Molecular characterization of cetacean poxviruses along the coast of mainland Portugal.

Cetacean poxvirus (CePV) is the causative agent of tattoo skin disease (TSD) in dolphins, porpoises and whales, a condition characterized by pinhole, ring-like lesions or generalized tattoo-like skin lesions. This study genetically characterized cetacean poxviruses from stranded animals along mainland Portugal. Samples from skin lesions compatible with TSD were obtained from 4 odontocete species (Delphinus delphis, Stenella coeruleoalba, Phocoena phocoena, and Tursiops truncatus) and analyzed using a conventional PCR assay targeting the DNA polymerase gene partially. Among the positive samples (n = 29, 65.9%), a larger DNA polymerase gene fragment was obtained, allowing a robust phylogenetic analysis. Nineteen samples (43.2%) were successfully amplified and sequenced using Sanger sequencing. By combining 11 of these sequences with those from public databases, a maximum likelihood phylogenetic tree was constructed, revealing high heterogeneity within the group. These findings contribute to a better understanding of the genetic diversity, epidemiology, phylogenetics, and evolution of CePV.

Systematic Determination of Herpesvirus in Free-Ranging Cetaceans Stranded in the Western Mediterranean: Tissue Tropism and Associated Lesions.

The monitoring of herpesvirus infection provides useful information when assessing marine mammals' health. This paper shows the prevalence of herpesvirus infection (80.85%) in 47 cetaceans stranded on the coast of the Valencian Community, Spain. Of the 966 tissues evaluated, 121 tested positive when employing nested-PCR (12.53%). The largest proportion of herpesvirus-positive tissue samples was in the reproductive system, nervous system, and tegument. Herpesvirus was more prevalent in females, juveniles, and calves. More than half the DNA PCR positive tissues contained herpesvirus RNA, indicating the presence of actively replicating virus. This RNA was most frequently found in neonates. Fourteen unique sequences were identified. Most amplified sequences belonged to the Gammaherpesvirinae subfamily, but a greater variation was found in Alphaherpesvirinae sequences. This is the first report of systematic herpesvirus DNA and RNA determination in free-ranging cetaceans. Nine (19.14%) were infected with cetacean morbillivirus and all of them (100%) were coinfected with herpesvirus. Lesions similar to those caused by herpesvirus in other species were observed, mainly in the skin, upper digestive tract, genitalia, and central nervous system. Other lesions were also attributable to concomitant etiologies or were nonspecific. It is necessary to investigate the possible role of herpesvirus infection in those cases.

Molecular fossils illuminate the evolution of retroviruses following a macroevolutionary transition from land to water.

The ancestor of cetaceans underwent a macroevolutionary transition from land to water early in the Eocene Period >50 million years ago. However, little is known about how diverse retroviruses evolved during this shift from terrestrial to aquatic environments. Did retroviruses transition into water accompanying their hosts? Did retroviruses infect cetaceans through cross-species transmission after cetaceans invaded the aquatic environments? Endogenous retroviruses (ERVs) provide important molecular fossils for tracing the evolution of retroviruses during this macroevolutionary transition. Here, we use a phylogenomic approach to study the origin and evolution of ERVs in cetaceans. We identify a total of 8,724 ERVs within the genomes of 25 cetaceans, and phylogenetic analyses suggest these ERVs cluster into 315 independent lineages, each of which represents one or more independent endogenization events. We find that cetacean ERVs originated through two possible routes. 298 ERV lineages may derive from retrovirus endogenization that occurred before or during the transition from land to water of cetaceans, and most of these cetacean ERVs were reaching evolutionary dead-ends. 17 ERV lineages are likely to arise from independent retrovirus endogenization events that occurred after the split of mysticetes and odontocetes, indicating that diverse retroviruses infected cetaceans through cross-species transmission from non-cetacean mammals after the transition to aquatic life of cetaceans. Both integration time and synteny analyses support the recent or ongoing activity of multiple retroviral lineages in cetaceans, some of which proliferated into hundreds of copies within the host genomes. Although ERVs only recorded a proportion of past retroviral infections, our findings illuminate the complex evolution of retroviruses during one of the most marked macroevolutionary transitions in vertebrate history.

