Molecular Diagnosis of Cetacean Morbillivirus in Beaked Whales Stranded in the Canary Islands (1999–2017)

A retrospective survey for detecting the cetacean morbillivirus (CeMV) was carried out in beaked whales (BWs) stranded in the Canary Islands (1999–2017). CeMV is responsible for causing worldwide epizootic events with the highest mass die-offs in cetaceans, although the epidemic status of the Canarian Archipelago seems to be that of an endemic situation. A total of 319 tissue samples from 55 BWs (35 Cuvier’s BWs and 20 specimens belonging to the Mesoplodon genus) were subjected to the amplification of a fragment of the fusion protein (F) and/or phosphoprotein (P) genes of CeMV by means of one or more of three polymerase chain reactions (PCR). RNA integrity could not be demonstrated in samples from 11 animals. Positivity (dolphin morbillivirus strain (DMV)) was detected in the skin sample of only a subadult male Cuvier’s BW stranded in 2002, being the earliest confirmed occurrence of DMV in the Cuvier’s BW species. The obtained P gene sequence showed the closest relationship with other DMVs detected in a striped dolphin stranded in the Canary Islands in the same year. A phylogenetic analysis supports a previous hypothesis of a cross-species infection and the existence of the circulation of endemic DMV strains in the Atlantic Ocean similar to those later detected in the North-East Atlantic, the Mediterranean Sea and the South-West Pacific.


Introduction
The cetacean morbillivirus (CeMV; genus Morbillivirus, Paramyxoviridae family, order Mononegavirales), consisting of a single linear molecule of negative-sense single-stranded RNA, has been responsible for major epizootic diseases in cetaceans, causing many of the biggest mass die-offs worldwide in these species [1]. The main pathological findings described in infected cetaceans are broncho-interstitial pneumonia, lymphoid depletion and nonsuppurative meningoencephalitis, as well as an increased susceptibility to opportunistic infections [1][2][3][4][5]. According to the stage of the infection, four presentation forms of the disease have been recently described [1]: acute and subacute systemic diseases, chronic systemic infections and chronic localized CeMV encephalitis.

