Virology xxx (xxxx) xxx–xxxContents lists available at ScienceDirectVirologyjournal homepage: disease in ball pythons (Python regius) experimentally infectedwith ball python nidovirus⁎Laura L. Hoon-Hanksa, , Marylee L. Laytona, Robert J. Ossiboffb,1, John S.L. Parkerc,⁎⁎Edward J. Dubovib, Mark D. Stengleina,aDepartment of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USADepartment of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USAcBaker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, USAbA R T I C L E I N F OA B S T R A C TKeywords:Ball pythonNidovirusExperimental infectionRespiratory diseasePneumoniaTorovirinaeBarnivirusKoch's postulatesCircumstantial evidence has linked a new group of nidoviruses with respiratory disease in pythons, lizards, andcattle. We conducted experimental infections in ball pythons (Python regius) to test the hypothesis that ballpython nidovirus (BPNV) infection results in respiratory disease. Three ball pythons were inoculated orally andintratracheally with cell culture isolated BPNV and two were sham inoculated. Antemortem choanal, oroesophageal, and cloacal swabs and postmortem tissues of infected snakes were positive for viral RNA, protein,and infectious virus by qRT-PCR, immunohistochemistry, western blot and virus isolation. Clinical signs included oral mucosal reddening, abundant mucus secretions, open-mouthed breathing, and anorexia. Histologiclesions included chronic-active mucinous rhinitis, stomatitis, tracheitis, esophagitis and proliferative interstitialpneumonia. Control snakes remained negative and free of clinical signs throughout the experiment. Our findingsestablish a causal relationship between nidovirus infection and respiratory disease in ball pythons and shed lighton disease progression and transmission.1. ImportanceOver the past several years, nidovirus infection has been circumstantially linked to fatal respiratory disease in multiple python species,but a causal relationship has not been definitively established. Throughexperimental infections, our study fulfills Koch's postulates and confirms ball python nidovirus as a primary respiratory pathogen in thisspecies. Our findings will provide veterinarians valuable informationfor the diagnosis and management of this disease and lay the groundwork for continued scientific investigation of this sometimes fatal disease. Python nidoviruses are members of a growing group of virusesthat have been associated with severe respiratory disease, includingbovine nidovirus and shingleback lizard nidovirus. The establishment ofBPNV as a primary pathogen in pythons is an important step in understanding the pathogenic potential of this emerging group of viruses.2. IntroductionThe nidoviruses (order Nidovirales) are a large and diverse group ofviruses that includes notable human and veterinary pathogens (DeGroot et al., 2012; Graham et al., 2013; Lauber et al., 2012; Masters andPerlman, 2013; Snijder et al., 2013; Snijder and Kikkert, 2013). Thediscovery of a group of related nidoviruses in snakes, lizards, cattle, andnematodes has recently expanded the order (Bodewes et al., 2014;Dervas et al., 2017; Marschang and Kolesnik, 2017; O’Dea et al., 2016;Shi et al., 2016; Stenglein et al., 2014; Tokarz et al., 2015; Uccelliniet al., 2014). These novel nidoviruses cluster most closely with virusesin the subfamily Torovirinae within the Coronaviridae family of the Nidovirales order, and form a distinct clade from viruses in the Bafinivirusand Torovirus genuses, which infect ray-finned fish and mammals, respectively. Based on phylogenetic analysis, it has been proposed thatthe reptile nidoviruses be classified within a distinct genus namedBarnivirus, and that Torovirinae be classified as its own family due to thegrowing evidence of the paraphyly of Coronaviridae, though theseviruses have not yet been formally classified (Adams et al., 2017; Battset al., 2012; Gonzalez et al., 2003; Nga et al., 2011; Stenglein et al.,2014). Toroviruses share similar tissue tropisms of the gastrointestinal(GI) and respiratory epithelium, ultrastructural features, and genome⁎Correspondence to: Colorado State University, 200 W Lake St. 2025 Campus Delivery, Fort Collins, CO 80523, USA.Co-corresponding author.E-mail addresses: [email protected] (L.L. Hoon-Hanks), [email protected] (M.D. Stenglein).1Current address: Department of Comparative, Diagnostic, and Population Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL, 008Received 29 September 2017; Received in revised form 1 December 2017; Accepted 11 December 20170042-6822/ 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license se cite this article as: Hoon-Hanks, L.L., Virology (2017),

