New insight into dolphin morbillivirus phylogeny and epidemiology in the northeast Atlantic: opportunistic study in cetaceans stranded along the Portuguese and Galician coasts

Background Screening Atlantic cetacean populations for Cetacean Morbillivirus (CeMV) is essential to understand the epidemiology of the disease. In Europe, Portugal and Spain have the highest cetacean stranding rates, mostly due to the vast extension of coastline. Morbillivirus infection has been associated with high morbidity and mortality in cetaceans, especially in outbreaks reported in the Mediterranean Sea. However, scarce information is available regarding this disease in cetaceans from the North-East Atlantic populations. The presence of CeMV genomic RNA was investigated by reverse transcription-quantitative PCR in samples from 279 specimens stranded along the Portuguese and Galician coastlines collected between 2004 and 2015. Results A total of sixteen animals (n = 16/279, 5.7 %) were positive. The highest prevalence of DMV was registered in striped dolphins (Stenella coeruleoalba) (n = 14/69; 20.3 %), slightly higher in those collected in Galicia (n = 8/33; 24.2 %) than in Portugal (n = 6/36; 16.7 %). Conclusions Phylogenetic analysis revealed that, despite the low genetic distances between samples, the high posterior probability (PP) values obtained strongly support the separation of the Portuguese and Galician sequences in an independent branch, separately from samples from the Mediterranean and the Canary Islands. Furthermore, evidence suggests an endemic rather than an epidemic situation in the striped dolphin populations from Portugal and Galicia, since no outbreaks have been detected and positive samples have been detected annually since 2007, indicating that this virus is actively circulating in these populations and reaching prevalence values as high as 24 % among the Galician samples tested. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0795-4) contains supplementary material, which is available to authorized users.

(Continued from previous page) distemper virus -1; PMV, Porpoise morbillivirus; PP, Posterior probability; PWMV, Pilot whale morbillivirus; RT-PCR, Reverse transcription PCR; RT-qPCR, Reverse transcription quantitative PCR; SC, Stenella coeruleoalba; SPVS, Sociedade portuguesa de vida selvagem; TT, Tursiops truncatus Background Morbillivirus infection affects mainly the upper respiratory tract, central nervous system and the immune system [1,2] and has been identified as a cause of death and stranding in marine mammals [3]. In odontecetes, infection has been associated with high mortality rates occurring during disease outbreaks in different parts of the world [4]. The mortality rate in striped dolphins from the Mediterranean Sea in the beginning of the nineties was the highest recorded so far [2,5,6]. Further studies are needed to deepen the knowledge about this disease. An integrated approach taking into consideration epidemiological and environmental parameters should provide a better picture of the ecology and evolution of Cetacean Morbillivirus (CeMV) in free-ranging cetaceans [2].
Morbilliviruses affecting cetaceans have been described in the last decades [2] after the initial detection of viral antigens in these species in the late eighties. The first evidence of morbillivirus infection in cetaceans occurred in 1988 during a PMV outbreak, when the viral antigen was detected in harbour porpoises (Phocoena phocoena) stranded in Ireland [11]. In the early nineties, dolphin morbillivirus (DMV) was isolated from striped dolphins from the Mediterranean [12,13]; in 2000 PWMV was first described in a long-finned pilot whale (Globicephala melas) from the US coast [14] and later, in 2011, from a short-finned pilot whale (Globicephala macrorynchus) in the Canary Islands [15].
Due to the virus pathogenic impact on cetacean populations, further information about morbillivirus infection in cetaceans worldwide is relevant to understand its epidemiology in these animals. Studying infectious diseases in these species is important, especially considering that additional non-infectious aggressions, mainly due to human activities, render these populations even more susceptible to disease. An annual average of 200 stranded cetaceans were registered between 2010 and 2012, considering the Algarve and the Northern region of the Portuguese continental coast [16] and fisheries bycatch was identified as the most significant cause of death. To this date, no molecular data was published on morbillivirus infection in animals stranded in Portugal or northern Spain. In 2014, dolphin morbillivirus infection was reported in a retrospective study affecting striped dolphins and a common dolphin from the Canary Islands [17], causing non-suppurative meningoencephalitis. Also, a fatal systemic morbillivirus infection was detected in a bottlenose dolphin stranded in 2005 in the Canary Islands [18]. It was suggested that DMV was not endemic in harbour porpoises and common dolphins (Delphinus delphis) from the NE Atlantic (British Isles) in the period 1996-1999 [19], as low antibodies titres were detected in animals from Spain and the North Sea.
DMV infection apparently did not persist as an endemic infection in Mediterranean striped dolphins after the 1990-92 epidemic [19]. Both epidemics in the Mediterranean Sea (1990-92 and 2006-07) started near the Gibraltar Strait [20] and it has been suggested that DMV-infected cetaceans may have entered the Strait of Gibraltar and infected striped dolphins, the most common cetacean at the time [7,21]. Pilot whales had been already proposed as reservoirs in 1995 [22,23]. Later, in 2006 several long-finned pilot whales were found stranded along the coast of the Alborean Sea,and morbillivirus infection was detected [24]. In this epidemic, deaths were first detected close to the Gibraltar Strait and spread further into the Mediterranean Sea. Recently described sequences found in striped dolphins from the Canary Islands show high identity with sequences from the Mediterranean outbreaks, indicating the possible circulation of viruses between the Atlantic and the Mediterranean [17]. The role of other cetacean species as reservoirs needs to be further assessed.
The objective of the present study was to clarify not only the prevalence of DMV in cetacean populations from the eastern Atlantic, but also to investigate the relationship between the dolphin morbillivirus circulating in the eastern Atlantic and elsewhere in the world, especially in the Mediterranean.  The animals were assigned a decomposition code (1 to 5) according to already established protocols [25]. Animals with a score ranging from to 1 to 3 (fresh to moderate decomposition) were surveyed in the present study. During necropsy, tissue samples were collected from 279 cetaceans: brain, lung, pulmonary lymph node, mesenteric lymph node, spleen, kidney and liver, whenever possible. For animals from Galicia the only available sample was the lung. Samples collected in Portugal were stored in vials with RNAlater® at −20°C and samples collected in Galicia were frozen at −20°C. All samples were kept in the marine animals' tissue banks (MATBs) of SPVS and CEMMA.

