3.1. Cell Lines
Cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) except as indicated. Trypsin (Gibco, Grand Island, NY, USA, cat. No. 25300-054) was used for passaging all adherent cell lines, except the insect cell lines, which were dislodged by scraping. Each cell line was passaged at least three times before the cells were harvested at the passage number indicated below and washed twice with 1× PBS (Quality Biologicals, Gaithersburg, MD, USA, cat. No. 114-058-101). After washing, 2 × 106 cells were pelleted at 1200 rpm at 4 °C (Allegra™ 6KR centrifuge, Beckman Coulter, Danvers, MA, USA) and frozen at −80 °C for further nucleic acid extraction.
MDCK (Madin-Darby canine kidney, passage 64; ATCC, cat. No. CCL-34), HEK-293, (human embryonic kidney, passage 42; ATCC, cat. No. CRL-1573), CV-1, (African green monkey kidney, passage 39; ATCC, cat. No. CCL-70), HeLa (human cervical carcinoma, passage 100–112; ATCC cat. No. CCL-2), MRC-5 (human diploid fetal lung, passage 23; ATCC, cat. No. CCL-171), and VERO (African green monkey kidney, passage 127; ATCC, cat. No. CCL-81) were grown in Eagle’s minimal essential medium (EMEM, Mediatech, Manassas, VA, USA, cat. No. 15-010-CV) supplemented with 10% fetal bovine serum (FBS, heat-inactivated at 56 °C for 30 min; Hyclone, Logan, UT, USA, cat. No. SH30071.07) or 5% FBS in case of VERO, 100 U penicillin per mL and 100 µg streptomycin per mL (Quality Biological, cat. No. 120-095-721), 2 mM l-glutamine (Quality Biological, cat. No. 118-084-721), 1× nonessential amino acids (MEM-NEAA, Quality Biological, cat. No. 116-078-721), and 1 mM sodium pyruvate (Quality Biological, cat. No. 116-079-060). 293T/17 (human fetal kidney, passage 21; ATCC, cat. No. CRL-11268) and A549 (human lung carcinoma, passage 84; ATCC, cat. No. CCL-185) were grown in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA, cat. No. 11995-065) supplemented with 10% FBS, 100 U of penicillin per mL and 100 µg of streptomycin per mL, and 2 mM l-glutamine. Cf2Th (neonate canine thymus, passage 62; ATCC, cat. No. CRL-1430) were grown in DMEM, 20% FBS, 100 U of penicillin per ml and 100 µg of streptomycin per ml, 2 mM l-glutamine, and 1× MEM-NEAA.
Raji (human lymphoblastoid lymphoma, FDA passage 5; ATCC, cat. No. CCL-86) was grown in RPMI 1640 (Quality Biological, cat. No. 112-024-101), 10% FBS, 100 U penicillin per mL and 100 µg streptomycin per mL, 2 mM l-glutamine, and 1× MEM-NEAA. A204 (human rhabdomyosarcoma, passage 84; ATCC, cat. No. HTB-62) was grown in RPMI 1640, 10% FBS, 100 U penicillin per mL and 100 µg streptomycin per mL, 2 mM l-glutamine, and 1× MEM-NEAA.
Sf9 (Spodoptera frugiperda ovary, passage 25; ATCC, cat. No. CRL-1711) and High Five (Trichopulsia ni ovary, FDA passage 5; Invitrogen, Grand Island, NY, USA, cat. No. P/N51-4005) were grown in Grace’s Insect Medium (Invitrogen, cat. No. 11605-094), supplemented with 10% FBS (FBS, heat-inactivated at 56 °C for 30 min; certified for insect cells Hyclone, cat. No. SH30070.03), 100 U penicillin per mL and 100 µg streptomycin per mL, and 2 mM l-glutamine. Schneider’s Drosophilia line 2 (SL2) (Drosophila melanogaster embryo, passage 21; D.Mel.(2); ATCC, cat. No. CRL-1963) was grown in Schneider’s Drosophila Medium (Invitrogen, cat. No. 21720-024), supplemented with 10% FBS (certified for insect cells), 100 U penicillin per mL and 100 µg streptomycin per mL, and 2 mM l-glutamine.