Specific capture and whole-genome phylogeography of Dolphin morbillivirus.

Dolphin morbillivirus (DMV) is considered an emerging threat having caused several epidemics worldwide. Only few DMV genomes are publicly available. Here, we report the use of target enrichment directly from cetacean tissues to obtain novel DMV genome sequences, with sequence comparison and phylodynamic analysis. RNA from 15 tissue samples of cetaceans stranded along the Italian and French coasts (2008-2017) was purified and processed using custom probes (by bait hybridization) for target enrichment and sequenced on Illumina MiSeq. Data were mapped against the reference genome, and the novel sequences were aligned to the available genome sequences. The alignment was then used for phylogenetic and phylogeographic analysis using MrBayes and BEAST. We herein report that target enrichment by specific capture may be a successful strategy for whole-genome sequencing of DMV directly from field samples. By this strategy, 14 complete and one partially complete genomes were obtained, with reads mapping to the virus up to 98% and coverage up to 7800X. The phylogenetic tree well discriminated the Mediterranean and the NE-Atlantic strains, circulating in the Mediterranean Sea and causing two different epidemics (2008-2015 and 2014-2017, respectively), with a limited time overlap of the two strains, sharing a common ancestor approximately in 1998.

A novel real-time PCR to detect Cetacean morbillivirus in Atlantic cetaceans.

Cetacean morbillivirus (CeMV, family Paramyxoviridae) is a re-emergent pathogen associated with severe epizootic outbreaks causing high mortality among cetaceans worldwide. Recently, CeMV caused an unusual mortality event of Guiana dolphins (Sotalia guianensis) in Brazil. Partial sequence of the viral phosphoprotein (P) gene showed that the Guiana dolphin morbillivirus (GDMV) might represent a new lineage of CeMV. This study aimed to develop a molecular technique to detect the most common CeMV strains known to circulate in the Atlantic Ocean: GDMV, Dolphin morbillivirus (DMV) and Pilot-whale morbillivirus (PWMV). A sensible real-time reverse transcription polymerase chain reaction (RT-qPCR) method based on intercalating dye, targeting the P gene was described. This assay successfully detected GDMV, PWMV and DMV from field samples. Its performance was compared to a RT-qPCR method that specifically detects GDMV. Both assays had high sensibility and excellent intra- and inter-assay reproducibility. A total of 109 field samples from 32 Guiana dolphins were screened for CeMV by conventional RT-PCR in parallel with the RT-qPCR assay. The detection rate increased from 32% to 60% by use of the novel RT-qPCR. The RT-qPCR assay described herein allows rapid and sensitive detection of Atlantic CeMV strains, and is potentially suitable for screening of CeMV globally.

Viral emergence in marine mammals in the North Pacific may be linked to Arctic sea ice reduction.

Climate change-driven alterations in Arctic environments can influence habitat availability, species distributions and interactions, and the breeding, foraging, and health of marine mammals. Phocine distemper virus (PDV), which has caused extensive mortality in Atlantic seals, was confirmed in sea otters in the North Pacific Ocean in 2004, raising the question of whether reductions in sea ice could increase contact between Arctic and sub-Arctic marine mammals and lead to viral transmission across the Arctic Ocean. Using data on PDV exposure and infection and animal movement in sympatric seal, sea lion, and sea otter species sampled in the North Pacific Ocean from 2001-2016, we investigated the timing of PDV introduction, risk factors associated with PDV emergence, and patterns of transmission following introduction. We identified widespread exposure to and infection with PDV across the North Pacific Ocean beginning in 2003 with a second peak of PDV exposure and infection in 2009; viral transmission across sympatric marine mammal species; and association of PDV exposure and infection with reductions in Arctic sea ice extent. Peaks of PDV exposure and infection following 2003 may reflect additional viral introductions among the diverse marine mammals in the North Pacific Ocean linked to change in Arctic sea ice extent.