Total RNA extraction
Total RNA was extracted from a pool of tissue homogenates using RNeasy mini kit (Qiagen, GmbH, Germany), according to the manufacturer's instructions. The pool included, whenever possible: lung, brain, pulmonary lymph node and mesenteric lymph node. Total RNA quantification and purity was determined using a Nanodrop 2000C spectrophotometer (ThermoScientific, USA) and stored at −80°C until used.
Detection of dolphin morbillivirus genomic RNA by reverse transcription-quantitative PCR (RT-qPCR) The detection of viral RNA for the DMV strain of CeMV was performed by RT-qPCR in a StepOnePlus thermocycler (Applied Biosystems), using primers (Stabvida genomics lab, Portugal) and probe (Eurogentec), targeting the N gene of DMV, as previously described [26] (Table 2). A previously detected positive sample for DMV was used as a positive control of the PCR reaction. Negative reaction controls were always included.
One step RT-qPCR assays were performed using 100 ng of the template RNA, in a total reaction volume of 20 μL containing: 10 μL of 1-step qPCR-ROX Mix (2×); 1 μL of RT enhancer; 0,2 μL of Verso Enzyme Mix (Verso 1-Step qRT-PCR ROX kit, ThermoScientific®); 0.4 μM of each primer and 0.25 μM of probe. For positive samples, total RNA was extracted individually for each of the available organs and the infection was evaluated individually in the different organs. The amplified DMV fragment was cloned into a plasmid vector (Pgem Teasy -Promega) and serial tenfold dilutions of the recombinant plasmid DNA were used to construct the standard curve (Fig. 1). The results showed a high correlation (R 2 = 0.997) with a calculated efficiency of 81 %. The primers and probe could detect viral RNA copies down to 10 2 , and the limit of detection was 224 copies.

Conventional PCR for amplification of DMV genes
Additional sequences were amplified from the positive samples by conventional reverse transcription-PCR  Striped dolphin (SC) 1 - (RT-PCR) using previously described primers (Table 3) purchased from Stabvida genomics lab (Portugal). Primers were used in different combinations, targeting different genomic regions (Fig. 2). The obtained amplicons were used to perform a phylogenetic analysis of the DMV sequences, along with sequences retrieved from NCBI for the same genes. L and M genes were not targeted in the conventional RT-PCR since very few sequences were available at the NCBI database.
The amplicons were directly sequenced by Sanger sequencing at Stabvida, Portugal and the specificity of the nucleotide sequences was compared by Blast analysis http://blast.ncbi.nlm.nih.gov/Blast.cgi with CeMV sequences available in the GenBank.

