrophoresis (1.5%, 1 × TBE), stained with Midori green (Nippon Genetics, Japan) and fluorescence visualized over UV light. Photographs were recorded using an E-BOX-VX2 /20 M (E & M, Japan).For direct sequencing, the PCR products were purified using a QIAquick PCR Purification Kit (Qiagen). The purified DNA was sequenced using BigDye Terminator Kit ver. 3.1 (Applied Biosystems, USA) and performed by the 3700 DNA analyzer (Applied Biosystems). The obtained sequences were analyzed using MEGA X software28 and the G haplotype was identified by the presence of the (A→G) point mutation in the COI region as previously described9.Virus detection in Palauan populationUsing the same samples as above, virus detection was carried out by PCR. The following primer pair was used to amplify a 944 bp fragment of the OrNV-gp054 gene (GrBNV-gp83-like protein): OrNV15a (5′-ATTACGTCGTAGAGGCAATC-3′) and OrNV15b (5′-ATGATCGATTCGTCTATGG-3′)29. PCR amplifications were performed as above.Transmission electron microscopy (TEM) was also used for detection of OrNV within a subset of PCR positive CRB tissue samples. After washing in phosphate-buffered saline (PBS), midgut and fat body samples of Palauan CRB adults from Melekeok and Aimeliik (respectively; two each), were subjected to following resin fixation as described previously30: tissues were fixed in 5% glutaraldehyde for 1 h, rinsed 4 times with Millonig’s phosphate buffer (0.18% NaH2PO4 × H2O, 2.33% Na2HPO4 × 7H2O, 0.5% NaCl, pH 7.4), post-fixed and stained in 1% OsO4 for 2 h and dehydrated in an ethanol series. Following the final dehydration step, the ethanol was replaced by QY-1 (Nisshin EM, Tokyo), and the tissues were embedded in epoxy resin comprising 47% TAAB EPON812, 19% DDSA, 32% MNA and 2% DMP30 (Nisshin EM, Tokyo). Then, they were cut into 70 nm thick sections with a diamond knife on an Ultracut N ultramicrotome (Leica, Vienna, Austria), attached to grids and observed using TEM (JEM-1400Plus, JEOL, Japan).Isolation of OrNV from Palauan samples and infectivity to Japanese CRB larvaeVirus isolation was carried out using a modification of a method previously described23. The frozen tissues of two virus positive CRB-G from Melekeok were washed with PBS twice, and after grounding with 1 ml PBS by pestles, centrifuged at 6,000 g × 5 min at 4 °C. The supernatant was filtered by 0.45 µm pore sized filter (Merck, USA) and transferred to a 1.5 ml ultracentrifuge tube in a clean bench. Virus was pelleted by centrifugation at 4 °C, 98,600 g for 30 min using a TLA55 rotor. After separation, the supernatant was discarded and the pellet was suspended in 500 μl of PBS and designated as “virus solution”. A portion of this solution (30 µl/larva) was intrahemocoelically injected into 82nd instar CRB to evaluate its infectivity. This experiment had no biological replicates due to the very small amount of inoculum available. Intrahemocoelically injected larvae were reared in the insect rearing mat at 25 °C for two weeks. Following death, larval cadavers were immediately dissected to collect midgut for following RNA extraction to detect expression of a viral gene, and electron microscopy observation. Total RNA was extracted from larval tissue samples using ISOGEN (Nippon Gene Co. Ltd., Tokyo, Japan), as described in the manufactural protocol. The total RNA samples were treated with RNAse-free recombinant DNAse I (TaKaRa, Japan) to remove the contaminating DNAs. The DNAse I treated total RNA samples (approximately 100 ng/µl) were used as templates for cDNA synthesis using a TaKaRa RNA PCR Kit (AMV) ver. 3.0 (TaKaRa, Japan). PCR reactions were conducted as above using OrNV15a and b primers (detects gene GrBNV-gp83-like gene). This experiment was conducted in triplicate.Inoculum preparation using FRI-AnCu-35 cellsOrNV isolates were propagated using the FRI-AnCu-35 (AnCu35) cell line for further analyses following methods previously described for the DSIR-Ha-1179 cell line system9,12. AnCu35 was a Coleopteran cell line readily available in Japan, and was inoculated with the Palau OrNV solution prepared above and the OrNV-X2B isolate which was provided by AgResearch, New Zealand. When the