Host-pathogen dynamics of squirrelpox virus infection in red squirrels (Sciurusvulgaris)

C. Fiegnaa, M.P. Dagleisha, L.Coultera, E. Milneb, A.Meredithb,J. Finlaysona, A. Di Nardoc,d& C.J. McInnesa,*

a Moredun Research Institute, Pentlands Science Park, Penicuik, Edinburgh, EH26 0PZ, Scotland, UK

bThe Royal (Dick) School of Veterinary Studies and The Roslin Institute, University of Edinburgh, Easter Bush Campus, Roslin, Midlothian, EH25 9RG, Scotland, UK

cThe Pirbright Institute, Pirbright, Woking, Surrey, GU24 0NF, UK

dInstitute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, Scotland, UK

* Corresponding author and contact details: C.J. McInnes,Moredun Research Institute, Pentlands Science Park, Penicuik, Edinburgh, Scotland, UK. E-mail address: ;Tel:0131 445 5111

Abstract

To improve our understanding ofsquirrelpox virus (SQPV) infection in the susceptible host, three red squirrels were challengedwith wild-type SQPV via scarification of the hind-limb skin.All squirrelsseroconvertedto the infectionby the end of the experiment (17 days post-challenge).Challenged animals suffered disease characterised by the development of multiple skin and oral lesions with rapid progression of skin lesions at the infection site by day 10 post-challenge. Nointernal pathological changeswere found atpost-mortem examination. A novel SQPV Taqman®Real-time PCRdetected viral DNA from multiple organs, with the largest amountsconsistentlyassociated with the primary and secondary skin and orallesions where viral replication was most likely occurring. Immunohistochemistryclearly detected viral antigen in the stratified squamous epithelium of the epidermis, tongue and the oropharyngealmucosa-associated lymphoid tissue and wasconsistently associated with histological changes resulting from viral replication. The lack of internal pathological changes and the detection of relatively low levels of viral DNA when compared with primary and secondary skin lesions argue against systemic disease, although systemic spread of the virus cannot be ruled out. This study allowed a comprehensive investigation of the clinical manifestation and progression of SQPV infection with a quantitative and qualitative analysis of virus dissemination and shedding. These findingssuggesttwo separate routes of SQPVtransmission under natural conditions,with both skin and salivaplaying key roles in infected red squirrels.

Keywords:

squirrelpox virus, SQPV, red squirrel, experimental infection, viral shedding, pathogenesis

1. Introduction

The native red squirrel (Sciurusvulgaris) populations of the UKand Eire have declined dramatically over the last 100 years andarenow heading towards extinction with just a few isolated populations remaining (Bosch and Lurz, 2012). In contrast, the grey squirrel(Sciuruscarolinensis),first introduced from North America in 1876,has greatly expanded its range.The reasons for the decline in the red squirrel population can be attributed to many variables(Bosch and Lurz, 2012), and althoughan earlier report suggested diseaseintroduced with the grey squirrel(Middleton, 1930), the connection betweenepidemic disease in red squirrels and the presence of the imported grey squirrels was debated throughout the 20th century (Edwards, 1962; Keymer, 1974; Vizoso, 1968).It was only latterly that a viral agent (initially described as parapoxvirusvirus) responsible for disease outbreaks in red squirrels was identified by transmission electron microscopy (TEM) of the eyelid skin from a diseased red squirrel(Scott et al., 1981). Even then it took several more years before researchestablishedthe role of the grey squirrel as a reservoir species for the virus (Reynolds, 1985; Sainsbury et al., 2000)and emphasisedthe crucial importance of this epizootic disease in the wider context of disease-mediated competition with grey squirrels(Rushton et al., 2006, 2000; Tompkins et al., 2003).Today, conservation strategies for the red squirrel take account of squirrelpox virus (SQPV) as a major contributing factor in the threat to red squirrels from the grey squirrel.