3.2. Preparation of XMRV and SFV-1 RNA Panels with and without Background Sf9 Total Nucleic Acids and RT-PCR Assays
XMRV and SFV-1 virus stocks were prepared using LNCaP and Mus dunni
cells, respectively. The infectious titer for XMRV was 104.5
per mL [39
] and for SFV-1 was 105.5
per mL. For both viruses, 10-fold serial dilutions (range 100
) were made from the original virus stock and the number of particles per µL was determined based upon RT activity using a modified STF-PERT assay (designated as two-step fluorescent-PERT; TSF-PERT). The number of particles in the original stock was determined as the average of the calculated particles at each dilution based upon a standard curve using HIV-1 RT enzyme (Worthington Biochemical Corporation Lakewood, NJ, USA, cat. No. LS05006; lot No. X1H2839), which was determined to have 262 pU of RT activity per particle [40
]. The STF-PERT one-step assay [40
] was modified into a two-step assay that no longer requires the use of AmpliWax®
PCR Gem 50, a product that has been discontinued. In addition, the TSF-PERT assay was designed as a partially automated process compatible with the Eppendorf epMotion 5070 robot (Eppendorf, Hauppauge, NY, USA, cat. No. 960000111). The RT master mix, standards, and samples were manually added to the 96-well plate as previously described [40
] and the RT reaction was carried out using an Eppendorf MasterCycler ProS (Eppendorf, cat. No. 950030020). Once complete, the RT plate was placed on a 96-well cold Thermoblock (Eppendorf, cat. No. 960002083) and the PCR master mix was placed on a TMX 24 × SafeLock Thermorack (Eppendorf, cat. No. 960002070). The Eppendorf epMotion 5070 robot dispensed 25 µL of the PCR master mix into each well. Finally, the plate was sealed and processed for Taqman qPCR. The results indicated that XMRV had 3.12 × 105
particles per µL and SFV-1 had 4.02 × 105
particles per µL.
Total viral RNA was extracted from each dilution using the QIAamp viral RNA mini kit (Qiagen, Valencia, CA, USA, cat. No. 52904) in combination with the RNase-free DNase I set (Qiagen, cat. No. 79254) according to the manufacturer’s instructions. Briefly, 200 µL of each dilution was processed as specified by the QIAamp viral RNA mini kit with an added on-column DNase I treatment. The RNA was eluted in 200 µL of DNase/RNase-free water. Concentration and purity were determined by using UV absorbance.
Viral RNA panels were made in the presence and absence of background Sf9 cell nucleic acids. Total cell nucleic acid was extracted from Sf9 cell pellets using the QIAamp® MinElute® Virus Spin Kit (Qiagen, cat. No. 57704). Briefly, cell pellets (2 × 106 cells) were resuspended in 200 µL buffer and processed as specified in the QIAamp® MinElute® Virus Spin Kit followed by elution in 50 µL of DNase/RNase-free water. RNA panels (10−2–10−6) were initially made in a background of 106 cell equivalents of Sf9 total nucleic acids or in water to create 10-fold and 100-fold dilutions for testing in a background of 105 and 104 cell equivalents of Sf9 total nucleic acid. Selected samples were tested in different assays.
cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA, cat. No. 170-8890) according to the manufacturer’s instructions. An XMRV gag fragment (500 bp) was amplified by a nested PCR assay using viral cDNA (2 µL) with an annealing temperature of 63 °C for the outer primers, as previously described [41
]. An SFV-1 gag fragment (548 bp) fragment was also amplified by a nested PCR assay. The first amplification was done with primers SFVgagF1 (5'-AACCTAGGTGGAGAGCTGAAGG-3') and SFVgagR1 (5'-ATGGAGAGGGTAAGAACCATGGG-3') to generate a 947 bp fragment. This was followed by a second amplification with primers SFVgagF2 (5'-ATGATGCACTTTGGCAGCCATTGG-3') and SFVgagR2 (5'-ACCTGCTGAATGTTGATTCTGTGC-3') to generate a 578 bp fragment. Viral cDNA (2 µL) was used in a 50 µL reaction containing 1.5 U Taq DNA Polymerase (Roche Applied Science), 0.5 mM primers, 0.2 mM dNTP mix, and 1× PCR buffer with 1.5 mM MgCl2
(Roche Applied Science, Indianapolis, IN, USA). The PCR conditions used were: 94 °C for 3 min, (94 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min) × 35 cycles, 72 °C for 10 min, and a 4 °C hold for infinity. In each case a control sample without reverse transcriptase (RT) was included.
