Detection and Preliminary Characterization of Phocine Distemper Virus in a Stranded Harp Seal (Pagophilus groenlandicus) from the Gulf of St. Lawrence, Canada Open Access

Phocine morbillivirus is one of the seven currently accepted species within the genus Morbillivirus, family Paramyxoviridae (Kuhn et al. 2019). The virus, phocine distemper virus (PDV), was first recognized in 1988 when it was isolated from dead eastern Atlantic harbor seals (Phoca vitulina vitulina) in the North Sea (Osterhaus and Vedder 1988; de Vries et al. 2013). Phocine distemper was responsible for epizootics among harbor seals and grey seals (Halichoerus grypus) in northern Europe in 1988 (Kennedy et al. 1988) and again in 2002 (Jensen et al. 2002). In both epizootics, tens of thousands of harbor seals died within a few months (Härkönen et al. 2006); however, genetic sequencing has shown that the PDV strains responsible for each epizootic were different (Nielsen et al. 2009). This discovery has led to speculation, supported by serologic surveys, that PDV strains are circulating enzootically among various seal species inhabiting coastal zones in the North Atlantic regions of North America and in the Arctic, particularly harp seals (Pagophilus groenlandicus), which number in the millions (Duignan et al. 2014). Retrospective studies have detected morbillivirus-binding antibodies in a number of seal species in Canada as far back as the 1970s (Henderson et al. 1992), and a high proportion of serum samples collected from harp, hooded (Cystophora cristata), and ringed (Pusa hispida) seals throughout the Canadian Arctic and the western Atlantic between 1988 and 1994 were PDV neutralization–positive, indicating exposure to the virus (Duignan et al. 1997).

Eastern North America has sporadic reports of moribund PDV-infected seals, including a harp seal stranded on the shore of Prince Edward Island (PEI), Canada (Daoust et al. 1993), and harbor seals stranded along the coast of New England, US (Duignan et al. 1993), suggesting that some infected seals do succumb but most survive the infection (Duignan et al. 2014). Additionally, a dermatitis caused by PDV was identified in a hooded seal and a harp seal found stranded on the New Jersey coast, US, in 1998 (Lipscomb et al. 2001). In 2006, an increase in stranded western Atlantic harbor and grey seals along the New England coast was observed, and PDV was isolated from a harbor seal from Maine, indicating that PDV outbreaks are reoccurring in New England (Earle et al. 2011). However, because of the rarity of cases identified in harp seals, PDV has not yet been genetically characterized from this species. Herein, we report the gross and microscopic pathology, virology, and molecular genetic findings from a second harp seal from PEI infected with PDV.

A juvenile (a few years old; standard length, 138 cm) male western Atlantic harp seal in poor body condition was found stranded at South Rustico, PEI (coordinates 46°24′57″N, 63°17′30″W), in June 2017, displaying clinical signs of lethargy and difficulty moving. It was euthanatized by injection of an overdose of barbiturate and a full postmortem examination was performed (Canadian Department of Fisheries and Oceans, Gulf Region, license SG-RHQ-17-010). Gross lesions were not evident, except for pulmonary congestion. Representative tissue samples were fixed in 10% neutral buffered formalin and processed routinely for light microscopic examination. Portions of brain and lung were also frozen at –80 C. Histologically, lesions were confined to the brain, particularly the medullary region of the brainstem, and consisted of marked perivascular cuffing and marked hypercellularity of the parenchyma caused by an infiltration of large numbers of lymphocytes, plasma cells, and macrophages (Fig. 1A). Swollen axons and myelin sheets were also common (demyelination). However, cellular necrosis was not evident, and viral intracytoplasmic or intranuclear inclusion bodies were not found. An immunohistochemical assay was used to demonstrate the presence of morbillivirus antigen in formalin-fixed, paraffin-embedded sections of the brain; other tissues were not examined immunohistochemically. Epitope retrieval was performed in a Tris-ethylenediaminetetraacetic acid (pH 9) buffer at 97 C for 20 min. A commercially available mouse monoclonal antibody against canine distemper virus nucleoprotein (mouse anti-canine distemper virus, clone DV2-12, Custom Monoclonals International, West Sacramento, California, USA) that has been shown to detect PDV (Stanton et al. 2004), was applied for 30 min at a dilution of 1:4,000. Binding of this primary antibody was detected with a horseradish peroxidase–labeled polymer detection reagent (EnVision+System-HRP Labelled Polymer, Agilent Technologies Canada, Mississauga, Ontario, Canada). Staining with immunohistochemical revealed morbillivirus antigen multifocally in the brain, including a large cluster in the medullary region of the brainstem (Fig. 1B).

