Fresh fecal samples were submitted to the Clinical Virology Laboratory (University of Tennessee Veterinary Medical Center) for electronmicroscopic examination. Samples were processed as described in
. Briefly, samples were centrifuged in distilled water at approximately 15,000×g for one hour. The supernatant was discarded and pellet resuspended in 2–3 ml distilled water. 100ul of the resuspended material was added to 1 ml distilled water containing 3% phosphotungstic acid. The solution was aerosolized onto a carbonate-coated copper grid and examined by EM at 30,000 and 300,000X.
Nucleic acid extraction
Total nucleic acid was extracted from stool samples using the Qiagen QiaAmp DNA mini kit, which purifies RNA and DNA, according to the manufacturer’s protocol. Additionally, the EM VLP-positive stool sample (sample ID 10–2208) was diluted in PBS, clarified by centrifugation at 10,000 g for 1 minute, and passed through a 0.22 um filter (Millipore). The Filtrate was treated with microccoal nuclease (MNase; NEB) in order to degrade non-encapsidated (or otherwise not protected) nucleic acid. MNase reactions containing 1x reaction buffer, 100 μg/ml BSA, and 4, 1, 0.2, or 0 units MNase were incubated at room temperature for 15 minutes. MNase was inactivated by addition of EGTA to 20 mM.
For virochip analysis, randomly-primed libraries were prepared from total nucleic acid. RNA was reverse transcribed in reactions containing 1x reaction buffer, 5 mM dithiothreitol, 1.25 mM dNTPs, 20 pmoles primer MDS-4 (Table
2), 100 U Superscript III (Invitrogen), and 100 ng template. Following reverse transcription, Sequenase reaction buffer and 2 U of Sequenase DNA polymerase (Affymetrix) were added to samples for second strand synthesis. The Sequenase reactions were performed twice so that starting DNA templates would be converted into end-tagged library molecules. The resulting libraries were amplified by PCR using primer MDS-189 (Table
2). PCRs contained 1x reaction buffer, 2 μM primer, 0.25 mM dNTPs, 2 U taq DNA polymerase, and 2 μl library template. Thermocycling was: 95°C for 2 min; 2 cycles of 95°C for 30 sec, 40°C for 30 sec, and 72°C for 1 min, then 25 cycles of 95°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min, with a final extension of 72°C for 5 min.
For microarray hybridzation, a fraction of each library was amplified by PCR as above but with a modified dNTP mixture including 5-(3-aminoallyl)-dUTP (Ambion) in lieu of 75% of the dTTP normally in the mixture. The resulting amino-allyl-containing DNA was purified using a DNA Clean and Concentrator-5 column (DNA-CC-5; Zymo Research). The eluate was heat denatured at 95°C for 2 min, cooled briefly on ice, then fluorescently labeled in reactions containing 100 mM sodium bicarbonate pH 9, 10% DMSO, and 667 μM Cy3 mono NHS ester (GE Healthcare) for 1 hour at 25 °C. Labeled DNA was purified using the DNA-CC-5 columns and added to hybridization reactions containing 3xSSC, 25 mM HEPES pH 7.4, and 0.25% SDS. Hybridization mixtures were heated at 95°C for 2 minutes then applied to microarrays and hybridized overnight at 65 °C. Following hybridization, arrays were washed twice in 0.57xSSC and 0.028% SDS and twice in 0.057x SSC, then scanned on an Axon GenePix 4000B microarray scanner. Three tools were used to analyze Virochip data: E-predict
, Z-score analysis
, and cluster analysis
Oligonucleotide sequences used to amplify the rabbit astrovirus genome are listed in Table
2. PCRs contained 1x reaction buffer, 2 μM primer, 0.25 mM dNTPs, 2 U Taq DNA polymerase, and 2 μl library template. Thermocycling was: 95°C for 2 min, then 30 cycles of 95°C for 30 sec, 58°C for 30 sec, and 72°C for 2 min, with a final extension of 72°C for 5 min. Amplicons were purified from agarose gels using the PureLink gel extraction kit (Invitrogen) and cloned into the pCR2.1 TOPO vector (Invitrogen) according to the manufacturer’s protocols. Cloned amplicons were sequenced on an ABI 3700 instrument. For diagnostic testing, primers MDS-119 and −120 (Table
1) were used with reaction and thermocycling as described above.
