Sample collection
We tested 124 BCCH (28 individuals with AKD and 96 asymptomatic control individuals) for the presence of Poecivirus using cloacal swabs. To obtain samples, individuals were captured using funnel traps and mist nets in Anchorage and the Matanuska-Susitna Valley, Alaska, during the non-breeding season in 2015 (March–April), 2016 (March–April, October–December), and 2017 (March, April). Standard beak measurements were used to classify individuals as AKD-affected or unaffected [1]. Briefly, an individual was considered AKD-affected if it had a nares-to-tip length (chord measurement from anterior end of the right nare to the tip of the upper beak) ≥8.2 mm, an overbite or underbite of > 1.0 mm, or, in the case of specimens for which we had beak samples, if its beak exhibited evidence of hyperkeratosis at the cellular level [7]. We chose to use cloacal swabs because they are non-destructive and easily obtained, and we previously showed cloacal swabs to contain relatively high viral load [7]. In addition, we tested buccal swabs, blood samples, and fecal samples from a subset of individuals to compare the efficacy of a variety of non-terminal sample types for Poecivirus detection. We tested buccal swabs from 21 of the individuals with AKD and 75 of the control individuals. We tested blood samples collected by brachial venipuncture and fecal samples obtained opportunistically from subsets of individuals with AKD (N = 13 and N = 4, respectively). Swab, blood, and fecal samples (collected opportunistically if a bird defecated during handling) were placed in Longmire buffer [13] and stored frozen at − 80 °C until processed.
For viral load determination, in-situ hybridization, and gene expression assay, we used subsets of samples remaining from our previous study [7], for which we obtained beak tissue samples from 28 individuals. Nineteen of these exhibited AKD and were trapped using funnel traps and mist nets in Anchorage and the Matanuska-Susitna Valley, Alaska, during the non-breeding season from 2001 to 2015. Data on infection status of these individuals have been published previously. The 10 individuals with AKD that were collected from 2001 to 2010 were euthanized upon capture with isoflurane using the open-drop method and stored frozen at − 20 °C; the remaining 9 specimens were captured in the winter of 2014 and spring of 2015, euthanized, and stored overnight at 4 °C prior to necropsy, at which time portions of tissues were frozen at − 80 °C and additional samples were placed in formalin. From 2 of these, we collected cloacal and buccal swabs prior to euthanasia; swabs were stored in Longmire buffer [13]. Nine individuals not affected by AKD were collected opportunistically between 1995 and 2010 and stored frozen at − 20 °C; tissues from a subset of these individuals were used as negative controls in the gene expression assay measuring viral replication. Of the tissue samples collected from our previous study, we had adequate samples remaining from 17 individuals that tested positive for Poecivirus; of these 16 exhibited AKD, and one was asymptomatic.
All work was conducted with the approval of the U. S. Geological Survey (USGS) Alaska Science Center Institutional Animal Care and Use Committee (Assurance #2016–14) and under appropriate state and federal permits.
Testing for an association between Poecivirus and avian keratin disorder
RNA was extracted from cloacal and buccal swab samples using a Zymo viral RNA kit. For each sample, 75 μl of Longmire buffer was mixed with 225 μl of viral RNA buffer and the extraction proceeded as described in the manufacturer’s protocol. RNA from tissue samples was extracted using a Zymo quick RNA miniprep kit as previously described [7]. Following extraction, 200 ng of RNA were reverse transcribed in 10 μl reactions containing 100 pmol random hexamer, 1 Χ reaction buffer, 5 mM dithiothreitol, 1.25 mM (each) deoxynucleoside triphosphates (dNTPs), and 100 U Superscript III (Life Technologies); mixtures were incubated at 25 °C for 5 min, 42 °C for 60 min, and 70 °C for 15 min. Following reverse transcription, cDNA was screened via PCR using Poecivirus-specific primers (Additional file 1). Samples were initially screened via qPCR using primers Poeci_7F and Poeci_7R; samples with a positive result via qPCR were than subjected to PCR using the additional primer pairs to obtain longer amplicons that were Sanger sequenced to confirm the presence of Poecivirus. Quantitative PCR mixtures contained 1X LC480 Sybr Green master mix (Roche), 0.1 μM Poeci_7F, 0.1 μM Poeci_7R, and 5 μl of 1:20-diluted cDNA. PCR mixtures contained 1Χ iProof master mix, 0.5 μM primer, and 5 μl sample template. Thermocycling consisted of 98 °C for 30 s, then 40–45 cycles of 98 °C for 10 s, 58 °C for 10 s, and 72 °C for 30 s, then a 5 min elongation step of 72 °C. Amplicons were visualized using agarose gels; those in the correct size range were purified using a DNA Clean and Concentrator kit or Zymoclean Gel DNA recovery kit (Zymo) and Sanger sequenced (Quintara Biosciences). We used likelihood ratio chi-square tests to test whether there was a difference in likelihood of infection with Poecivirus between AKD and control individuals.