Phylogenetic analysis
The nucleotide sequences of the Portuguese and Galician sequence datasets available in the GenBank (National Center for Biotechnology (Table 4), according to their primary structure similarity using the multiple alignment ClustalW program [27].
Six sets of alignments were considered for the phylogenetic analysis: nucleotide sequence alignments for genes N, P, F and H, composed by sequences of 218, 342, 449 and 316 base pairs, respectively; concatenated sequence of amino acids (540 aa) and nucleotides (1446 bps). Due to heterogeneity of the available DMV sequences it was not feasible to maintain the same set of DMV sequences in the alignment for each gene. In the concatenated alignment only the sequences with all partial genomic regions were included. The multiple sequence alignments were manually corrected with Jalview, Version 2.0.1 [28] removing long internal gaps and unmatched ends to maximize genetic similarities and phylogenetic trees were inferred by Bayesian methods (MrBayes v.3.2.1) [29,30].
For the Bayesian analysis a Markov chain Monte Carlo (mcmc) simulation technique was carried out to approximate the PP of trees [30]. The evolutionary GTR (nucleotides) and LG (amino acids) models were selected with gamma-distributed rate variation across sites and a proportion of invariable sites (rates = invgamma). The analysis was initiated using a random tree from the dataset with four chains running simultaneously for 20 × 10 6 generations, sampling every 100 generations. The first 25 % trees were discarded and a majority rule consensus tree was generated from the remaining trees.
The graphical representation and edition of the phylogenetic tree were performed with FigTree v1.3.1. Only support values equal or greater than 0.70 of PP are shown in the trees.

Statistical analysis
Chi-square test of association was performed to assess if the difference in prevalence was statistically significant between different species (DD and SC) and between  For the nucleotide sequences of each genomic region, phylogenetic trees were inferred by Bayesian methods. All trees exhibited a similar sequence topology, supported by robust PP values, regardless of the total number of sequences in each tree.
In the tree of the concatenated nucleotide sequences (Fig. 3) the CeMV sequences were distributed in three  In the nucleotide tree for the F gene (Additional file 1) additional available sequences from the early nineties were included. Samples collected in the Atlantic during the 2011-2013 period clustered in the same branch; samples from the nineties clustered in a separate branch, and samples from the Mediterranean from 2007 clustered in a third branch, together with the sample SC/15/ 2007, similarly to the distribution of the concatenated trees. The PWMV was included in a unique branch. All branches were supported with a high PP values.   The nucleotide tree for the P gene (Fig. 5) contained the higher number of sequences (35). One sequence from a guiana dolphin (Sotalia guianensis) collected in 2010 in Brazil appeared to be a distinct strain from the already characterized strains of CeMV (PMV, PWMV and DMV. The two PWMV samples clustered in the same branch and the only PMV included in the tree was isolated from all the other sequences. All these strains were supported by high PP values. The DMV sequences included in this tree were all similar, including sequences from distinct geographic origins, such as Germany, Taiwan or the Mediterranean. Two sequences obtained from white-beaked dolphins in Germany and the