Canine and Phocine Distemper Viruses: Global Spread and Genetic Basis of Jumping Species Barriers.

Canine distemper virus (CDV) and phocine distemper (PDV) are closely-related members of the Paramyxoviridae family, genus morbillivirus, in the order Mononegavirales. CDV has a broad host range among carnivores. PDV is thought to be derived from CDV through contact between terrestrial carnivores and seals. PDV has caused extensive mortality in Atlantic seals and other marine mammals, and more recently has spread to the North Pacific Ocean. CDV also infects marine carnivores, and there is evidence of morbillivirus infection of seals and other species in Antarctica. Recently, CDV has spread to felines and other wildlife species in the Serengeti and South Africa. Some CDV vaccines may also have caused wildlife disease. Changes in the virus haemagglutinin (H) protein, particularly the signaling lymphocyte activation molecule (SLAM) receptor binding site, correlate with adaptation to non-canine hosts. Differences in the phosphoprotein (P) gene sequences between disease and non-disease causing CDV strains may relate to pathogenicity in domestic dogs and wildlife. Of most concern are reports of CDV infection and disease in non-human primates raising the possibility of zoonosis. In this article we review the global occurrence of CDV and PDV, and present both historical and genetic information relating to these viruses crossing species barriers.

Marine Morbilliviruses: Diversity and Interaction with Signaling Lymphocyte Activation Molecules.

Epidemiological reports of phocine distemper virus (PDV) and cetacean morbillivirus (CeMV) have accumulated since their discovery nearly 30 years ago. In this review, we focus on the interaction between these marine morbilliviruses and their major cellular receptor, the signaling lymphocyte activation molecule (SLAM). The three-dimensional crystal structure and homology models of SLAMs have demonstrated that 35 residues are important for binding to the morbillivirus hemagglutinin (H) protein and contribute to viral tropism. These 35 residues are essentially conserved among pinnipeds and highly conserved among the Caniformia, suggesting that PDV can infect these animals, but are less conserved among cetaceans. Because CeMV can infect various cetacean species, including toothed and baleen whales, the CeMV-H protein is postulated to have broader specificity to accommodate more divergent SLAM interfaces and may enable the virus to infect seals. In silico analysis of viral H protein and SLAM indicates that each residue of the H protein interacts with multiple residues of SLAM and vice versa. The integration of epidemiological, virological, structural, and computational studies should provide deeper insight into host specificity and switching of marine morbilliviruses.

Comparative histopathologic and viral immunohistochemical studies on CeMV infection among Western Mediterranean, Northeast-Central, and Southwestern Atlantic cetaceans.

Cetacean morbillivirus (CeMV) is a major natural cause of morbidity and mortality in cetaceans worldwide and results in epidemic and endemic fatalities. The pathogenesis of CeMV has not been fully elucidated, and questions remain regarding tissue tropism and the mechanisms of immunosuppression. We compared the histopathologic and viral immunohistochemical features in molecularly confirmed CeMV-infected Guiana dolphins (Sotalia guianensis) from the Southwestern Atlantic (Brazil) and striped dolphins (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus) from the Northeast-Central Atlantic (Canary Islands, Spain) and the Western Mediterranean Sea (Italy). Major emphasis was placed on the central nervous system (CNS), including neuroanatomical distribution of lesions, and the lymphoid system and lung were also examined. Eleven Guiana dolphins, 13 striped dolphins, and 3 bottlenose dolphins were selected by defined criteria. CeMV infections showed a remarkable neurotropism in striped dolphins and bottlenose dolphins, while this was a rare feature in CeMV-infected Guiana dolphins. Neuroanatomical distribution of lesions in dolphins stranded in the Canary Islands revealed a consistent involvement of the cerebrum, thalamus, and cerebellum, followed by caudal brainstem and spinal cord. In most cases, Guiana dolphins had more severe lung lesions. The lymphoid system was involved in all three species, with consistent lymphoid depletion. Multinucleate giant cells/syncytia and characteristic viral inclusion bodies were variably observed in these organs. Overall, there was widespread lymphohistiocytic, epithelial, and neuronal/neuroglial viral antigen immunolabeling with some individual, host species, and CeMV strain differences. Preexisting and opportunistic infections were common, particularly endoparasitism, followed by bacterial, fungal, and viral infections. These results contribute to understanding CeMV infections in susceptible cetacean hosts in relation to factors such as CeMV strains and geographic locations, thereby establishing the basis for future neuro- and immunopathological comparative investigations.

