Quantitative PCR (qPCR) qPCR was performed on pooled samples from each month. Equivalent amounts of RNA (10 mg) from each hive sample (monitor hives 1–20) were pooled according to the month in which they were collected (April 2009 to January 2010). Pooled RNA was further purified using Qiagen RNAeasy columns, including on column DNase Treatment (Qiagen). cDNA synthesis reactions were performed with SuperScriptIII (Invitrogen) according to manufacturer’s instructions. In brief, RNA from each pooled sample (5 mg), random hexamer (1.25 mg) and dNTPs (0.5 mM each) were combined in a 50 mL reaction volume, incubated at 65uC (5 min), cooled on ice (1 min) and subsequently combined with 50 mL of 26 First-Strand Buffer containing SSIII (1000 U), DTT (5 mM), and RNaseOUT (200 U). Reverse transcription reactions were incubated for 12 hours at 42uC followed by inactivation of the reaction (70uC, 15 min). qPCR was performed in triplicate wells using 2 mL of cDNA as template in 20 ml reactions composed of HotStartTaq 26 Mastermix (Denville), 16 SYBR Green (Invitrogen), MgCl2 (3 mM), and forward and reverse primers (600 nM each) (Table S2) on a LightCycler480 (Roche). The qPCR thermo-profile consisted of a single pre- incubation 95uC (10 min), 35 cycles of 95uC (30 s), 60uC (30 s), and 72uC (30 s). No RT control reactions using pooled RNA as the template for qPCR were performed in triplicate on each plate. Target qPCR amplicons were cloned into pGEM-T (Promega) or TOPO CR 2.1 (Invitrogen) vectors and sequence verified. Plasmid standards, containing from 109 to 102 copies per reaction, were used as qPCR templates to assess primer efficiency and generate the pathogen-specific standard curves used to quantify the viral genome or rRNA copy number. The linear standard equations generated by plotting the crossing point (Cp) versus the log10 of the initial plasmid copy number for each primer set were as follows: BQCV Cp = 25.676+59.44, R2 = 0.975; SBV Cp = 25.34 6+56.33, R2 = 0.976; ABPV Cp = 24.036+43.7, R2 = 0.995; LSV1 Cp = 24.216+46.56, R2 = 0.993; LSV2 Cp = 23.66 6+40.76, R2 = 0.998; ALP-Br Cp = 22.916+34.76, R2 = 0.980; BSRV Cp = 23.286+36.93, R2 = 0.999; Nosema ceranae Cp = 27.036+69.43, R2 = 0.975; Crithidia rRNA Cp = 23.13 6+36.44, R2 = 0.994 (LightCycler 480 Software, Abs Quant/2nd Derivative Max, high sensitivity mode, Roche). The detection limits of each qPCR primer set were as follows: Crithidia and ALP- Br 2102 copies, LSV2 and BSRV 2103 copies, BQCV, SBV, ABPV, LSV1 and Nosema 2104 copies. Specific qPCR amplicons had Cp values of ,30. Pathogen copy number data were reported per RT-qPCR reaction (Figure 4). Values obtained from the no RT control reactions, all below the detection limit of the assays, were subtracted from the total pathogen copy number for each month. An estimate of the number of viral genomes per bee can be obtained by multiplying the reported qPCR copy number values by 500. This estimate is based on the following: typical RNA yield was approximately 50 mg per bee, each qPCR reaction was performed on cDNA generated from 100 ng RNA, therefore each well represents 1/500th of an individual bee. We choose to represent the raw data, since each monthly-pooled sample was composed of variable bee numbers due to differential sampling frequency each month. In addition, qPCR with a host primer set, Apis m. Rpl8, was performed using 1 mL cDNA template on each qPCR plate to ensure consistency and cDNA quality. qPCR products were analyzed by melting point analysis and 2% agarose gel electrophoresis (Figure S1). LSV Northern Blot Honey bees from two LSV positive honey bee colonies were homogenized in 500 mL phosphate buffered saline (PBS, UCSF Temporal Analysis of the Honey Bee Microbiome Cell Culture Facility) with a sterile