Materials and Methods
The availability of the number of animals for this study was possible thanks to the permission for the management of stranded cetaceans granted by the Spanish Ministry of the Environment. Moreover, no animal was sacrificed and no experiments were performed with live animals, so ethical review and approval were waived.
Fifty-five BWs stranded along the coasts of the Canary Islands from November 1999 to May 2017 were included in this study, from which 35 specimens were Cuvier's BWs and 20 specimens belonged to the Mesoplodon genus: one True's BW (Mesoplodon mirus), two Sowerby's BWs (Mesoplodon bidens), seven Blainville's BWs and ten Gervais' BWs (Mesoplodon europaeus). A map indicating stranding location of each animal (indicated by its case number and species) was created by the software ArcMap [38] and is shown in Figure 1. The information about each stranding (date, location with coordinates and type) and decomposition stage (grade 1: extremely fresh carcass; grade 2: fresh carcass; grade 3: moderate decomposition; grade 4: advanced decomposition and grade 5: mummified or skeletal remains) [39] is compiled in Table 1, as well as life history data (species, age category, sex and body condition). The nutritional status was classified as good, moderate, poor or emaciated in consonance with the anatomical parameters, such as the observable presence of marked bony process and prominent bones through the skin (the transverse and spinous vertebral processes and ribs), the observable presence of dorso-axial muscular mass and the presence or distribution of fatty tissue in several organs, taking into consider-Vet. Sci. 2022, 9,121 3 of 18 ation the species and the age of the animal [22,25]. A complete postmortem examination, following standardized necropsy protocols [39,40], was performed on all of the animals from the study. Collected samples were fixed in 10% neutral buffered formalin solution, embedded in paraffin blocks, sectioned at 5 µm, stained with haematoxylin and eosin (HE) and examined under a light microscope. All the cases included in the present study were diagnosed during routine pathological and cause-of-death analyses of stranded cetaceans at the Division of Histology and Animal Pathology of the Institute for Animal Health (IUSA), Veterinary School, Universidad de Las Palmas de Gran Canaria. Immunohistochemistry was performed in molecular positive samples following a previously described standardized protocol. The tissue sections were incubated with a mouse monoclonal antibody against the nucleoprotein antigen of canine distemper virus (CDV, 1:200 dilution; CDV-NP MAb, VMRD Inc.), for which cross-reactivity with CeMV has been previously reported [41]. Positive controls included laryngeal tonsil from a CeMV-positive striped dolphin stranded in the Canary Islands in 2019. Virological analyses were performed in kept frozen samples (−80 • C). mals from the study. Collected samples were fixed in 10% neutral buffered formalin solution, embedded in paraffin blocks, sectioned at 5 µm, stained with haematoxylin and eosin (HE) and examined under a light microscope. All the cases included in the present study were diagnosed during routine pathological and cause-of-death analyses of stranded cetaceans at the Division of Histology and Animal Pathology of the Institute for Animal Health (IUSA), Veterinary School, Universidad de Las Palmas de Gran Canaria. Immunohistochemistry was performed in molecular positive samples following a previously described standardized protocol. The tissue sections were incubated with a mouse monoclonal antibody against the nucleoprotein antigen of canine distemper virus (CDV, 1:200 dilution; CDV-NP MAb, VMRD Inc.), for which cross-reactivity with CeMV has been previously reported [41]. Positive controls included laryngeal tonsil from a CeMV-positive striped dolphin stranded in the Canary Islands in 2019. Virological analyses were performed in kept frozen samples (−80 °C).
According to the availability in each case, a total of 319 tissue samples were analyzed: the skin ( Table 1. Frozen kept samples were thawed and mechanically macerated for a subsequent simultaneous extraction of DNA and RNA by means of a QuickGene R Mini 80 nucleic acid isolation instrument with the DNA Tissue Kit S (QuickGene, Kurabo, Japan) according to the manufacturer's instructions with some modifications: an RNA carrier (Applied Biosystems TM , Thermo Fisher Scientific Waltham, MA, USA) was added during the lysis step, as previously published [45].
Molecular detection of CeMV was performed using one or more of three polymerase chain reaction (PCR) methods. (1) was a modified conventional one-step reverse transcription polymerase chain reaction (RT-PCR), which amplifies a fragment of 426 base pairs (bp) from a conserved region of the phosphoprotein (P) gene. Primers and PCR protocol used were the following: ((DMV C: ). The obtained amplicons were analyzed by means of a 2% agarose gel horizontal electrophoresis [42][43][44]. (2) was a real-time one-step reverse transcription polymerase chain reaction (RT-qPCR) amplifying a size region of 192-bp from the fusion protein (F) gene using the following primers: (DMVFuF: 5 -GGCACCATAATTAGCCAGGA-3 /DMVFuR: 5 -GCCCAGATTTGTGCCTACAT-3 ) and the PCR protocol (30 [45] and (3) was a PAN RT-qPCR method based on SYBRN ® green dye [46] that successfully detects GDMV, PWMV and DMV strains. The primer set (Forward PAN-F (5 -CCTCTAACAGGGGATCT(A/G)CTC-3) and Reverse PAN-R (5 -CCTGTGCCCTTTTTAATGGA-3)) amplifies 205 bp from a region of the phosphoprotein (P) gene. The PCR protocol used was as follows: 50 . The information about which PCR method was used for the detection of CeMV in each animal is specified in Table 1. A negative control (non-template) and an amplification-positive control (known cetacean morbillivirus RNA previously obtained in our laboratory) were added in each ADN/ARN extraction and PCR protocols described above. Genomic DNA digestion was performed in the total RNA/DNA extractions followed by a second purification following the same protocol as previously described. RNA extractions were then subject to a one-step RT-qPCR that amplifies the housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as previously described [47], in order to ensure the high quality of the RNA [48]. In addition, all the samples were checked for the presence of herpesvirus by means of a pan-herpesvirus conventional nested PCR based on the DNA polymerase gene [35,49].
Previous to the Sanger sequencing method, the PCR products were purified using Real Clean spin kit (REAL ® , Durviz, S. L., Valencia, Spain). BLAST algorithm (www.ncbi. nlm.nih.gov/blast/Blast.cgi/ (accessed on 10 November 2021)) [50] was used to compare the obtained amplicons with other somewhat similar sequences published in GenBank. The sequences were aligned using ClustalW algorithm through software MEGA X [51]. A total of 102 CeMV nucleotide sequences based on the P gene were recovered from GenBank to construct the phylogenetic tree, where three canine distemper virus (CDV) sequences were used to root the phylogram as an outgroup. The phylogenetic tree was constructed using the Maximum Likelihood Method and the Kimura 2-parameter model with a discrete Gamma distribution to model the evolutionary rate differences among sites (5 categories (+G, parameter = 0.7797)). The Bootstrap method (500 replicates) was applied to assess the reliability of the tree.
A phylogenetic tree based on the F gene was also created, which was constructed using the Maximum Likelihood Method and the Tamura 3-parameter model with a discrete Vet. Sci. 2022, 9, 121 7 of 18 Gamma distribution to model the evolutionary rate differences among sites (5 categories (+G, parameter = 0.5319)). A Bootstrap test from 500 replicates was also implemented. This analysis involved 52 nucleotide sequences, where two Peste des petits ruminants virus (PPRV) and two Phocine distemper virus (PDV) sequences established the root of the tree.
The DMV sequences from P gen and F gen were identified from this study and were deposited in GenBank under accession nos. OM055653 and OM055654, respectively.