5′ and 3′ RACE
5′ and 3′ RACE were performed essentially as described
[64, 65], with primers listed in Table
2. RACE amplicons were cloned and sequenced as described above.
Ion Torrent metagenomic sequencing
Randomly primed cDNA was prepared in reverse transcription reactions containing 1x reaction buffer, 5 mM dithiothreitol, 1.25 mM dNTPs, 20 pmoles random hexamer primer, 100 U Superscript III (Invitrogen), and 100 ng RNA template. Second strand DNA was synthesized as above using Sequenase DNA polymerase. cDNA was adapted for Ion Torrent sequencing using the Ion Xpress Library Kit (catalog # 4468987) according to the manufacturer’s protocols. Although this protocol includes PCR amplification, identical reads were removed during analysis to eliminate possible PCR duplicates. Sequencing was performed on an Ion Personal Genome Machine on one model 314 chip and one model 316 chip according to the manufacturer’s protocols.
100 μl of stool positive for rabbit astrovirus RNA by RT-PCR was diluted 1:10 in PBS and filtered through a 0.22 μm filter (Millipore). Filtrate was used to inoculate cultures of RK, Vero, HT-29, or Caco-2 cells, which were grown in DMEM supplemented with 10% fetal bovine serum and 50 units/ml penicillin and 50 μg/ml streptomycin at 37°C and 5% CO2. Cultures were supplemented with 0, 1, 10, or 100 μg/ml trypsin, which has been shown to be necessary for astrovirus infectivity in tissue culture. Culture supernatant was harvested and replaced at the indicated time points and stored at −80°C until processing. RNA was extracted from supernatant and reverse transcribed as described above. PCR using primers MDS-119 and −120 for rabbit astrovirus and MDS-156 and −157 for rabbit rRNA (as a positive RT-PCR control) was used to detect viral RNA, with reaction conditions as described above (See Table
Astrovirus protein sequences for all astroviruses with complete genome sequences were downloaded from GenBank. The accessions of these sequences are: AB308374, AF260508, AY179509, AY720891, AY720892, DQ028633, DQ070852, DQ344027, EU111742, FJ222451, FJ402983, FJ434664, FJ755402, FJ755403, FJ755404, FJ755405, FJ919225, FJ919226, FJ919227, FJ919228, FJ973620, GQ415660, GQ495608, GQ891990, GU985458, HM237363, HM450380, HQ398856, HQ916313, HQ916314, HQ916316, HQ916317, JF414802, JF755422, L23513, NC_001943, NC_002469, NC_002470, NC_003790, NC_004579, NC_005790, NC_010646, NC_011400, NC_012437, NC_013060, NC_013443, NC_014320, NC_015935, and Y15937. Sequences were aligned using Clustal (version 2.0.12) using default parameters
. Alignments were manually inspected and trimmed to the point of clear homology. The ORF2 (capsid) alignment is based on the first ~420 amino acids of the protein sequences, which correspond to the relatively conserved core region of the capsid protein. Neighbor joining trees were constructed using PhyML software (PhyML plugin for Geneious version 2.0.12) using 100 bootstrap replicates and default parameters
. The sequence of the rabbit astrovirus has been deposited in GenBank, with accession JF729316.
The BLAST alignment software (version 2.2.25+) was used to taxonomically categorize the sequences in the Ion Torrent dataset
. The NCBI non-redundant nucleotide database was searched using the blastn algorithm with an expect value cutoff of 1e-6. For each query producing an alignment, the taxonomic ID of the best alignment was determined and tallied. The Paired-Read Iterative Contig Extenion (PRICE, version 0.13) de novo assembler (freely available at:
http://derisilab.ucsf.edu/software/price/index.html) was used to assemble the rabbit astrovirus genome from the Ion Torrent dataset.
Several tools were combined to determine coverage, quality, and error metrics. First, CD-HIT was used to collapse identical sequences (cd-hit-est version 4.5.4 run with parameter –c 1), which may be PCR duplicates
. Then, the bowtie2 aligner was used to map unique reads to the Sanger-verified viral genome assembly (version 2.0.0 run in –local mode with otherwise default parameters;
). The number of gap openings (XO field) and mismatches (XM field) and the total number of aligned bases in the bowtie2 SAM output were tallied to determine the frequency of indels and mismatches. Average per base quality scores were determined directly from the Ion Torrent FASTQ output.