Virology xxx (xxxx) xxx–xxxL.L. Hoon-Hanks et al.medium changes. At 70% confluence, monolayers were passed using0.25% trypsin first into T25, and then into T75 tissue culture flasks(Corning). At 100% confluence, T75 flasks were trypsinized, washed incomplete cell growth medium, and resuspended in 1 ml of complete cellgrowth medium with 20% irradiated FBS and 10% DMSO for storage inliquid nitrogen in 1.2 ml cryovials (Corning).organization (Batts et al., 2012; Pradesh et al., 2014; Schutze et al.,2006), and represent a group of emerging pathogens of unknown, andpossibly underestimated, significance in veterinary and human medicine.The snake-associated nidoviruses were first discovered in ball pythons (Python regius) and Indian rock pythons (P. molurus) with severerespiratory disease that had tested negative for known snake respiratorypathogens (Bodewes et al., 2014; Stenglein et al., 2014; Uccellini et al.,2014). Postmortem findings in sick pythons included stomatitis, sinusitis, pharyngitis, tracheitis, esophagitis, and proliferative pneumoniawith significant mucus secretion in affected tissues; secondary bacterialinfections were also noted within the respiratory tract or systemically insome snakes. In 2017, similar findings were detected in green treepythons (Morelia [M.] viridis) infected by a related nidovirus (Moreliaviridis nidovirus) (Dervas et al., 2017). Additionally, nidoviruses havebeen detected in antemortem oral swabs (or rarely blood) from a Burmese python (P. bivittatus), ball pythons, Indian rock pythons, greentree pythons, a carpet python (M. spilota), and boa constrictors (Boaconstrictor) with or without documented respiratory signs (Marschangand Kolesnik, 2017). Related nidoviruses associated with respiratorydisease in wild shingleback lizards and cattle have also been recentlydescribed (O’Dea et al., 2016; Tokarz et al., 2015).Reports of nidovirus in multiple python species are highly suggestive of, but do not definitively establish, a causal relationship betweenviral infection and respiratory disease. This study sought to fulfillKoch's postulates through experimental infections of ball pythons withball python nidovirus (BPNV). The goal was to conclusively establish acausative relationship between infection and respiratory disease as wellas further characterize the clinical course of disease, describe usefuldiagnostic techniques, and to investigate possible routes of transmission.3.2. Isolation of BPNVOral swabs were collected from a ball python with upper respiratorydisease that was part of a colony with a documented history of BPNVinfections (Uccellini et al., 2014). Swabs were placed in 1.5 ml of viraltransport medium (MEM/EBSS, 0.5% bovine serum albumin, 200 Upenicillin, 200 μg streptomycin, 0.25 μg fungizone, and 10 μg ciprofloxacin; Gibco) prior to inoculation on diamond python heart (DPHt)cells. Briefly, 1 ml of the swab extracts were added to DPHt cells in T25culture flasks. After a 3 h incubation at 30 C, monolayers were rinsedand cell growth medium added (MEM/EBSS, 10% irradiated FBS, 200 Upenicillin, 200 μg streptomycin, 0.25 μg fungizone, and 10 μg ciprofloxacin; Gibco). Cultures were maintained at 30 C and monitoreddaily for cytopathic effects. At 7 days post inoculation cells were frozenat 70 C, thawed, and were re-inoculated onto new DPHt monolayers. The study challenge virus (deemed BPNV-148) was a passage 2preparation.3.3. Plaque assayDPHt cells were incubated in complete cell medium [MEM/EBSS(HyClone), 10% irradiated FBS (HyClone), 10% Nu-Serum1 (Corning),and 2x penicillin-streptomycin solution (HyClone)] in a 6-wellCELLSTAR cell culture plate (Greiner Bio-one) at 30 C in 5% CO2 until90% confluence was attained. BPNV-148 stock was diluted in serumfree MEM/EBSS to