Genetic Data Associated with Avian Keratin Disorder and Poecivirus in Black-capped Chickadees, Alaska, 2001-2017

We tested 124 BCCH (28 individuals with AKD and 96 asymptomatic control individuals) for the presence of Poecivirus using cloacal swabs. Samples were collected as part of this study and by Zylberberg et al. (2016). 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 (April), 2016 (March-April, October-December), and 2017 (March, April). We chose to use cloacal swabs because they are non-destructive, are easily obtained, and contain relatively high viral load. 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 (Longmire 1988) and stored frozen at -80°C until processed.

Standard beak measurements and microscopy were used to classify individuals as AKD-affected or unaffected (Handel et al. 2010). 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.

RNA was extracted from cloacal and buccal swab samples using a Zymo viral RNA kit as previously described (Zylberberg et al. 2016). 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. 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. Samples were initially screened via qPCR using primers Poeci_7F and Poeci_7R (for primer information see Cross Reference of metadata); 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 5μl 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 sec, then 40–45 cycles of 98°C for 10 sec, 58°C for 10 sec, and 72°C for 30 sec, 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).

For attributes “Cloacal swab, Poecivirus detected”, “Buccal swab, Poecivirus detected”, “Fecal sample, Poecivirus detected”, and “Blood sample, Poecivirus detected”, samples were initially screened via qPCR; samples with a Ct value lower than a negative control were subjected to PCR and visualized on an agarose gel; amplicons obtained via PCR were Sanger sequenced to confirm the presence of Poecivirus.

Quantitative PCR was used to determine viral load in beak samples from Poecivirus-positive individuals. For each sample, the cyclic threshold (Ct) value obtained using primers Poeci_7F and Poeci_7R (CtPoeci; for primer information see Cross Reference section of metadata) 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).

For mRNA in situ hybridization, we examined tissue from 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.

The presence of negative strand virus was evaluated in Poecivirus-positive individuals using the NanoString nCounterTM Elements system (NanoString, Seattle, WA) gene expression assay. Negative and positive strand virus genome segments were made using Gibson assembly cloning followed by transcription with T7 polymerase; 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 65ºC for 16 hours 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, 1,000, 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.