Squirrelpox virusis now classified as the sole member of an unclassified genus within the Poxviridaefamily (Thomas et al., 2003;McInnes et al., 2006;Darby et al., 2014).The origin of SQPV is still unknown, although it is thoughtthat it was imported with grey squirrels from North America.Whilethe virus has never been described in the USA, serological samples collected from grey squirrelsin North America have tested positive for anti-SQPV antibodies(McInnes et al., 2006).Fatal cases of disease resulting from SQPV infection have been described solely in red squirrels in the UK and the Ireland (Sainsbury and Ward, 1996;Tompkins et al., 2002; Thomas et al., 2003;Sainsbury et al., 2008;McInnes et al., 2009; LaRose et al., 2010; McInnes et al., 2013;Naulty et al., 2013)with the exception ofone report of a grey squirrel showing clinical signs of SQPV disease which was confirmed by TEM(Duff et al., 1996). Otherwise,grey squirrels are considered to be asymptomatic or sub-clinically affectedby the virus(Atkin et al., 2010; Tompkins et al., 2002).In red squirrels,the infection causesmultifocal skin lesions characterised by erythematous and ulcerative dermatitis which progress to haemorrhagic scabs and the disease causes significant mortality(Carroll et al., 2009; Chantrey et al., 2014; Duff et al., 2010; LaRose et al., 2010; McInnes et al., 2013, 2009; Sainsbury and Gurnell, 1995; Sainsbury and Ward, 1996; Tompkins et al., 2002). To confirm that thedisease is clinically asymptomatic in the grey squirrel,an experimental infection with SQPV was performed(Tompkins et al., 2002).Grey and red squirrels were inoculated simultaneously via skin scarification and subcutaneous routes.All the red squirrels developed typical,severe SQPV-associated dermatitis and their health deteriorated rapidly in contrast to grey squirrels which showed no clinical signs of infection and remained healthy throughout.

Despite the disease and the potential carrier role of the grey squirrel being recognised for the last 15 years, remarkably little has been discoveredabout the pathogenesis or the transmission route(s) of the virus either withinred or grey squirrels orbetween the species.To date, there have beenno detailed studies on the temporal development of SQPV infection in red squirrels focusing on the incubation period, the occurrence and developmentof lesions or which tissues or organs support viral replication and,potentially,shedding. Furthermore, what is known has been derived primarily from epidemiological studies of field cases.As part of a wider study to investigate the feasibility of producing a vaccine against SQPV it was necessary first to establish, under experimental conditions,an infection of red squirrels resemblingSQPV infection in the wild.To investigate this and provide additional information on the pathogenesis of SQPVwe developed a specific and sensitiveSQPV Taqman®qPCR assay to compare the viral load from a broad range of tissues from SQPV positivered squirrels bothnaturally and experimentallyinfected.We have correlatedthese results with those from a variety of diagnostic techniques such as post mortem examination, histopathology anda novel SQPV-specific immunohistochemical method (IHC) to determine which tissues harbour the virus and which are most likely permissive for viral replication and are therefore likely to be involved in virus transmission and shedding of virus.

2. Materials and methods

2.1 Experimentally-infected animals and related procedures

All experimental protocols involving infection of red squirrels were approved by the Moredun Research Institute Animal Experiments & Ethical Review Committee and adhered strictly tothe requirements of the UK Animals (Scientific Procedures) Act 1986.Threeadult red squirrels (twomale, sq. 01/12 and sq. 06/12; one female, sq. 04/12)were individually housed in1m × 0.75 m × 0.75 m cages which were furnished with a nest box with a removable lid. Food (mixed nuts, sunflower seeds, whole corn and fruit) and water were provided ad libitum. All squirrels were allowed to adjust to their environment for 14 days before the start of the procedures. All handling procedures were performed under general anaesthesia. Anaesthesia was induced with gaseous 5% isoflurane (IsoFlo, Abbott Animal Health, UK) in oxygen administered within an anaesthetic chamber and maintained by 1.5-4% isoflurane in oxygen via a face mask.

2.2 Preparation of inocula,experimental infection and clinical observations

Dry skin lesions and scabs were collected from dead free-ranging red squirrels in the UK found with clinical signs typical of SQPV disease and confirmed positive by either SQPV qPCR or TEM. Scabs were ground with sterile sand in sterile PBS,approximately 6% v/v penicillin/streptomycin solution (100 units/mL and 100mg/mL, respectively) was added andthe inoculum clarified by centrifugation at 2,000 x g for 5 min. The inoculumwas dispensed into multiple aliquots and stored at -70°C. SQPV DNA concentration was quantified by qPCR and found to be approximately 2.5×1010virusgenome equivalents/mL. The presence of intact SQPV virion particles was confirmed by TEM (courtesy of David Everest, Animal and Plant Health Agency, Weybridge, UK). Mock inoculum was prepared using the antibiotic solutions added to PBS.

Inocula were applied topically onto a previously shaved and scarified 2 × 2 cm area of skin on the lateral aspect of the squirrel thighs. Scarification was with the tip of a 16G needle in a cross hatched pattern with scratches approximately 0.5 cm apart. Each squirrel was challenged with 100 μl of SQPVinoculumon the right thigh and 100 μlof mock inoculum on the left thigh. Animals were monitored daily for clinical signs of diseasewith the skin lesions distant from the challenge sites being fully assessed and recorded at the time of post-mortem examination (PM).The weight of each squirrel was measured at the time of the virus challenge and estimatedevery day thereafter by weighing the squirrels within their nest boxes. Clinical scores were recorded daily using a modification of that used previously to assess the impact of squirrelpox disease on red squirrels(Tompkins et al., 2002). A total clinical score of 6on three consecutive days was the designated humane end-point at which an individual animal would be removed from the experiment. All three animals were euthanised,while under general anaesthetic, by intracardiac injection of pentobarbitone sodium B.P. (approx. 200 mg/kg) 17 days post challenge (DPC) in line with this clinical scoring procedure.