cell culture reached 25% confluency, a 100 µl aliquot of virus solution was inoculated and incubated at 25 °C. The virus-treated cells were observed by optical microscope.Quantification of viral copy number using qPCR was conducted as follows. To measure the amount of OrNV virus produced by the AnCu35 cell line, DNA was extracted as described above for tissue samples from 1.5 ml of the virus treated cell’s suspension at 10 dpi (3 suspensions per each virus isolate). The extracted DNA was subjected to quantitative PCR (qPCR) following previously described methods31. The primer pair for qPCR was designed from the genome sequence of the P74 homolog of OrNV, a viral structural protein that is conserved widely among nudiviruses, polydnaviruses and baculoviruses32, to amplify a region of 82 bp of OrNV-X2B-gp120 (OrNV-p74_f2026: 5′-ATCGCCGGTGTGTTTATGG-3′, OrNV-p74_r2107: 5′-AGAGGGCTAACGCTACGAC-3′). The qPCR reaction was performed by using Step One Plus Real-Time PCR System (Life Technologies, USA). The reaction mixture contained 10 ng of template DNA, 5 µl of FastStart Universal SYBR Green Master Mix (ROX) (Roche, Switzerland), 0.3 µl forward primer (10 µM), 0.3 µl reverse primer (10 µM), and 3.4 µl Milli-Q water. The qPCR cycle condition was as follows: 95 °C 10 min; 40 cycle of 95 °C 15 s, 60 °C 1 min. At the end of the cycles, a dissociation curve analysis of the amplified product was performed as follows: 95 °C 15 s, 60 °C 1 min, 95 °C 15 s. The Ct value of each sample DNA was measured twice using two wells as technical replicates. The quantity of the viral genome (ng) in each sample was calculated from a standard curve generated from 29.7 to 29.7 × 10–5 ng of purified PCR amplicon from the OrNV P74 gene. The viral copies in 1 ng of sample DNA was estimated from the molecular weight of qPCR target region (p74). The virus titer was determined from average copy numbers of three virus suspensions as follows. The p74 qPCR amplicon was 83 bp, and the molecular weight of the amplicon was calculated as the length of dsDNA (83 bp) × 330 daltons × 2 nt/bp = 54,780 daltons (g/mol). DNA weight of 1 copy of virus genome was calculated as 54,780 g/mol/Avogadro constant (6.023 × 1023 molecules/mol) = 9.095 × 10–20 g/ molecule. Amplicons of the above region was purified by QIA quick PCR purification kit (Qiagen) and 29.7 ng/ul of DNA was obtained for use as a quantification standard. This is equivalent to 3.266 × 1011 copies of p74 gene (because the amplicon is 9.095 × 10–20 g/copy). Based on qPCR using the serial dilutions (× 10 – 105) of the standard DNA prepared above, Ct values were examined by each concentration of viral DNA. Ct-value = − 3.3112x – 1.4219 (x: diluton factor of 10x). Accordingly, copy number of p74 = 3.266 × 1011+x. Viral copy number (copy number of p74 genes) was calculated from Ct-value from the above formula.Viral replication in CRB larvae by time course and killing speedField collected CRB-G larvae from Japan were inoculated with the OrNV-Palau1 and -X2B isolates to examine establishment of infection over time using qPCR. The inoculum was prepared from supernatant collected from OrNV infected AnCu35 cell cultures at 10 dpi, passed through a 0.45 µm filter, and preserved at 4 °C until use.Second instar CRB was inoculated intrahemocoelically with 30 μl of the virus solution prepared from cell-culture per larva using a microinjector (Kiya Kogyo Seisakusho, Japan) fitted with a micro-syringe (Ito Seisakusho, Japan). The virus doses of OrNV-Palau1 and -X2B strains used for inoculation were confirmed to be comparable by absolute quantification using the above qPCR method (Palau1: 3.1 × 105 copies/ng, X2B: 3.3 × 105copies/ng; the mean titer of 3 DNA templates, respectively). As a mock treatment, CRB was injected with 30 µl PBS. The inoculated larvae were kept individually in plastic containers with a rearing mat in a 25 °C incubator. The samples were collected at 3, 6, and 9 dpi (25–30 larvae per time point) into 15 ml tubes and stored at − 30 °C until the DNA was extracted as above. Total DNA was extracted from whole, individual larvae which were dissected to remove midgut contents to prevent interference