Discussion
In this study we surveyed 279 animals and our results indicate a higher prevalence of DMV among stranded striped dolphins (20.6 %) when compared to stranded common dolphins (1 %) from the Atlantic based populations. Similar results had been previously described in the Mediterranean during the 1990-92 and 2006-08 CeMV breakouts, when striped dolphins presented higher death and stranding rates than other species [2,31,32]. Several theories have been hypothesized for this higher mortality rate amongst striped dolphins in the Mediterranean: they were the most numerous species in the Mediterranean and serological studies suggested that, prior to the 2006-08 outbreak, antibody levels were low in this population rendering them more susceptible to the CeMV infection [19]; also, the fact that they are highly gregarious and tend to live in large pods could contribute to the spread of CeMV infection [33]; high polychlorinated biphenyl (PCB) levels were also detected in the affected animals, leading to the hypothesis that an impaired immune system might have facilitated the infection by CeMV; finally, genetic susceptibility as a result of inbreeding in the Mediterranean population [33], which had already been reported as relatively isolated from the Atlantic populations [34].
Prevalence among striped dolphins from Galicia was 24.2 % while prevalence in striped dolphins stranded in Portugal was 16.7 %. Although this difference was not statistically significant, it is important to highlight that prevalence among striped dolphin samples from Galicia was probably underestimated since only lung samples were tested. Samples from the Portuguese coastline allowed testing several organs and antigen was only detected in brain samples of four individuals out of the 6 positive striped dolphins. It is therefore possible that the prevalence in striped dolphins from Galicia is being strongly underestimated. Previous studies from the Atlantic based populations were performed in the western part of the Atlantic, along the USA coast, and bottlenose dolphins were the most affected cetaceans in that area.
In the Canary Islands a retrospective study was published in 2014 and 6 animals were positive for CeMV (5 striped dolphins and 1 common dolphin) [17]. In this study striped dolphins seem to be the most affected species sampled from the East Atlantic.
In four animals it was not possible to amplify viral genomic fragments by conventional RT-PCR. These samples recorded high CT values in the RT-qPCR, corresponding to a low target copy number (ranging from 105 to 943 copies), which would present a downside using a less sensitive conventional assay. Also, three of the four samples were collected in animals from Galicia originally stored at −20°C, which may possibly imply RNA degradation hampering the amplification of longer genomic fragments, by conventional RT-PCR.
The genetic distances between samples were low among all sequences included in the phylogenetic trees. Nonetheless, PP values were high and consistent in all trees particularly in the DNA concatenated tree, adding robustness to the phylogenetic arrangement.
In the phylogenetic trees for the concatenated nucleotides the grouping of viral sequences followed a temporal arrangement, with samples collected since 2007 forming different clades. When a higher number of sequences was added to the trees (P gene tree) a phylogeographic arrangement becomes clear: all samples from Portugal and Galicia cluster together (with isolates ranging from 2007 to 2014), further away from the samples from the Mediterranean. The only exception seems to be the sequence from the animal SC/15/2007,clustering with samples from the Mediterranean, as well as with samples from the Canary Islands. Even samples from animals stranded in the south of Portugal (Algarve), such as SC/11/2013, clustered separately from samples obtained in the Mediterranean. This suggests that these populations may be relatively isolated from each other, which is supported by previous findings by other authors [34]. It is worth noticing that only one sample from a striped dolphin collected in the Canary Islands clusters closer to the Portuguese and Galician samples. All the other samples from the Canary Islands are closer to Mediterranean samples.
Positive  [2], morbillivirus infection seems to be endemic in the population of striped dolphins from the Atlantic. This correlates to the serological survey conducted in 2011 in which 21.6 % (n = 37) of the analysed cetaceans cross reacted with Canine Distemper Virus antigen in a commercially available ELISA kit (unpublished observations Bento, C.). To date, the harbour porpoise was reported as the most affected species with morbillivirus infection in the north-eastern Atlantic, although infection is probably not endemic considering porpoises' solitary behaviour [2]. Large populations are needed to maintain morbillivirus infections as endemic [19] and although striped dolphin abundance has increased over the last years in the Portuguese Continental coast it is still a rather small population if compared to the common dolphin population (Araújo, H. personal communication). Notwithstanding, evidence suggests an endemic situation rather than an epidemic, since no outbreaks have been detected in the striped dolphin population of the Atlantic. Moreover, positive samples have been detected annually since 2007, indicating that this virus is actively circulating in this population reaching prevalence values as high as 24 % in the Galician samples. In 1999, dolphins stranded along the Atlantic coast of Spain had low antibody titres for CeMV. Considering the results obtained in this study, further serological studies are needed to deepen the knowledge about the epidemiology of this disease in striped dolphins.
Unlike striped dolphins, the prevalence of stranded common dolphins positive for viral antigen is much lower (1 %). The difference in CeMV prevalence between stranded common and striped dolphins needs to be fully assessed and further studies are needed to clarify the virus impact on cetacean populations and why do striped dolphins appear to be more susceptible to DMV infection. New approaches should be considered: viral enrichment and random amplification techniques associated with next generation sequencing could contribute to deepen the knowledge on this virus and its interaction with other pathogens.
Surveys are a unique tool to provide information on viral epidemiology, especially in free-ranging cetaceans.

Conclusion
Our results suggest that DMV infection is endemic in striped dolphin populations of the Eastern Atlantic. Since it was first reported in cetaceans in the early nineties subtle but consistent changes in the reported viral sequences suggest that the Atlantic and the Mediterranean populations are relatively isolated from each other, as suggested by other authors. The prevalence of infection in stranded common dolphins is very low when compared to striped dolphins, and our results are in agreement with previous reports that point to a higher susceptibility of striped dolphins to CeMV. Reasons for differences in susceptibility to this viral infection in different species should be further investigated and serological surveys should also be performed to assess their protection level towards CeMV infection.

Additional files
Additional file 1: Phylogenetic tree for the F gene nucleotidic sequences. Phylogenetic tree generated with the aligned sequences for the F gene, inferred by Bayesian methods. Sequences for the outgroup taxa were retrieved from NCBI for