Evolutionary evidence for multi-host transmission of cetacean morbillivirus.

Cetacean morbillivirus (CeMV) has emerged as the pathogen that poses the greatest risk of triggering epizootics in cetacean populations worldwide, and has a high propensity for interspecies transmission, including sporadic infection of seals. In this study, we investigated the evolutionary history of CeMV by deep sequencing wild-type viruses from tissue samples representing cetacean species with different spatiotemporal origins. Bayesian phylogeographic analysis generated an estimated evolutionary rate of 2.34 × 10-4 nucleotide substitutions/site/year and showed that CeMV evolutionary dynamics are neither host-restricted nor location-restricted. Moreover, the dolphin morbillivirus strain of CeMV has undergone purifying selection without evidence of species-specific mutations. Cell-to-cell fusion and growth kinetics assays demonstrated that CeMV can use both dolphin and seal CD150 as a cellular receptor. Thus, it appears that CeMV can readily spread among multiple cetacean populations and may pose an additional spillover risk to seals.

Novel and highly sensitive SYBR® Green real-time pcr for poxvirus detection in odontocete cetaceans.

Poxviruses are emerging pathogens in cetaceans, temporarily named 'Cetaceanpoxvirus' (CePV, family Poxviridae), classified into two main lineages: CePV-1 in odontocetes and CePV-2 in mysticetes. Only a few studies performed the molecular detection of CePVs, based on DNA-polymerase gene and/or DNA-topoisomerase I gene amplification. Herein we describe a new real-time PCR assay based on SYBR® Green and a new primer set to detect a 150 bp fragment of CePV DNA-polymerase gene, also effective for conventional PCR detection. The novel real-time PCR was able to detect 5 up to 5 × 106 copies per reaction of a cloned positive control. Both novel PCR methods were 1000 to 100,000-fold more sensitive than those previously described in the literature. Samples of characteristic poxvirus skin lesions ('tattoo') from one Risso's dolphin (Grampus griseus), two striped dolphins (Stenella coeruleoalba) and two Guiana dolphins (Sotalia guianensis) were all positive to both our novel real time- and conventional PCR methods, even though three of these animals (a Risso's dolphin, a striped dolphin, and a Guiana dolphin) were previously negative to the conventional PCRs previously available. To our knowledge, this is the first real-time PCR detection method for Cetaceanpoxvirus, a much more sensitive tool for the detection of CePV-1 infections.

Transmission of morbilliviruses within and among marine mammal species.

Transmission of morbilliviruses within and among marine mammal species has been documented in a variety of marine habitats. Cetacean morbillivirus spreads between cetacean species in the aquatic environment whereas both phocine distemper virus and canine distemper virus have been associated with transmission within and between pinniped and terrestrial carnivore species in their natural habitat and at the aquatic-terrestrial interface. Periodically these viruses have caused large epizootics involving thousands of animals, due to sustained intra-species virus transmission. Social behavior of host species, marine habitat, geographical barriers and virus-host adaptations all likely contribute toward modulating virus spread. In combination with increased surveillance and whole genome sequencing, further research into ecological and host factors will be pivotal in better understanding the global transmission dynamics of marine morbilliviruses.