zinc-coated steel ball bearing (5 mm) using a TissueLyzer II (Retsch) for 4 minutes at 30 Hz; 5 bees per micro-centrifuge tube. Lysates were centrifuged for 10 minutes at 12,0006 g and RNA was extracted from both the supernatant and the bee carcass containing pellet using TRIzol Reagent (Invitrogen) according to manufacturer’s instructions. RNA was further purified using RNAeasy columns, including on column DNase Treatment (Qiagen). RNA (15 mg per lane) was combined with glyoxal-based loading dye (Northern-MaxH sample loading dye, Ambion) and denatured at 50uC for 30 min prior to gel electrophoresis on an ethidium bromide containing 1.5% agarose BPTE gel using BPTE running buffer. BTPE buffer is composed of 10 mM PIPES, 30 mM Bis-Tris, 1 mM EDTA, pH 6.5. The gel was imaged using a UV lightbox and then soaked in 0.05 N NaOH for 20 min prior to overnight transfer to a membrane using the NytranH SuPerCharge TurboblotterTM system and 206 SSC. Following transfer the membrane was washed in 26 SSC (265 min), dried and UV crosslinked using a Stratalinker (Stratagene). LSV specific primers were used to amplify sequences corresponding approximately to the 59, middle, and 39 regions of the viral genome (primers listed in Table S2). The PCR products were column purified using the MinElute Reaction Cleanup Kit (Qiagen), labeled with {a´32P} dCTP using Ready-To-GoTM DNA Labelling Beads (-dCTP) (Amersham; GE Healthcare) and used as LSV-specific Northern blot probes. The membrane was cut into three pieces and incubated, while rotating, in ULTRAhyb buffer (Ambion) at 42uC for 30 min prior to the addition of the radiolabled probes (106 counts per minute per mL hybridization buffer). Following overnight hybrization at 42uC, the membranes were washed at 42uC (265 min in 26 SSC 0.1% SDS; 2615 min in 16 SSC 0.1% SDS, 2615 min in 0.16 SSC 0.1%SDS). Phosphoimaging was performed using a Typhoon 9400 imager (GE Healthcare) (Figure S4). Negative strand-specific RT-PCR LSV strain 1 and 2 positive samples were analyzed for the presence of negative-strand RNA, which is indicative of virus replication, using strand-specific RT-PCR [39,64,65]. RNA from select samples (e.g. pooled July sample) was further purified using Qiagen RNAeasy columns, including on column DNase Treat- ment (Qiagen). cDNA synthesis reactions were performed with SuperScriptIII (Invitrogen) according to manufacturer’s instruc- tions using negative strand-specific LSV1 and 2 primers tagged with an additional 21 nt of sequence (59- GGCCGTCA- TGGTGGCGAATAA) at their 59 end [65]; the tag sequence shares no homology with LSV nor to the honey bee genome (primer sequences listed in Table S2). In brief, RNA from each sample (1 mg), tagged-negative strand specific LSV primer (10 pmole) or random hexamers (50 ng) and dNTPs (0.5 mM each) were combined in a 10 mL reaction volume, incubated at 65uC (5 min), cooled on ice (1 min) and subsequently combined with 10 mL of 26 First-Strand Buffer containing SSIII (200 U), DTT (5 mM), and RNaseOUT (40 U). Reverse transcription reactions were incubated for 1 hour at 50uC followed by inactivation of the reaction (70uC, 15 min). Unincorporated primers present in the RT reactions were digested with exonuclease I (Fermentas), 0.1 Units per reaction which corresponds to a 10-fold excess of enzyme relative to the initial primer concentration, at 37uC for 30 min followed by heat inactivation at 80uC for 15 minutes. PCR was performed using 2 mL of exonuclease I treated cDNA template in 25 ml reactions containing 10 pmol each of a tag-specific forward primer (TAGS) and an LSV-specific reverse primer using the following cycling conditions: 95uC for 5 min; 95uC for 30 s, 58uC for 30 s, 72uC for PLoS ONE | www.plosone.org 14 June 2011 | Volume 6 | Issue 6 | e20656