Results
The presence of CeMV was detected in one animal (1/55; 1.82%), a subadult male Cuvier's BW stranded to death in Fuerteventura in September 2002 in a good state of preservation (code 2, CET 182). Positivity was achieved in the skin sample (1/319; 0.3%). All the tested tissue samples from this animal were negative for the herpesvirus infection [35].
This Cuvier's BW was one of the 14 BWs (designed as BW-3) stranded on the beaches of Fuerteventura and Lanzarote islands on 24 September 2002, temporally associated with naval exercises involving acoustic (sonar) activities (manoeuvers called Neo-Tapon 2002) [19]. Macroscopically, no systemic, inflammatory or neoplastic processes were noted, although the carcass showed severe diffuse congestion and haemorrhages, especially around the acoustic jaw fat, ears, brain and kidneys. Gas bubble-associated lesions and fat embolism were observed in the vessels and parenchyma of vital organs in the histopathological study (DCS) [19]. No pathogens were identified in routine aerobic bacterial cultures of the brain, lungs and spleens from this specimen [19].
The housekeeping gene was not amplified in samples from 11 animals of the 55 analyzed in the present study (20%). The RNA from these negative samples was too degraded for causes determining causes due to descomposition stages or the time elapsed since it was taken. Thus, only 44 animals were considered validated for the CeMV molecular analyses.

Phylogenetic Analyses
The nucleotide phylogenetic analysis based on the P gen showed that the obtained tree presents five main branches, where DMV, BWMV, PWMV, PMV and GDMV strains are clearly clustered in their respective clades ( The phylogenetic nucleotide sequence analysis based on the F gene ( Figure 3) showed a DMV clade supported by a Bootstrap value of 81 conformed by a polyphyletic group of 42 sequences detected in seven cetacean species and one harbour seal (Phoca vitulina). No other F gene sequences obtained from BWs are available in GenBank. The obtained BW sequence from this study was exclusively clustered with a sequence detected in a Risso's dolphin (Grampus griseus) stranded in the Central-East Atlantic Ocean (Canary Islands) in 2008 (GenBank acc. no. KX512308).

Histopathology, Immunohistochemistry
To date, only frozen skin tissue samples from this case remained available for the histopathological study. At the microscopic level, neither CeMV-associated lesions nor immunostainings for CDV were observed, although ballooning, interpreted as a freeze artefact, was present in most of the keratinocytes of the stratum spinosum ( Figure 4A,B). The immunolabelling of CDV was observed in the epithelial cells from the positive control (laryngeal tonsil sample from a CeMV-positive striped dolphin stranded in the Canary Islands in 2019, Figure 4C). genetic tree consists of 102 sequences from reported cases of cetacean morbillivirus. To construct the tree, were designed the Neighbour-Join and BioNJ algorithms along with the Kimura 2-parameter model and Gamma distribution to model the evolutionary rate differences among sites [5 categories (+G, parameter = 0.7797)]. The Bootstrap method was performed to resample 500 replicates and evaluate the reliability of the tree. The accession number from GenBank, the host, the sample of detection, the date of collection, and the geographic area of stranding were used to identify the nucleotide sequences. Abbreviations: ENoAt (Northeast Atlantic Ocean); WAt (West Atlantic Ocean); CeAt (Central Atlantic Ocean); SoAt (South Atlantic Ocean); Me (Mediterranean Sea); Pa (Pacific Ocean); NPa (North Pacific Ocean); No (North Sea); CaspS (Caspian Sea). To construct the tree, we designed the Neighbour-Join and BioNJ algorithms along with the Tamura 3-parameter model and Gamma distribution to model the evolutionary rate differences among sites (five categories (+G, parameter = 0.5319)). The Bootstrap method was performed to resample 500 replicates and evaluate the reliability of the tree. The accession number from GenBank, the host, the sample of detection, the date of collection and the geographic area of each stranding were used to identify the nucleotide sequences. Abbreviations: ENoAt (North-East Atlantic Ocean); WAt (West Atlantic Ocean); CeAt (Central Atlantic Ocean); Me (Mediterranean Sea); No (North Sea).