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.

A simultaneous diagnosis and genotyping method for global surveillance of cetacean morbillivirus.

Cetacean morbillivirus (CeMV) is considered one of the most important viral pathogens in cetaceans. CeMV outbreaks of lethal disease have repeatedly been observed in Europe, the Americas, and Australia, while large herds of gregarious species were found to be the likely reservoirs and sources of CeMV infection to susceptible species in the Atlantic and Pacific Oceans. Furthermore, three new strains were detected recently in Hawaii, Brazil and Australia. To clarify the real global distribution of CeMV and possible carriers, we showed a novel technique successfully diagnosing and distinguishing different virus strains (DMV, PWMV and novel CeMVs) using FFPE samples from 1996 to 2011. This efficient method that combines qRT-PCR and high resolution melting (HRM) could be applied to the future retrospective global studies for better understanding of different prevalence and outbreak conditions among ocean basins and the mechanism of variable host response to pathogens.

Molecular analysis of dolphin morbillivirus: A new sensitive detection method based on nested RT-PCR.

Cetacean Morbillivirus (CeMV) has been identified as the most pathogenic virus for cetaceans. Over the past three decades, this RNA virus has caused several outbreaks of lethal disease in odontocetes and mysticetes worldwide. Isolation and identification of CeMV RNA is very challenging in whales because of the poor preservation status frequently shown by tissues from stranded animals. Nested reverse transcription polymerase chain reaction (nested RT-PCR) is used instead of conventional RT-PCR when it is necessary to increase the sensitivity and the specificity of the reaction. This study describes a new nested RT-PCR technique useful to amplify small amounts of the cDNA copy of Cetacean morbillivirus (CeMV) when it is present in scant quantity in whales' biological specimens. This technique was used to analyze different tissues (lung, brain, spleen and other lymphoid tissues) from one under human care seal and seven cetaceans stranded along the Italian coastline between October 2011 and September 2015. A well-characterized, 200 base pair (bp) fragment of the dolphin Morbillivirus (DMV) haemagglutinin (H) gene, obtained by nested RT-PCR, was sequenced and used to confirm DMV positivity in all the eight marine mammals under study. In conclusion, this nested RT-PCR protocol can represent a sensitive detection method to identify CeMV-positive, poorly preserved tissue samples. Furthermore, this is also a rather inexpensive molecular technique, relatively easy to apply.

Initial characterization of novel beaked whale morbillivirus in Hawaiian cetaceans.

Cetacean morbillivirus (CeMV) is a causative factor in epizootics that have resulted in thousands of deaths throughout the Atlantic and Mediterranean since 1987, but less is known of its presence and significance in the Pacific. The first case of CeMV reported in Hawai'i was in a Longman's beaked whale that stranded in 2010. The initial CeMV sequence from this individual indicated the possibility of a novel strain. To address this, archived samples from cetaceans that stranded in Hawai'i between 1997 and 2014 were screened for CeMV. The beaked whale morbillivirus (BWMV) was detected in 15 individuals representing 12 different species (24% of Code 1 and 2 stranded cetaceans). The earliest detected case was a humpback whale that stranded in 1998. Sequence comparisons of a 2.2 kb sequence spanning the phosphoprotein (P) and nucleocapsid (N) genes strongly suggest that the BWMV represents a novel strain of CeMV present in Hawai'i and the Central Pacific. In contrast to recently reported isolates from Brazil and Australia that may represent a distinct clade, BWMV appears to be more closely related to known strains of CeMV (dolphin morbillivirus; porpoise morbillivirus; and pilot whale morbillivirus). Detection rates with repeat sampling of positive lymph nodes were between 2 and 61%, illustrating the extreme heterogeneity that can occur in affected tissues. Taken together, these results suggest that BWMV may be common and established in Hawaiian cetacean populations. BWMV will be important for understanding CeMV and health threats in the relatively understudied cetaceans of the Pacific.