generate 5 dilutions of 1 10 2 through 1 10 6.For cell inoculation, all medium was removed and 900 μl of each dilution was placed on the cells, with serum-free MEM/EBSS added to thelast well as a negative control. Cells were incubated at 30 C in 5% CO2for 1 h, after which the infected medium was removed and an agaroseoverlay was placed [complete cell medium with 0.8% UltraPure LMPAgarose (Invitrogen)]. Assays were incubated at 30 C in 5% CO2 for 6days, at which time 1 ml of 4% paraformaldehyde (EM grade; ElectronMicroscopy Sciences) in DPBS (Corning) was added to each well andincubated for an additional hour. The agarose overlay was removed,cells were rinsed with DPBS, and an additional 1 ml of paraformaldehyde mixture was added. Cells were placed at 4 C overnight. The formaldehyde was removed, cells were rinsed with sterile water, and 100μl of crystal violet (0.5% crystal violet in 25% methanol and 75% sterilewater) was added and incubated for 10 min at room temperature.Crystal violet was rinsed off with sterile water, assays were dried, andplaques were counted. Plaque assays were also performed using samples collected during experimental infection studies; the same protocolwas utilized.3. Materials and methods3.1. Generation of a diamond python cell lineA non-immortalized cell line was generated from heart tissue collected from a diamond python (Morelia spilota). Multiple 2 mm cubesof myocardium were collected from a diamond python directly following humane barbiturate overdose euthanasia for chronic vertebraldisease. Tissues were collected within 2 h of euthanasia and placed in1.5 ml, ice-cold, sterile phosphate buffered saline (PBS) in 2 ml microcentrifuge tubes for transport to the laboratory. Tissue samples wereindividually transferred to a 6-well cell culture plate (Corning), washedthree times with ice cold PBS, and manually minced with a sterilescalpel blade in 1.5 ml PBS with 0.25% trypsin (Gibco) and 1 mMethylenediaminetetraacetic acid (EDTA). Samples were incubated at37 C with gentle agitation every 20 min (m) for a total of 60 m.Following incubation, 0.5 ml of the digested product was added perwell of a 12-well cell culture plate (Corning) along with 2 ml of complete cell growth medium [Minimum Essential Medium with Earle'sBalanced Salts, L-Glutamine, and Nonessential Amino Acids (MEM/EBSS; Hyclone); 10% irradiated fetal bovine serum (FBS; HyClone); 100U penicillin; 100 μg streptomycin; 0.25 μg amphotericin B (Cellgro);and 50 μg gentamicin (Cellgro)] and placed at 30 C in a humidified 5%CO2 atmosphere. Wells were monitored regularly for evidence of celladherence and replication. Partial ( 50%) medium changes wereperformed weekly. When cell monolayers reached 70% confluence,monolayers were washed twice with room temperature sterile PBS, 1 mlenzyme free cell dissociation buffer (Gibco) was added to each well,and the samples were incubated for 5 m at 30 C. Cell monolayers weredisrupted by gently pipetting samples up and down, and the cell/dissociation buffer mixture was transferred to a 60 mm tissue culture dish(Corning) with 7 ml of complete cell growth medium and returned to a30 C, humidified, 5% CO2 atmosphere. The cells were monitored regularly for evidence of cellular replication with weekly, partial ( 50%)3.4. Experimental infectionFive captive-bred ball pythons (BP A-E; 4 males and one undetermined sex) were acquired, each approximately 6 weeks old andvarying in size from 77 to 106 g. All pythons were housed and treatedaccording to the IACUC protocol (15–6063A) and Colorado StateUniversity Laboratory Animal Resources standards. Infected snakeswere housed in a cubicle with separate HEPA-filtered air supply fromcontrol snakes and all snakes were housed in separate cages withoutdirect contact. Uninfected snakes were always handled prior to infectedsnakes to prevent fomite transmission. Physical exams were performedand all snakes were deemed clinically healthy at the time of acquisition.Pre-infection choanal (CHS), oroesophageal (OES), and cloacal swabs(CLS) were collected and tested by qRT-PCR (see below) for BPNV. One2