Histopathology, Immunohistochemistry
To date, only frozen skin tissue samples from this case remained available for the histopathological study. At the microscopic level, neither CeMV-associated lesions nor immunostainings for CDV were observed, although ballooning, interpreted as a freeze artefact, was present in most of the keratinocytes of the stratum spinosum (Figure 4 A, B). To construct the tree, we designed the Neighbour-Join and BioNJ algorithms along with the Tamura 3-parameter model and Gamma distribution to model the evolutionary rate differences among sites (five categories (+G, parameter = 0.5319)). The Bootstrap method was performed to resample 500 replicates and evaluate the reliability of the tree. The accession number from GenBank, the host, the sample of detection, the date of collection and the geographic area of each stranding were used to identify the nucleotide sequences. Abbreviations: ENoAt (North-East Atlantic Ocean); WAt (West Atlantic Ocean); CeAt (Central Atlantic Ocean); Me (Mediterranean Sea); No (North Sea). The immunolabelling of CDV was observed in the epithelial cells from the positive control (laryngeal tonsil sample from a CeMV-positive striped dolphin stranded in the Canary Islands in 2019, Figure 4C).

Discussion
CeMV was detected in one of the 55 BWs analyzed for the presence of this pathogen Immunohistochemistry. CDV immunostaining was not observed. Immunohistochemistry stain, X 10. Inset: detail of lack of specific immunostaining against CDV in keratinocytes (*). Immunohistochemistry stain, ×40. (C) Immunohistochemistry. Positive control for CDV antibody in laryngeal tonsil sample. Immunostaining was observed in the cytoplasm of epithelial cells. Immunohistochemistry for morbillivirus using an antibody to CDV nucleoprotein., ×20. Inset: detail of specific immunostaining against CDV in epithelial cells (*). ×60.