to Taq polymerase, and subjected to qPCR as above. Changes in viral copy number within the same virus strain over time were analyzed by one-way, nonparametric Steel–Dwass tests using JMP@ 9.0.0 software (SAS Institute, Cary, NC). Differences in virus copy number between strains were analyzed in the same way, but to correct for errors in the test values due to multiple comparisons, Bonferroni’s correction was used to set the α-value for the test at 0.008333. Ten larvae were inoculated and examined per each treatment-time point with three replications.To estimate killing speed, CRB-G larvae from Japan were inoculated with the OrNV-Palau1 and -X2B isolates as described previously. Intrahemocoelically inoculated larvae were reared individually in plastic containers with a rearing mat in a 25 °C incubator. Mortality of inoculated larvae were observed every day. Forty larvae were examined in a replicate with three replications carried out for virus treatments (total 120 larvae). The mock PBS inoculation treatment was done only once (total 37 larvae).Genome sequencingGenome sequencing of the OrNV-Palau1 isolate and X2B isolate was conducted. For obtaining high quality DNA, virus particles were purified, from 3 mL of AnCu35 culture supernatant collected six days after inoculation with OrNV. Virus containing supernatant was transferred to Ultra-Clear polyallomer tubes (Beckman Coulter, USA) with a 20–50% (w/w) sucrose density gradient and subjected to ultracentrifugation at 72,100 g, 4 °C, for 1 h. After ultracentrifugation, the white virus band was collected in a 1.5 ml tube. The solution was then subjected to ultracentrifugation at 110,000 g, 4 °C for 1 h to precipitate the viral particles33. Then, DNA was extracted from purified OrNV virions as described above. For the sequencing analysis, DNA libraries were prepared using the Nextera XT DNA Library Prep Kit (Illumina, USA). Amplified libraries were sequenced on Illumina HiSeq 2500 instrument using paired-end 2 × 150 bp chemistry which was performed by Novogene (Beijing, China). Contigs of each strain from NGS reads were generated by assembly using Unicycler (version 0.4.8)34. The gaps between contigs were further closed with Sanger sequences obtained by PCR direct sequencing using appropriate specific primers, and the sequence was aligned by minimap2 (version 2.17)35. The assembly and sequences of contigs were also confirmed by mapping to the OrNV isolate Solomon Islands genome sequence (GenBank accession no. MN623374.1) with NGS reads and Sanger sequences using minimap2. The mapped reads (SAM files) were converted to BAM format using SAMtools (version 1.10)36. After the sorting and indexing of BAM files, the consensus sequences were generated using bcftools (version 1.10.2)37.ORFs of at least 50 codons in size that possessed significant amino acid sequence similarity with ORFs from OrNV-Ma07 were identified with Lasergene GeneQuest (DNAStar, v. 17) and BLASTp. ORFs with no significant matches to other sequences also were selected for annotation if (a) they did not overlap a larger ORF by > 75 bp, and (b) they were predicted to be protein-encoding by both the fgenesV0 (http://www.softberry.com/berry.phtml?topic=index&group=programs&subgroup=gfindv) and Vgas38 programs.OrNV genome sequences were compared by pairwise alignment using the Martinez/Needleman-Wunsch method as implemented in Lasergene MegAlign 15. Pairwise sequence identities were determined from these alignments as previously described39. Differences in ORF content and distribution of selected OrNV genomic regions were visualized with Mauve version 2015022640.Phylogenetic inferenceTo infer the relationships among OrNV isolates on the basis of nucleotide sequence alignments, the DNA polymerase ORFs of completely sequenced isolates (Table 2), OrNV-PV50516, and a set of nine isolates from Indonesia17 were aligned by MUSCLE as implemented in Lasergene MegAlign Pro v. 17 (DNAStar). Phylogeny was inferred by maximum likelihood using MEGA X28 with the Tamura-Nei (TN93) model41, with ambiguous data eliminated prior to analysis. Tree reliability was evaluated by bootstrap with 500 replicates. More