Virology xxx (xxxx) xxx–xxxL.L. Hoon-Hanks et 200 pmol of a random pentadecamer oligonucleotide (MDS-911;Table 1) and incubated for 5 min at 37 C; a water template control wasalso used. Reverse transcription reaction mixture containing 1x SuperScript III FS reaction buffer (Invitrogen), 5 mM dithiothreitol (Invitrogen), 1 mM each deoxynucleoside triphosphates (dNTPs), and 100U SuperScript III reverse transcriptase enzyme (Invitrogen) was addedto the RNA-oligomer mix (12 μl total reaction volume) and incubatedfor 30 min at 42 C, then 30 min at 50 C, then 15 min at 70 C.Quantitative reverse transcription polymerase chain reaction (qRTPCR) was performed using 1x HOT FIREPol DNA Polymerase (SolisBioDyne), 3 μM of each degenerate nidovirus primer (MDS-918 andMDS-919; Table 1), and 5 μl of diluted (1:10) cDNA in a 30 μl reaction.Reaction mixtures were placed in a TempPlate semi-skirted 96-wellPCR plate and were run in a Roche LightCycler 480 II with the following cycle parameters: 95 C for 15 min; 95 C for 10 s, 60 C for 12 s,and 72 C for 12 s with 40 cycles; and a melting curve. All samples wererun in duplicate, Ct values were averaged and standard deviations werecalculated. The PCR reaction efficiency for each primer-pair was measured using a dilution series of positive samples (BP-B terminal OES fornidovirus primers and BP-B trachea/esophagus for GAPDH primers);the dilution series samples were run in duplicate. Relative viral RNA forall CHS, OES, and CLS samples was determined by comparison of eachsample Ct to the sample with the highest Ct (lowest viral RNA) at thefirst collection time point following inoculation (BP-B OES at week 1PI). Relative viral RNA from tissues was determined by normalization tosnake GAPDH within each sample (same qRT-PCR conditions withMDS-921and MDS-923 primers; Table 1).week after arrival, three snakes were inoculated with BPNV infectedDPHt cell culture supernatant discussed above (BP-A, B, and C) and twowere sham inoculated (BP-D and E). Inoculation was performed bothorally (200 μl) and intratracheally (100 μl) for each snake with 1.1 10 5 PFU in 300 μl for the infected snakes and a similar volume ofuninfected cell culture medium for the control snakes. Snakes weremonitored daily, weights were taken weekly, and CHS, OES, and CLSwere collected weekly from all snakes using PurFlock Ultra sterileflocked 6″ plastic-handle swabs (Puritan Diagnostics). Swabs wereplaced in 2 ml Bacto brain-heart infusion medium (Becton, Dickinsonand Company), incubated at room temperature (RT) for approximately30 min, vortexed, and then stored at 80 C. BP-C was euthanized at 5weeks post infection (PI) as a demonstration of early infection. BP-Awas euthanized at 10 weeks PI and BP-B at 12 weeks PI based onclinical signs and established euthanasia criteria. BP-D and E were euthanized at 12 weeks to end the study. Final CHS, OES, and CLS andculture swabs of the oral cavity (BBL CultureSwab plus Amies gelwithout charcoal; Becton, Dickinson and Company) were collected atthe time of euthanasia. Sections of the glottis, nasal and oral cavity,cranial, middle, and caudal trachea and esophagus, lungs, heart, liver,kidneys, gallbladder, spleen, pancreas, stomach, small intestine, colon,feces, blood, urates, gonads, head and vertebrae with brain and spinalcord were saved fresh and/or placed in 10% neutral buffered formalin.3.5. RNA extractionRNA from swabs and fresh-frozen tissues (lung, cranial trachea/esophagus, liver, kidney, heart, stomach, small intestine, colon, feces,urates) was extracted using a combination of TRIzol (tissue; AmbionLife Technologies) or TRIzol LS (swabs in BHI; Ambion LifeTechnologies) with RNA clean and concentrator columns (CC-5; ZymoResearch). Approximately 100 mg of tissue was added to 1 ml of TRIzoland 250 μl of BHI swab medium was added to 750 μl of TRIzol LS andincubated at room temperature (RT) for 5 min. Tissue samples weremacerated using a single sterile metal BB shaken in a TissueLyzer(Qiagen) at 30 Hz for 3 min. Then, 200 μl of chloroform (SigmaAldrich) was added, shaken for 15 s by hand, and incubated at RT for2 min. Samples were spun at 12,000 RPM for 10 min at RT. The aqueous phase was removed (approximately 450 μl) and was added to amixture of 450 μl of RNA binding buffer (CC-5; Zymo Research) and450 μl of 100% ethanol (EtOH). This was added to an RNA