Discussion
CeMV was detected in one of the 55 BWs analyzed for the presence of this pathogen in the Canary Islands. Specifically, the virus (DMV strain) was present in the skin sample of a subadult male Cuvier's BW, representing the second molecular confirmation of a DMV infection in this species. The detection of DMV in a Cuvier's BW was first reported in Italy in the lung sample of a male calf stranded in 2015 [15], 13 years after the sample from our study was collected. Therefore, the retrospective analysis of archived tissue samples carried out in our study exposed that this was the earliest confirmed occurrence of DMV in the Cuvier's BW species.
Positivity was achieved in the skin sample by means of two real-time PCRs but not with the conventional one. The skin sample had high cycle-threshold values in both qPCRs and therefore low viral loads, which could be only detected with test methods with higher sensitivities when compared to more traditional assays [52]. CeMV has been previously detected in the skin samples of marine mammals with and without associated lesions [41,53,54]. No lesions were observed in the skin sample of the Cuvier's BW from our study. In cetaceans, the effects of CeMV are widely variable from causing epidemics to subclinical infections. According to previous studies about CeMV in BWs, even if a study does not describe histopathological findings in detail, it seems that the infection does not cause severe pathological effects in these species [13][14][15]. The only exception is the first published case of a BWMV infection in a Longman's BW stranded in Hawaii in 2010, which presented an associated nonsuppurative encephalitis but which was also co-infected with the herpesvirus [9]. In addition to the herpesvirus, secondary infections or co-infections with other viruses, bacteria, fungi, protozoans or parasites have been frequently described [2,48,[55][56][57][58][59]. In our study, the presence of bacterial pathogens and a herpesvirus infection was ruled out [19,35].
The level of detection of CeMV in stranded BWs in the Canary Islands (1.82%; 1/55) in an 18-year period (1999-2017) is low compared with previous studies in other geographical regions, which range from 5.7% to 48.1% (Table 2). This variability in the level of detection is partially due to the fact that some of those retrospective studies included eligibility (inclusion) criteria or morbillivirus-related lesions, species classically considered DMV hosts, such as striped and bottlenose dolphins and/or areas and years in which some epizootic events or outbreaks were involved [1,48,60]. However, the absence of DMV detection in other samples from our study does not rule out the presence of DMV in such samples. The assessment of RNA integrity is a critical first step for detecting false-negative samples. Current epidemiological knowledge of CeMV in Canarian waters indicates the presence of some inter-epizootic sporadic endemic presentations of the infection with the detection of the virus in 1996, 2002, 2005, 2007-2009, 2011-2012, 2015 [44,59,61,62], 2016 and 2018-2020 (unpublished data). The exception is an unusual mortality event in 2015 involving short-finned pilot whales and the PWMV strain [63]. The isolate from this study is one of the only two earliest CeMV sequences available in GenBank detected after the first epizootic event on the Spanish Mediterranean coast in 1990-1992 [73], both of them detected in the same year in the Canary Islands in two different cetacean species and highly similar. The individual from our study stranded in 2002, and the obtained sequences showed the closest relationship with another DMV detected in cetaceans from the Canary Islands (with a striped dolphin stranded in the same year with a systemic chronic infection (GenBank acc. no. KJ139451) [61] according to the nucleotide sequence alignment and phylogenetic analysis of the P gene) and with a Risso's dolphin stranded in 2008 (with chronic localized CeMV encephalitis (GenBank acc. no. KX512398) [66] according to the phylogenetic analysis of the F gene). This information seems to support a previous hypothesis of a cross-species infection [15,44,61,66,67,69,74,75]. The P gene nucleotide analysis also showed that the isolate is highly similar to one detected in a striped dolphin stranded on the North-East coast of Portugal in 2007 (GenBank acc. no. KP835995) [64] in the middle of the second epizootic event on the Spanish Mediterranean coast [55,76,77], which also clustered with samples from the Mediterranean Sea from 2007 to 2013. The F gene nucleotide analysis showed that very similar sequences were detected in the Mediterranean Sea in the first and second Mediterranean epizootic events. Taken together, these results are consistent with the previous supported idea that CeMV is endemic in the Central and North-East Atlantic and that interanimal transmissions might occur through the Strait of Gibraltar. This viral exchange could cause Mediterranean outbreaks and unusual mortality events in that region due to the relatively high density of animals, specifically striped dolphins, and their gregarious behaviour [57,61,64,76,[78][79][80][81][82][83][84][85]. In addition, the P gene sequence from Portugal (GenBank acc. no. KP835995) is clustered with the DMV isolate detected in a Longman's BW from New Caledonia (GenBank acc. no. KR704575) as previous studies have indicated [64]. Thus, both DMV sequences detected in BWs from the Central East Atlantic in 2002 and the South-West Pacific in 2013, respectively, show high similarities to the sequence detected in a striped dolphin from the coast of Portugal in 2007, which is also similar to sequences from the Mediterranean from 2007 to 2013. It has been previously reported that undefined cetacean species, due to their extensive patterns of migration and their interactions with certain dolphin populations, may mediate the trans-oceanic spread of CeMV, driving the dispersal and evolution of this virus [72,75,86]. The Cuvier's BW species has a worldwide distribution, featured in almost all temperate, sub-tropical, tropical, sub-polar and polar waters [87]. Moreover, this species presents migrating behaviours in the Atlantic Ocean, and it could probably act as a reservoir spreading the disease into more susceptible populations as previously proposed for fin whales [75,88,89].

Conclusions
We present the first molecular confirmation of a DMV infection in a Cuvier's BW, although new retrospective studies worldwide could add further evidence of prior cases of infection in this species. The isolate dates from 2002, 10 years after the first Mediterranean epizootic event took place. This is also the second DMV sequence available since then, both of them detected the same year (2002) and collected from stranded cetaceans of different species in the Central East Atlantic Ocean (Canary Islands). These two sequences were identical, indicating that the cross-species infection is not as new as previously suggested.
The epidemic status of the archipelago is that of an endemic situation of several cetacean species rather than an epidemic, although the circulation of strains similar to those later detected in the North-East Atlantic, the Mediterranean and the South-West Pacific indicate that the migration pattern of some of these species could disseminate the virus to other areas and cause epizootic or unusual mortality events. Funding: Part of this research work was supported through the Canary Islands Government, which has funded and provided support to the stranding network. The MARCET (MAC/1.1b/149) and MARCET II (MAC2/4.6c/392) projects, and the Research National Project "Embolic Pathology (gas/fat) in cetaceans," which is supported by the Ministerio de Ciencia e Innovación del Gobierno de España (CGL2015-71498-P), Ministerio de Ciencia, Innovación y Universidades (PGC2018-101226-B-100).

Institutional Review Board Statement:
The availability of the number of animals for this study was possible thanks to the permission for the management of stranded cetaceans granted by the Spanish Ministry of the Environment (SGPM/BDM/AUTSPP/70/2019). Moreover, neither animal was sacrificed and not experiments were performed with live animals, so ethical review and approval were waived.

Informed Consent Statement: Not applicable.
Data Availability Statement: The DMV obtained sequences from a Cuvier's beaked whale have been deposited in GenBank (under accession numbers: OM055653, OM055654).