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.

Comparative sequence analysis of morbillivirus receptors and its implication in host range expansion.

SLAM (CD150) and nectin-4 are the major morbillivirus receptors responsible for virus pathogenesis and host range expansion. Recently, morbillivirus infections have been reported in unnatural hosts, including endangered species, posing a threat to their conservation. To understand the host range expansion of morbilliviruses, we generated the full-length sequences of morbillivirus receptors (goat, sheep, and dog SLAM, and goat nectin-4) and tried to correlate their role in determining host tropism. A high level of amino acid identity was observed between the sequences of related species, and phylogenetic reconstruction showed that the receptor sequences of carnivores, marine mammals, and small ruminants grouped separately. Analysis of the ligand binding region (V region; amino acid residues 52-136) of SLAM revealed high amino acid identity between small ruminants and bovine SLAMs. Comparison of canine SLAM with ruminants and non-canids SLAM revealed appreciable changes, including charge alterations. Significant differences between feline SLAM and canine SLAM have been reported. The binding motifs of nectin-4 genes (FPAG motif and amino acid residues 60, 62, and 63) were found to be conserved in sheep, goat, and dog. The differences reported in the binding region may be responsible for the level of susceptibility or resistance of a species to a particular morbillivirus.

Genetic heterogeneity of dolphin morbilliviruses detected in the Spanish Mediterranean in inter-epizootic period.

BACKGROUND: In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the three dolphin morbillivirus (DMV) epizootics of 1990-1992, 2006-2008 and 2011 that affected Mediterranean striped dolphins (Stenella coeruleoalba). Systemic infection caused by DMV in the Mediterranean has been reported only during these outbreaks. RESULTS: We report the infection of five striped dolphins (Stenella coeruleoalba) stranded on the Spanish Mediterranean coast of Valencia after the last DMV outbreak that ended in 2011. Animal 1 stranded in late 2011 and Animal 2 in 2012. Systemic infection affecting all tissues was found based on histopathology and positive immunohistochemical and polymerase chain reaction positive results. Animal 3 stranded in 2014; molecular and immunohistochemical detection was positive only in the central nervous system. Animals 4 and 5 stranded in 2015, and DMV antigen was found in several tissues. Partial sequences of the DMV phosphoprotein (P), nucleoprotein (N), and hemagglutinin (H) genes were identical for Animals 2, 3, 4, and 5, and were remarkably different from those in Animal 1. The P sequence from Animal 1 was identical to that of the DMV strain that caused the epizootic of 2011 in the Spanish Mediterranean. The corresponding sequence from Animals 2-5 was identical to that from a striped dolphin stranded in 2011 on the Canary Islands and to six dolphins stranded in northeastern Atlantic of the Iberian Peninsula. CONCLUSIONS: These results suggest the existence of an endemic infection cycle among striped dolphins in the Mediterranean that may lead to occasional systemic disease presentations outside epizootic periods. This cycle involves multiple pathogenic viral strains, one of which may have originated in the Atlantic Ocean.

Candidate gene molecular markers as tools for analyzing genetic susceptibility to morbillivirus infection in stranded Cetaceans.

Morbilliviruses, such as Cetacean morbillivirus (CeMV) or Phocine distemper virus (PDV), represent a growing threat for marine mammals on both hemispheres. Because free-ranging animal populations strongly rely on natural resistance mechanisms, innate immunity-related genes and virus cell entry receptor genes may represent key factors involved in susceptibility to CeMV in Cetaceans. Using the next generation sequencing technology, we have sequenced 11 candidate genes in two model species, Stenella coeruleoalba and Phocoena phocoena. Suitable single nucleotide polymorphism markers of potential functional importance, located in genes coding for basigin (BSG, CD147), the signaling lymphocyte activating molecule (SLAMF1), the poliovirus-related receptor-4 (NECTIN4, PVRL4), toll-like receptors 3, 7, 8 (TLR3, TLR7, TLR8), natural resistance-associated macrophage protein (SLC11A1) and natural cytotoxicity triggering receptor 1 (NCR1), were identified in each model species, along with MHC-DQB haplotypes unique for each species. This set of molecular markers represents a potentially useful tool for studying host genetic variation and susceptibility to morbillivirus infection in Cetaceans as well as for studying functionally important genetic diversity of selected Cetacean populations.

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans.

Morbillivirus infection is a severe threat to marine mammals. Mass die-offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain-like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three-dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.

[Entry mechanism of morbillivirus family].

The genus Morbillivirus includes measles virus, canine distemper virus and rinderpest virus. These are highly contagious and exhibit high mortality. These viruses have the attachment glycoprotein, hemagglutinin (H), at the virus surface, which bind to signaling lymphocyte activation molecule (SLAM) and Nectin 4 as receptors for the entry. However, the molecular mechanism for this entry has been limitedly understood. Here we summarize the current topics, (1) newly identified receptor, Nectin 4, (2) crystal structures of H-receptor complexes and (3) detail biochemical studies of the H-F communication for the entry. These provide insight on the mechanism of morbillivirus entry event and furthermore drug developments.

Genetic comparison among dolphin morbillivirus in the 1990-1992 and 2006-2008 Mediterranean outbreaks.