clean andconcentrator column (CC-5; Zymo Research). The interphase and organic phase were discarded. The RNA column was washed with 400 μlRNA wash buffer and then incubated with 6 U DNase enzyme (NEB), 1xDNase buffer (NEB), and RNA wash buffer for 15 min. The column wasspun to remove DNase mixture and then washed with 400 μl RNA prepbuffer. An additional wash with 800 μl RNA wash buffer was performed, the column was dried with a 1 min high-speed spin, and thenRNA samples were eluted in 30 μl of RNase-free water.3.7. Antibody developmentThe predicted amino acid (aa) sequence for the ball python nidovirus 1 nucleocapsid protein (152 aa protein; GenBank: AIJ50569.1)and nidovirus nucleocapsid protein sequences isolated from green treepythons (unpublished data) were used by our lab to identify a relativelyconserved peptide sequence with predicted high immunogenicity andepitope exposure. The peptide (aa 136–152 of the N protein of a greentree python nidoviral isolate: Cys-RAFIPLKHEGAETEEEV) was submitted to Pacific Immunology (Ramona, CA) for synthesis and polyclonal anti-nidoviral nucleocapsid antisera (NdvNcAb) was developedin two rabbits.3.8. Histopathology/immunohistochemistryFormalin-fixed tissue was paraffin-embedded and 5 µm sectionswere stained by hematoxylin and eosin (H&E), Gram, periodic acidSchiff (PAS), and Ziehl-Neelsen acid fast for light microscopy (performed by Colorado State University Veterinary Diagnostic Laboratory;CSUVDL). Immunohistochemistry was also performed by CSUVDL usingthe Bond Polymer Redefine Red Detection kit (Leica) and a 10 minincubation with Epitope Retrieval Solution 1 (Leica). NdvNcAb(0.32 μg/ml) was used as the primary antibody and the slides werecounter stained with hematoxylin. Lung tissue from a green tree pythonthat was nidovirus positive (PCR and virus isolation) and that died ofrespiratory disease was used as a positive control (data not shown).3.6. Viral RNA detectionRNA extracted from swabs and fresh-frozen tissues was reversetranscribed into cDNA as follows. Five microliters of RNA were addedTable 1Primers. List of primers used during qRT-PCR for detection of BPNV or GAPDH and used for sequencing library generation. Forward (F); Reverse (R); N/A (not applicable).Primer nameTargetSequence (5′-3′)Direction DS-921MDS-923Sequencing library RF(Runckel et al., 2011)RandomORF1b python nidovirusPython GAPDH gene3N/A–(Stenglein et al., 2017)

Virology xxx (xxxx) xxx–xxxL.L. Hoon-Hanks et al.Samples were incubated at 37 C for 20 min followed by 94 C for2 min. Then, single-stranded cDNA was converted to double-strandedDNA by adding 2.5 U Klenow DNA polymerase (3′ to 5′ exo- NEB) in5 μl 1x SuperScript III FS reaction buffer and 2 mM each dNTPs andincubated at 37 C for 15 min. DNA was purified using SPRI beads at a1.4:1 bead/DNA volume ratio. DNA was eluted in 20 μl molecular gradewater (Sigma-Aldrich). The dsDNA concentration from each sample wasmeasured fluorometrically and 10 ng was used as a template in 6.5 μl of1x Tagment DNA buffer and 0.5 μl Nextera Tagment DNA enzyme(Illumina). The mixture was incubated at 55 C for 10 min and thenplaced directly on ice. Tagmented DNA was cleaned with SPRI beadsand used as a template (5.8 μl) in the addition of full-length adaptorswith unique bar-code combinations by PCR. The 25 μl PCR reactioncontained 1x Kapa real-time library amplification master mix (KapaBiosystems), 0.33 μM (each) MDS-143 and MDS-445 primers (Table 1),and 0.020 μM each of adaptor 1 and 2 bar-coded primers (Stengleinet al., 2015). Thermocycling conditions in consecutive order were 72 Cfor 3 min, 98 C for 30 s, and 8 cycles of 98 C for 10 s, 63 C for 30 s,and 72 C for 3 min. Relative concentrations of libraries were measuredin qRT-PCR reactions containing 1x qRT-PCR master mix [10 mM TrisHCl pH 8.6, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 5%glycerol, 0.08% NP-40, 0.05% Tween-20, 1x Sybr green (Life Technologies) and 0.5 U Taq polymerase] and 0.5 μM MDS-143 and MDS445 primers. Equivalent amounts of DNA from each sample werepooled and then cleaned using SPRI beads. The pooled libraries wererun on a 2% agarose gel and size selected (400–500 nucleotides) by gelextraction with a gel DNA recovery kit (Zymo