DNA fragments were visualized with ethidium bromide staining after running 10 µL of each PCR reaction on a 1.4% agarose gel. DNA sizes were determined using a 100 bp DNA ladder (New England Biolabs, Ipswich, MA, USA, cat. No. N3231L).
3.3. PCR/ESI-MS (PLEX-ID)
Cell pellets (2 × 106
cells) were processed in our laboratory or were sent to Athogen for processing [20
]. In addition, total nucleic acids were extracted in our laboratory from PBS, ultra-pure dH20, complete medium (DMEM supplemented with 10% FBS (Hyclone, Logan, UT, USA, cat. No. SH30071.07, lot. No. ATE32066), 100 U of penicillin per ml and 100 µg of streptomycin per mL, l
-glutamine and 1× MEM-NEAA), and trypsin (Gibco, Grand Island, NY, USA, cat. No. 25300-054, lot No 917992). Briefly, cell pellets were resuspended in 200 µL AVE buffer or 200 µL of liquid sample were processed using the QIAamp®
Virus Spin Kit (Qiagen, cat. No. 57704) as described by the manufacturer. Total nucleic acids were eluted in 200 µL of DNAse/RNAse-free water. All samples were analyzed using PLEX-ID Biopharma Viral Assay Kits (Ibis BioSciences, Abbott, Carlsbad, CA, USA): Bovine origin virus, Porcine origin virus, CHO cell virus, and Other virus, either in our laboratory using an on-site PLEX-ID or sent to Athogen for testing. In all cases, a one-step 50 µL RT-PCR reaction containing 5 µL of total nucleic acids extract was performed as previously described [42
]. All PCRs were conducted in a 96-well plate format using an Eppendorf Mastercycler under conditions previously described [14
]. Samples were processed by an automated carousel contained with the PLEX-ID machine, where PCR samples were desalted and injected into the ESI-TOF mass spectrometer. Once the signal was processed, genomic signatures were generated by Ibis software and compared against Athogen’s curated database.
3.4. LLMDA and Virohip
RNA, DNA, and total nucleic acid samples were sent to Lawrence Livermore National Laboratory (LLNL, Livermore, CA, USA) and University of California, San Francisco (UCSF, San Francisco, CA, USA) to be analyzed by LLMDA [4
] and ViroChip v.5 [2
], respectively. Briefly, each RNA sample (10 µL for the LLMDA and 11 µL for the Virochip) was reverse-transcribed to cDNA using random primers and PCR-amplified prior to labeling with Cy3 fluorescent dye and hybridization to the arrays [2
An updated LLMDA v.5 that was designed to detect around 5500 microbial species, which were sequenced through December 2011, was used for our studies. This study employed the 12-plex 135 K format of this array, which is restricted to pathogens associated with vertebrate infection, and includes 1856 viral, 1398 bacterial, 123 archaean, 48 fungal, and 94 protozoan species. In the LLMDA v.5 [4
], in order to expand the abilities to detect these viruses from cell lines probes were also designed to detect additional mammalian endogenous retroviruses and some insect viruses. These included: 6 eukaryotic sequences (Drosophila simulans
clone F pop-variant Chicharo (Portugal) retrotransposon tirant envelope protein (env
) gene partial cds (Genbank ID JN786103.1), Drosophila melanogaster
copia-like element 17.6 (Genbank ID X01472.1), Drosophila melanogaster
Idefix retroelement: gag pol
genes partial (Genbank ID, AJ009736.1), Drosophila melanogaster
transposable element 297 (Genbank ID, X03431.1), Drosophila melanogaster
clone 4.1 gypsy retrotransposon MDG4 complete sequence (Genbank ID, DQ887187.1), Homo sapiens
gypsy retrotransposon integrase 1 (GIN1) mRNA (Genbank ID, NM_017676.2); 9 partial sequences of Spodoptera frugiperda
insect viruses [33
], 1 baculovirus (Autographa californica
nuclear polyhedrosis virus (AcMNPV) mutant FP-D with incorporated Trichoplusia ni
retrotransposon TED (Tn368) and three open reading frames (Genbank ID M32662.1); and 3 endogenous retroviruses (Odocoileus hemionus
endogenous virus Cervid endogenous retrovirus CrERVg complete genome (Genbank ID JN592050.1), Simian retrovirus 1 isolate SRV_Vero_Assembled gag pseudogene partial sequence; pol protein (pol) gene partial cds; and env pseudogene complete sequence (Genbank ID HM143845.1), and Python molurus
endogenous retrovirus Gag-Pro-Pol protein and Env genes complete cds (Genbank ID AF500296.1).