2.3 Post-mortem examination and collection of samples from experimental animals

Thedistribution of SQPV within 34different tissues, blood, faecal and urine sampleswas determined by qPCR analyses.Sterile nylon-flocked swabs (Thermo Scientific, Sterilin, Newport, UK), pre-wetted or not with sterile PBS as appropriate, were used to collect samples of oral and ocular secretions, samples from the skin surfaces associated with challenge sites and secondary lesions and the lids of the nest boxes.Immediately post-euthanasia, 3-5 mL of blood was collected by cardiac puncture using a 21G hypodermic needle. Approximately 2.5mLwas allowed to clot for serology to test for the presence of antibodies against SQPV using the enzyme-linked immunosorbent assay (ELISA) previously described (Sainsbury et al., 2000). Theremainderwas placed into paediatric EDTA tubes for extraction of nucleic acids. Individualsterile surgical instruments were used to harvest each tissue sample to avoid possible SQPV cross-contamination between tissues. Scarified skin samples were collected first followed by other representative skin samples (eyelid, lip, chin, nose, ear, axilla, anterior and posterior digital and mock scarified skin).Harderian glands, submandibular lymph nodes (SM LN), submandibular salivary glands (SM SG) andmucosa-associated lymphoid tissue (MALT)presentbilaterally between the palatoglossal and palatopharyngeal arches on the lateral walls of the oropharynx (corresponding to the anatomical location of palatine tonsils in other mammals) were collected in sequence followed by tongue and parotid salivary glandsamples. Tissue samples were collected in duplicate withone stored at -70 °C and the other fixed in 10% v/v neutral buffered formalin solution. Due to their small size, bothright and left popliteal lymph nodes were collected for molecular analyses only, whereas MALT samples from both sidesof the oropharyngeal mucosa from squirrel 01/12 were collected for histopathology only. All major internal body organs were examined for evidence of macroscopic abnormalities followed, whenever possible,by an additional ~0.5 cm thick tissue sample processed as above. The brain was removed whole,fixed and processed as aboveafter a small section of the frontal cortex was collected and frozen for molecular studies. Urine samples were usually collected by cystocentesis during the PM and faecal samples were collected from the descending colon and rectum.A full list of tissues sampled appears in Table1.

2.4 Suspected naturally-infected red squirrels

Wild red squirrel carcasses (n=13) found by red squirrel conservation organizations, ranger services and members of the public were submitted to the Royal (Dick) School of Veterinary Studies or to the Moredun Research Institute. Where the condition of the carcasses permitted, a full diagnostic PM, including histology, was undertaken to establish the cause of death, and sera (or fluid from the body cavity) tested for the presence of SQPV antibodies(Sainsbury et al., 2000). SQPV qPCR analyses were performed on a panel of different tissues as indicated in Table 1. Where no skinlesions suspicious of SQPV disease were present, tissue samples were generally confined to eyelid, digital and lip skin. A skin lesion or scab sample was also taken from suspected SQPV-infected animals for confirmationof SQPV by TEM (data not shown).

2.5 Histology and immunohistochemistry

Tissue samples were processed routinely prior to embedding in paraffinwax. Sections (5 μm thick) were stained with hematoxylin and eosin (HE) for histology. For SQPV immunohistochemistry (IHC), sections (5 μm) were mounted on Superfrost™ slides (Menzel-Gläser, Braunschweig, Germany), dewaxed in xylene and takenthrough graded alcohols to 95% prior to quenching endogenous tissue peroxidase activity with 3% hydrogen peroxide in methanol (v/v) for 20 minutes at room temperature (~18-22°C). Slides werewashed in water for 5 minutes then transferred to PBS containing 0.05% v/v Tween20 (PBS-T). Antigen retrieval was performed by immersing sections in a solution of 0.01M Trizma® base, 0.001M EDTA, 0.05% Tween20 at pH 9.0 (all Sigma-Aldrich Co., Dorset, UK) for 10 minutes at 95C. Non-specific antigen binding was blocked by incubation with 25% normal rabbit serum (NRS), diluted in PBS-T, for 30 minutes at room temperature. The IgGfraction was purified from pooled sera from grey squirrels previously testing positive in the SQPV ELISA (Sainsbury et al., 2000). Approximately 0.5 ml of pooled sera was diluted to 5ml in PBS containing 0.02% sodium azide and applied to a 1ml Protein A/G cartridge (ThermoFisher Scientific Loughborough UK) prewashed with 10ml PBS. Chromatography was carried out on an AKTA FPLC (GE Healthcare Life Sciences, Little Chalfont UK).Bound IgG was washed with 20ml PBS, then eluted with Pierce IgG Elution Buffer (ThermoFisher Scientific, UK) and 1ml fractions collected into tubes containing 100µl 1MTris pH 8.4. The majority ofIgG, estimated by monitoring the OD280, was eluted in a 2ml volume which was dialysed overnight against a 2L volume of PBS at 4oC. Purified IgG was adjusted to 0.5mg/ml prior to use. It was then further diluted 1/500 in 25% NRS/PBS-T (final total IgG concentration 24μg/ml), applied to sections and the slides incubated overnight at 4°C prior to being washed in PBS-T. Primary antibodies were visualised using a rabbit anti-squirrel IgG: horse radish peroxidaseconjugate (kindly provided by David Deane, Moredun Research Institute) diluted 1/200 in 25% NRS/PBS-T and applied to sections for one hour at room temperature. Sections were washed in PBS-T,Nova red chromogen (Vector Laboratories,Peterborough, UK) added for 10 minutes, washed in tap water and counterstained with hematoxylin Z (Cellpath plc., Newtown, Powys, UK), blued-up with Scot’s tap water substitute.Negative control slides were prepared by substituting the primary antibody, at the same concentration, with purified squirrelIgG from the sera of SQPV antibody negative grey squirrels.