In 1990, dolphin morbillivirus (DMV) killed thousands of striped dolphins in the Mediterranean. Subsequently, the prevalence of the infection declined in this species. In 2006-2008, the virus killed not only numerous striped dolphins but also long-finned pilot whales. All partial sequences of the phosphoprotein and nucleoprotein genes obtained thus far from different host species during the 2006-2008 outbreak show 100% identity, suggesting that a single virus was involved, and these sequences are nearly identical to the 1990 Spanish strain. Here our first objective was to determine the sequence identity between the morbillivirus from the 2006-2008 outbreak and the 1990 Spanish strain by sequencing more extensive genomic regions of strains from one pilot whale and one striped dolphin stranded in 2007. The second objective was to investigate the relationship between the 1990 and 2007 strains by constructing a phylogenetic tree based on the phosphoprotein gene to compare several Cetacean morbilliviruses, and another tree based on the nearly complete genomes of Mediterranean DMV. The third objective was to identify the most variable regions in the DMV genomes. Results showed that the two 2007 Spanish strains were 99.9% identical over 9050 bp and should be considered the same virus, and that this virus is 99.3-99.4% similar to the 1990 Spanish strain. The phylogenetic trees, together with the common geographical area for the two outbreaks, suggest that the 2007 DMV strains evolved from the 1990 DMV strain. Pilot whales do not seem to have been exposed or infected during the 1990-1992 epidemic, since these populations appeared to be immunologically naïve in 2006-2008. Our results suggest that the virus may have evolved in striped dolphin populations prior to the 2006-2008 outbreak, after which it entered the long-finned pilot whale, perhaps aided by an alanine to valine mutation in the N-terminal domain of the fusion protein.

Morbillivirus infection in pilot whales: strict protein requirement drives genetic conservation.

Morbillivirus infection of marine mammals has been documented across all of the world's oceans. Whilst infection is generally demonstrated using a variety of histopathological and serological techniques, where possible, the use of molecular techniques is being used to enable accurate genetic typing of virus strains through sequence analysis. Here, we present genetic data from dolphins and pilot whales affected by morbillivirus infection in the recent outbreak in the Mediterranean Sea during a six-month period from the end of October 2006 to April 2007. To date, very few studies have looked at characterizing outbreaks of morbillivirus infections in whale species at the molecular level. Here, we provide a full sequence for the haemagglutinin (H) gene from material derived from both a dolphin and a pilot whale from the 2007 outbreak in the Mediterranean Sea and show this virus to be 100% identical across the region analysed. Furthermore, we compare partial sequence data from the nucleocapsid (N) gene of the pilot whale material with previously published data and show evidence for strong protein conservation between these different isolates. Finally, we discuss the current classification of cetacean morbilliviruses as a single species.

Histologic, immunohistochemical, and polymerase chain reaction studies of bottlenose dolphins from the 1987-1988 United States Atlantic coast epizootic.

Tissues from 95 bottlenose dolphins (Tursiops truncatus) that died during the 1987-1988 US Atlantic coast epizootic and 11 bottlenose dolphins that died along the Atlantic coast prior to 1987 were examined histologically and immunohistochemically. Polymerase chain reaction (PCR) testing was performed on 36 of the epizootic and all of the pre-1987 cases. Epizootic cases had syncytia and rare intranuclear and intracytoplasmic inclusion bodies within lung, lymph node, and spleen. Lymphoid depletion was present in lymph node, spleen, and gut-associated lymphoid tissue of epizootic cases. Pre-1987 cases did not have these pulmonary and lymphoid lesions. A larger percentage of epizootic than pre-1987 cases had bacterial and/or fungal infections (primarily pneumonias), pulmonary and lymphoid tissue histiocytosis, mucocutaneous ulcers, and evidence of negative energy balance. Immunohistochemically, 49/95 (52%) epizootic dolphins were positive for morbilliviral antigen. Morbilliviral antigen was detected in lung, lymph node, spleen, thymus, skin, tongue, esophagus, liver, pancreas, gastrointestinal tract, urinary bladder, oviduct, and mammary gland by immunohistochemistry. PCR testing identified morbilliviral RNA in 35/36 (97%) epizootic cases tested. Neither morbilliviral antigen nor morbilliviral RNA were detected in pre-1987 cases. Histologic, immunohistochemical, and PCR results provide strong evidence that morbillivirus infection was the primary cause of the 1987-1988 bottlenose dolphin epizootic.

Morbilliviruses and morbillivirus diseases of marine mammals.

In recent years, serious disease outbreaks among seals and dolphins were attributed to infection with established or newly recognized morbilliviruses. The first identification of a morbillivirus as causative agent of mass mortality among marine mammals was in 1988, when the previously unrecognized phocine distemper virus (PDV) caused the death of 20,000 harbor seals (Phoca vitulina) in northwestern Europe. A similar epizootic among Baikal seals (Phoca sibirica) in Siberia in 1987 was later attributed to infection with canine distemper virus (CDV). A morbillivirus isolated from stranded harbor porpoises (Phocoena phocoena) between 1988 and 1990 proved to be yet another new member of the genus Morbillivirus, distinct from PDV and CDV and more closely related to rinderpest virus and peste-des-petits-ruminants virus: porpoise morbillivirus. A similar virus, dolphin morbillivirus, was the primary cause of mass mortality among striped dolphins (Stenella coeruleoalba) in the Mediterranean from 1990 to 1992. In this review, current knowledge of the genetic and antigenic relationships of these viruses is presented, and the origin and epizootiological aspects of the newly discovered morbilliviruses are discussed. In addition, the possible contributory role of environmental contaminant-related immunosuppression in the severity and extent of the different disease outbreaks is discussed.