The ViroChip v.5 used in this study was an 8-plex 60K format and was updated to include 60,000 probes representing complete and partial sequences of all viral genomes in GenBank as of December 2010 [21
3.5. PCR Assay for RD114-Like Virus and Nucleotide Sequencing Analysis
A positive hit for RD114 by PLEX-ID was verified using a nested PCR assay and nucleotide sequencing. PCR primers were developed based upon the PLEX-ID broad-range forward and reverse primer set 3417 sequences (kindly provided by Rangarajan Sampath, Ibis BioSciences, Abbott, Carlsbad, CA): outer primers were 3417F (5'-TGGGGATTGATTGGAAATTAC-3') and 3417R (5'-TGTTCTATTCATTCTTTCTAC-3'); an inner primer set was designed: 3417F2 (5'-TATTCATTCTTTCTACCTGTCGTG-3') and 3417R2 (5'-ATTGATTGGAAATTACATTGTGC-3'). Total nucleic acids (5 µL) from MDCK cells were amplified in a PCR reaction of total volume 50 µL containing 1.5 U Taq DNA Polymerase (Roche Applied Science, cat. No. 11647687001), 0.5 mM primers, 0.2 mM dNTP mix, and 1× PCR buffer with 1.5 mM MgCl2 (Roche Applied Science, cat No 11647687001). The PCR conditions used were: 95 °C for 5 min (95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min) × 35 cycles, 72 °C for 10 min, and a 4 °C hold for infinity. A 74 bp fragment was amplified after the first round of PCR using the outer primers and a 64 bp fragment was amplified by a second amplification with the inner primers.
DNA fragments were visualized by ethidium bromide staining after running 20 µL of the PCR reaction on a 4% NuSieve GTG gel (Lonza, Rockland, ME, USA, cat. No. 50081). DNA sizes were determined using ΦX174 DNA/BsuR I (Hae III) marker (Fermentas, Inc., Glen Burnie, MD, USA, cat. No. SM0253). DNA fragments were purified from the gel fragment by using Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corp., Orange, CA, USA, cat. No. D4001) according to the manufacturer’s instructions.
The purified PCR fragments were cloned into pGEM-T Easy Vector (Promega, Fitchburg, WI, USA, cat. No. A1380) as per the manufacturer’s instructions and transformed into JM109 high efficiency competent cells. Several colonies were picked and were grown in S.O.C. medium (Invitrogen, Carlsbad, CA, USA, cat. No. 15544-034) at 37 °C overnight. Plasmid preps were made using QIAprep® Spin Miniprep kit (Qiagen, cat. No. 27106) and sequenced with the T7 primer using Big Dye v3.1 (Applied Biosystems, Foster City, CA, USA, cat. No. 4337455). Nucleotide sequences were generated on an ABI 3130xl Genetic Analyzer and sequence analysis was done using Vector NTI (Invitrogen). The identity of the PCR fragments amplified from the first and second PCR amplifications was confirmed by nucleotide sequencing; since both had similar overlapping sequences the larger 74 bp sequence was used for subsequent analysis.
Bioinformatic and phylogentic analysis were done using BLAST (National Center for Biotechnology Information, National Library of Medicine, NIH, Bethesda, MD, USA), CLC Genomics workbench (CLC Bio., Cambridge, MA, USA), RetroTector© (Uppsala Universitet, Uppsala, Sweden), and MEGA 5.1 (Molecular Evolutionary Genetics Analysis, Tempe, AZ, USA).