Samples of brain and lung held at –80 C were thawed, then ground with sterile sand and a mortar and pestle with added Hanks balanced salt solution plus penicillin (200 IU/mL), streptomycin (200 mg/mL), and gentamicin (50 mg/mL) to give a 10% (w/v) ratio of tissue suspension. After centrifugation (2,060 × G for 10 min), 0.5-mL aliquots were inoculated onto 25-cm² flasks containing Vero.DogSLAMtag cells (originally obtained from Dr. Yasuke Yanagi, Department of Virology, Kyushu University, Fukuoka, Japan), which have been shown to be useful in isolating PDV (Nielsen et al. 2008). The flasks were incubated at 37 C and observed daily. All flasks were subcultured at a 1:4 ratio weekly for a month and were negative by virus isolation.

We extracted RNA from lung and brain tissues using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, California, USA) according to manufacturer's instructions and subjected to a seminested reverse transcription PCR assay targeting a portion of the paramyxovirus RNA–dependent RNA polymerase gene using primers RES-MOR-HEN F1/F2 and RES-MOR-HEN-R (Tong et al. 2008). The reverse transcription PCR assay confirmed the presence of paramyxovirus RNA in the brain tissue, whereas the lung tissue sample was negative. The second round PCR product (494 base pairs [bp]) was purified with a QIAquick PCR Purification Kit (Qiagen) and sequenced in both directions on an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, California, USA). The primer sequences were removed before assembly and editing of the forward and reverse sequence reads in CLC Genomics Workbench V10 (Qiagen). BLASTN (National Center for Biotechnology Information 2016) searches of the edited sequence showed 99.5% (437/439 bp) and 99.8% (438/439 bp) identity to PDV strains isolated from harbor seals that stranded along the coastlines of the Irish (Ulster 88; GenBank accession no. Y09630) and Wadden (PDV/Wadden_Sea.NLD/1988; GenBank no. KC802221) seas, respectively, in 1988 (McIlhatton et al. 1997; de Vries et al. 2013) and 99.5% (437/439 bp) identity to a PDV isolate sequenced from a western Atlantic harbor seal that stranded in Maine (PDV/USA 2006; GenBank no. KY629928) in 2006. The PDV sequence obtained from the harp seal brain sample in this study has been deposited in GenBank under accession number MN581725.

Since the first epizootics of PDV in European seals in 1988 and again in 2002, biologists have sought to understand the role that arctic seal species play in the maintenance and periodic introduction of PDV to European and New England seals. Harp seals infected with PDV were first identified in 1991 (Daoust et al. 1993) and again in 2017 in the northwest Atlantic, indicating that PDV can cause severe disease in at least a few harp seals and supporting the hypothesis that harp seals are enzootically infected. The partial PDV genomic sequence from our 2017 case was found to be very closely related to genomic sequences previously determined for PDV strains recovered from moribund harbor seals in the Irish and Wadden seas in 1988 and in the northwest Atlantic in 2006. Full genome sequencing of the 2017 case compared with the other isolates would be needed to further support the hypothesis that harp seals play a role in the maintenance of PDV in pinniped populations widely distributed across the coastlines of North America and Europe. Nonetheless, much remains to be understood about the epidemiology of PDV, because movements of harp seals between their western and eastern populations appear to be limited (Sergeant 1991; Perry et al. 2000).

We thank the Department of Fisheries and Oceans, Canada, for funding the genetic sequencing portion of this investigation.