2.6 SQPV Taqman® Real­Time PCR

The SQPV Taqman® Real-Time PCR (qPCR) assay was based on the amplification of part of theSQPV_003 gene (Accession number: HE601899)(Darby et al., 2014; McInnes et al., 2006; Thomas et al., 2003) which encodes a protein predicted to contain an immunoglobulin-like domain. Primers: forward 5'-TCCTGCAGTCATCCATCGAA-3', reverse 5'TCGCTGATGTTGTAGATGAAGTTG-3' and probe 5'Fam-CTCCGATCCCCGTCGCAACCT-3'Tam were designed to amplify a 142 bp fragment of the SQPVgene. Reaction conditions were established for the ABIPRISM®7000 Sequence Detection System (Applied Biosystems, Warrington, UK) in a total reaction volume of 25 μl. The assay mixture contained 12.5 μl of 2 ×Taqman®Universal PCR Master Mix (Applied Biosystems, UK), 2.5 μl of 3 μM forward primer, 2.5 μl of 9 μM reverse primer and 2.5 μl of 2 μM probe and 5 μlRNase/DNase-free water (for the no-template control; NTC),5 μl of DNA template (40ng/ μl) or 5 μl SQPV uncut SQPV cosmid standard dilutions (40 ng/μl), as appropriate. All samples were tested in quadruplicate.For positive controls, squirrelpox viral genomic DNA was used. Total DNA extracted from each tissue was quantified by spectrophotometry at 260/280nmand adjusted to a final concentration of 40ng/μl. As blood, urine and swab samples generally contained lower concentrations of genomic DNA qPCR reactions were performed with 5 μlof purified DNA without adjusting the concentration. Thermal reaction conditions consisted of 2 minutes at 50°C, 10 minutes at 95°C and 45 cycles each consisting of 95°C for 15 seconds and 60°C for 1 minute. The meancycle threshold (Ct) value for each sample was determined for all reactions. When at least one of the quadruplicate NTCgave a positive reaction, the assay was considered to be invalid and therefore repeated. Unknown samples with all four replicates negative were considered SQPV-negative. Viral DNA concentrations were calculated for all those samples with 4/4 positive replicates and a Ct score ≤36.5. Ifthe standard deviation of the resultantmean was >1, the sample was retested.Samples with Ct scores ≤36.5 but with less than 4/4 positive replicateswere classified as borderline positiveas were thosewith at least 1/4 positive replicatesand amean Ct score ≥36.5.

2.7 qPCRvalidation and relative SQPV quantification in samples

Ten-fold dilutions of acosmidcontaining the SQPV_003 gene (McInnes et al., 2006),ranging from 3 to 3×107viral genome equivalents/reaction, were used to generate 23 consecutive and independent standard curves. Standard curves were generatedbyplotting the resulting Ct values versus the log10 of the cosmiddilutions . To mimic the concentration and composition of clinical samples, 200 ng of squirrel genomic DNA (containing no SQPV DNA) were added to each standard dilution.Values obtained from the 23 standard curves were employed to construct a reference grand mean calibration curve [i.e. the Grand Mean Standard curve (GMS)]for determining the amount of SQPV DNA in each clinical sample. Viral load was inferred from viral genome equivalent/μg of total DNA extracted (V/μg) for all tissue and faecal samples, viral genome equivalent/mL (V/mL) for urine and blood samples and viral genome equivalent/swab (V/s) for swab samples.