Research) according tothe manufacturer's protocol. Size-selected pooled libraries were amplified once more in a PCR mixture containing 1x Kapa real-time libraryamplification mix, 500 pmol of MDS-143 and -445 each, and 5 μl oflibrary template in a 50 μl total reaction volume. This PCR also includedsingle reactions of 4 separate fluorometric standards (Kapa). Thermocycler conditions were 98 C for 45 s and 14 cycles of 98 C for 10 s,63 C for 30 s, and 72 C for 2 min, which was the cycle at which thesample curve passed standard 1. DNA was purified using SPRI beads aspreviously described. Library quantification was performed with theIllumina library quantification kit (Kapa Biosystems) according to themanufacturer's protocol. Paired-end 2 150 sequencing was performed on an Illumina NextSeq. 500 instrument with a NextSeq. 500/550 Mid Output Kit v2 (300 cycles).3.9. Virus isolation and immunofluorescenceOroesophageal swabs collected at the time of euthanasia from allinfected and uninfected snakes were filtered (Merck MilliporeUltraFree-MC 0.22 µm centrifugal filters) and 40 μl was inoculated ontoDPHt cells at 80% confluence in 35 mm diameter glass-bottom plates(MatTek corporation). Cells were maintained in 2 ml of complete cellmedium and incubated at 30 C with 5% CO2; medium was refreshedevery other day. Cells infected with BP-A OES were formalin-fixed aspreviously described at 1, 12, 24, 48, 96, 144, and 192 h PI; all otherOES-infected cells (BP-B, C, D, and E) were formalin-fixed at 4 days PI.Approximately 50 mg of lung or feces from infected and uninfectedpythons was homogenized in 500 μl of DPBS, clarified, and then filtered(0.22 µm). Infection of cell culture was as previously described. Lunginfected cells were formalin-fixed at 10 days PI and fecal-infected cellsat 3 days PI.Fixed cells were washed 3 times with 1 ml of PBS. Cells were permeabilized in 0.1% Triton X-100 (reagent grade; Amresco) in PBS for5 min. Washes were repeated and then cells were incubated in blockingbuffer (1% bovine serum albumin (Fisher Scientific) in PBS) for 1 h. A1:2000 dilution of NdvNcAb rabbit serum (primary antibody) wasadded to the blocking buffer and incubated for an additional hour.Wash steps were repeated and then new blocking buffer with 5 μg/ml ofsecondary antibody (Alexa Fluor 488 goat anti-rabbit IgG antibodies;A11008 Life Technologies) was added and incubated for 1 h. Washsteps were repeated and then cells were stained with Hoechst 33342(1 μg/ml final concentration; Life Technologies) to stain DNA. Cellswere imaged on an Olympus IX81 motorized inverted system confocalmicroscope with FluoView 4.2 software. Images were processed inAdobe Photoshop CC (2017) and both infected and uninfected wereprocessed equally.3.10. Western blotDPHt cells inoculated with OES from all infected and uninfectedsnakes, as previously described, as well as a sham inoculated control(BHI only) were harvested at 4 days PI. Cells were lysed using equalvolumes of sample and SDS-based tissue lysis buffer (40 mM TrisCl pH7.6, 120 mM NaCl, 0.5% Triton X-100, 0.3% SDS, Roche completeprotease inhibitor cocktail tablet), mixed for 30 min at 4 C, and clarified by centrifugation at 4 C for 10 min at 10,000 rpm. Twelve microliters of sample or 4 μl of ladder with 8 μl of PBS (precision plusprotein western C; BioRad) were combined with 1x NuPage LDS samplebuffer (Life Technologies) and separated using a 4–12% polyacrylamidegel (Invitrogen). Protein was transferred to a nitrocellulose membraneusing a Trans-Blot turbo (low molecular weight protein transfer; BioRad). A 1 h incubation of the membrane in blocking buffer [1x PBS,0.05% Tween20, 1% Carnation nonfat dry milk, and 1:1000 KathonCG/ICP preservative (Dow Chemical)] was followed by a 1 h incubationwith 1.6 μg/ml NdvNcAb in blocking buffer. The membrane was washed (1x PBS and 0.05% Tween20) 3 times for 5 min each followed by a1 h incubation with a 1:50,000 dilution of goat anti-rabbit IgG antibodyconjugated to horseradish peroxidase (HrP; Pierce 31460 Invitrogen)and 1:4000 dilution of streptactin-HrP (ladder) in blocking buffer. Asecond wash was performed and the blot was developed using a 5 minincubation with clarity western ECL substrate (BioRad). Imaging wasvia