Retrospective seroepidemiological investigations against Morbillivirus, Toxoplasma gondii and Brucella spp. in cetaceans stranded along the Italian coastline (1998-2014).

This study reports the results of seroepidemiological investigations carried out against Morbillivirus, Toxoplasma gondii and Brucella spp. on blood serum samples collected from 70 cetacean specimens found stranded along the Italian coastline between 1998 and 2014. A total number of 23 serum samples (32.8%) obtained from Stenella coeruleoalba, Tursiops truncatus, Balaenoptera physalus and Globicephala melas harboured anti-Morbillivirus neutralizing antibodies. Ten sera (16%) collected from S. coeruleoalba and T. truncatus were found positive against T. gondii, while no antibodies against Brucella spp. were found. These data reveal that stranded cetaceans provide a unique opportunity for monitoring the health status of free-ranging animals living in the Mediterranean Sea, in order to investigate the level of exposure of cetacean populations to selected infectious agents representing a serious threat for aquatic mammals.

Identification of Novel Cetacean Poxviruses in Cetaceans Stranded in South West England.

Poxvirus infections in marine mammals have been mainly reported through their clinical lesions and electron microscopy (EM). Poxvirus particles in association with such lesions have been demonstrated by EM and were previously classified as two new viruses, cetacean poxvirus 1 (CePV-1) and cetacean poxvirus 2 (CePV-2). In this study, epidermal pox lesions in cetaceans stranded in South West England (Cornwall) between 2008 and 2012 were investigated by electron microscopy and molecular analysis. PCR and sequencing of a highly conserved region within the viral DNA polymerase gene ruled out both parapox- and orthopoxviruses. Moreover, phylogenetic analysis of the PCR product clustered the sequences with those previously described as cetacean poxviruses. However, taking the close genetic distance of this gene fragment across the family of poxviridae into account, it is reasonable to postulate further, novel cetacean poxvirus species. The nucleotide similarity within each cluster (tentative species) detected ranged from 98.6% to 100%, whilst the similarity between the clusters was no more than 95%. The detection of several species of poxvirus in different cetacean species confirms the likelihood of a heterogeneous cetacean poxvirus genus, comparable to the heterogeneity observed in other poxvirus genera.

Cetacean morbillivirus: current knowledge and future directions.

We review the molecular and epidemiological characteristics of cetacean morbillivirus (CeMV) and the diagnosis and pathogenesis of associated disease, with six different strains detected in cetaceans worldwide. CeMV has caused epidemics with high mortality in odontocetes in Europe, the USA and Australia. It represents a distinct species within the Morbillivirus genus. Although most CeMV strains are phylogenetically closely related, recent data indicate that morbilliviruses recovered from Indo-Pacific bottlenose dolphins (Tursiops aduncus), from Western Australia, and a Guiana dolphin (Sotalia guianensis), from Brazil, are divergent. The signaling lymphocyte activation molecule (SLAM) cell receptor for CeMV has been characterized in cetaceans. It shares higher amino acid identity with the ruminant SLAM than with the receptors of carnivores or humans, reflecting the evolutionary history of these mammalian taxa. In Delphinidae, three amino acid substitutions may result in a higher affinity for the virus. Infection is diagnosed by histology, immunohistochemistry, virus isolation, RT-PCR, and serology. Classical CeMV-associated lesions include bronchointerstitial pneumonia, encephalitis, syncytia, and lymphoid depletion associated with immunosuppression. Cetaceans that survive the acute disease may develop fatal secondary infections and chronic encephalitis. Endemically infected, gregarious odontocetes probably serve as reservoirs and vectors. Transmission likely occurs through the inhalation of aerosolized virus but mother to fetus transmission was also reported.