Viral load
Quantitative PCR was conducted as described above and was used to determine viral load in beak samples from Poecivirus-positive individuals (n = 15). For each sample, the cyclic threshold (Ct) value obtained using primers Poeci_7F and Poeci_7R (CtPoeci) was normalized to the Ctref value obtained using primers to avian cellular RNA (avi_8F, avi_8R). Viral load was calculated using the equation viral load = 2(CtPoeci – Ctref). We used linear regression to test for a relationship between beak length and viral load in Poecivirus-positive individuals.
Virus localization of mRNA via in situ hybridization
For mRNA in situ hybridization, we examined tissue from four of the individuals with the highest qPCR measured levels of Poecivirus in beak tissue. Beak tissue was collected and stored frozen at − 80 °C until processed. Tissue was fixed overnight in 4% paraformaldehyde at room temperature, and then decalcified for 2.5 weeks in 0.5 mM EDTA at 4 °C. Decalcified tissue was frozen in Optimal Cutting Temperature (OCT) embedding media with dry ice and cut into 10 μm sections on a cryostat. In situ detection of positive strand viral RNA was conducted using the RNAscope 2.5 High Definition (HD) Brown assay kit (Advanced Cell Diagnostics, Inc., Hayward, CA). The assay was conducted following the manufacturer’s recommendations, with the exception that sections underwent a 45 min protease plus treatment in place of the recommended 30 min treatment. Images were captured with a Nikon Ti-E microscope fitted with a Nikon DS-Ri2 camera.
Detection of viral replication
The presence of negative strand virus was evaluated in Poecivirus-positive individuals using the NanoString nCounter™ Elements system (NanoString, Seattle, WA) gene expression assay. Eight oligonucleotide probes were designed to target negative strand virus evenly distributed across the viral genome, 2 oligonucleotide probes were designed to target positive strand virus, and an additional 2 oligonucleotide probes were designed to target a BCCH housekeeping gene, NADH ND2 (GenBank: KF183899.1); oligonucleotide probes were obtained from IDT (Coralville, IA). Negative and positive strand virus genome segments were made using Gibson assembly cloning followed by transcription with T7 polymerase [14]; equimolar pools of this RNA served as positive controls of probe activity. A 50 ng sample was hybridized with Tagsets (NanoString) and oligonucleotide probes at 65 °C for 16 h and the excess probes were washed away; in addition, a water negative control was run in one lane, and positive control RNA was run in each of four lanes, with 100, 1000, 10,000, or 100,000 copies of target RNA added to control lanes 1–4, respectively. The digital counts were captured by nCounter Digital Analyzer and the data were analyzed by nSolver Analysis Software (NanoString). Following standard NanoString analysis methods, individuals were considered to have a positive result for any single probe if that probe exhibited levels 2 standard deviations or greater above that of negative controls. We used linear regression to test for a relationship between beak length and viral RNA count in Poecivirus-positive individuals. Statistical analyses were conducted using JMP 7.0.1 by SAS (SAS Institute Inc., Cary, NC).