chemiluminescence for 60 s (BioRad Gel Doc).3.12. Sequence analysisSequences were trimmed using Cutadapt (version 1.9.1) in order totrim adaptor sequences and low-quality bases, and remove trimmedsequences that were shorter than 80 nucleotides (nt) long (Martin,2011). Quality base was set to 33 (default) and quality cutoff was set to30 for the 5′ and 3′ ends. The first base of each sequence was alsotrimmed. The CD-HIT-DUP sequence clustering tool was then used tocollapse reads with 99% global pairwise identity, leaving unique reads(Li and Godzik, 2006). Python-derived sequences were then filteredusing the Bowtie2 alignment tool (version 2.2.5) (Langmead andSalzberg, 2012). First, a bowtie index was generated from the hostgenomic sequence [Python bivittatus (Burmese python) genome assembly (NC 021479.1)] and then sequences aligning with a –local modealignment score greater than 60 were removed. SPAdes genome assembler (version 3.5.0) was used to generate contiguous sequences(contigs) (Bankevich et al., 2012). Then, to taxonomically categorizesequences, the NCBI nt database was queried with all contigs greaterthan 150 nt using the BLASTn alignment tool (version 2.2.30 )(Altschul et al., 1990; Camacho et al., 2009). Any hit with an expectvalue less than 10 8 was assigned taxonomically according to the sequence with the highest alignment score (Altschul et al., 1990; BLAST Command Line Applications User Manual, n.d.). Additionally, to attempt to categorize contigs that were too divergent to produce a highscoring nt-nt alignment, the NCBI nr database was queried using3.11. Metagenomic sequencingShotgun libraries were generated from total RNA extracted from BPA, B, C, D, and E lung and cranial trachea/esophagus and BPNV-148inoculum. Library preparation was as follows: Ten microliters of undiluted cDNA (see polymerase chain reaction for cDNA preparation)was treated with 1 U RNase H (NEB) diluted in 5 μl 1x SuperScript III FSreaction buffer and 160 pmol MDS-911 to degrade RNA templates.4

Virology xxx (xxxx) xxx–xxxL.L. Hoon-Hanks et al.criteria. Grossly, oral cavities of each snake were similar to BP-C butwith significantly more mucinous exudate. BP-A also had a focal ulceration of the glottis, the caudal esophagus adjacent to the lungs wasmarkedly dilated with air and mucus, and the cranial 1/3 of the lungswere wet and red (Fig. 2). BP-B lungs were slightly reddened and wet inthe cranial portion but the caudal esophagus was grossly normal. Histologically, both snakes had similar but more severe lesions in the upperrespiratory tract (URT) and cranial esophagus as compared to BP-C(Fig. 3A and B). Additionally, there were regions of erosion and ulceration in areas of inflammation as well as individual epithelial cellnecrosis and regions of marked epithelial proliferation. The caudalesophagus of BP-A also had similar inflammatory infiltrates to that inthe cranial esophagus but these were significantly milder. Lumina of theURT, cranial esophagus, central lumen of the lung, and faveoli contained mucus, necrotic debris, heterophils, hemorrhage, and occasionalcolonies of short Gram-negative bacterial rods. Both snakes (BP-Agreater than BP-B) had a mild interstitial pneumonia of the cranial lungfield with pneumocyte proliferation (Fig. 3C). Lesions were characterized by multifocal hyperplasia of respiratory epithelial cells lining thecentral lumen, hypertrophy and hyperplasia of faveolar pneumocytes(predominately in the luminal 1/3 of the faveoli), and expansion of theinterstitium by edema and similar inflammatory cells to that in theURT.Sham inoculated snakes (BP-D and E) did not show clinical signs norhave histologic lesions of the respiratory tract or esophagus (Fig. 3D-F).Both the infected and control snakes had moderate to severe lymphoplasmacytic and heterophilic, non-ulcerative colitis of unknown originand mild lymphohistiocytic to granulomatous embolic hepatitis, whichare considered un

pythons (Morelia [M.] viridis) infected by a related nidovirus (Morelia viridis nidovirus) (Dervas et al., 2017). Additionally, nidoviruses have been detected in antemortem oral swabs (or rarely blood) from a Bur-mese python (P. bivittatus), ball pythons, Indian rock pythons, gree