Genome sequence of Fraser’s dolphin morbillivirus isolated from a stranded Fraser’s dolphin (Lagenodelphis hosei) in Hawaiʻi

Abstract

Over the past three decades, cetacean morbilliviruses (CeMVs) have emerged as significant pathogens affecting cetacean populations worldwide. Here, we report the genome sequence of a new CeMV, designated Fraser’s dolphin morbillivirus (FDMV), isolated from a stranded Fraser’s dolphin (Lagenodelphis hosei) in Hawaiʻi in 2018. Next-generation sequencing facilitated recovery of the nearly complete FDMV genome (15,685 bp), and annotation indicated a genomic organization similar to that of other morbilliviruses, including six open reading frames. Genetic analysis of the FDMV RNA-dependent RNA polymerase gene sequence showed 87–89.6% nucleotide sequence identity compared with other CeMV strains. Phylogenetic analysis demonstrated that FDMV is a distinct member of the genus Morbillivirus, branching as the most basal member of the CeMV clade, which may represent a new species.

Body text

Cetacean morbilliviruses (CeMVs) are enveloped, negative-sense RNA viruses classified within the family Paramyxoviridae [1]. The genus Morbillivirus comprises ten recognized species, including M. ceti, within which several CeMV strains have been identified: dolphin morbillivirus (DMV), porpoise morbillivirus (PMV), pilot whale morbillivirus (PWMV), and beaked whale morbillivirus (BWMV), as well as two more divergent strains detected in Indo-Pacific bottlenose dolphins (Tursiops aduncus) and Guiana dolphins (Sotalia guianensis), further expanding the recognized genetic diversity and host range of CeMVs worldwide [1–5]. These strains infect a wide range of cetacean species [2], as well as other marine mammals, including Eurasian otters (Lutra lutra) and common seals (Phoca vitulina) [6, 7]. CeMV infection can result in severe immunosuppression through lymphoid depletion, increasing susceptibility to secondary viral, bacterial, fungal, and parasitic infections and thereby exacerbating disease severity and mortality [2]. At the population level, these pathogenic effects have translated into substantial morbidity and mortality during epizootic events, with the first documented outbreak of M. ceti resulting in the deaths of hundreds of bottlenose dolphins (Tursiops truncatus) along the Atlantic coast of the United States between 1987 and 1988 [8]. Additional epizootic events associated with CeMV strains have been reported in the Gulf of Mexico, southeastern Brazil, the coast of South Australia, and the Mediterranean Sea [9–13].

On January 17, 2018, a stranded Fraser’s dolphin (Lagenodelphis hosei) was found dead on a beach near Olowalu, Maui [14]. The carcass was subsequently retrieved and transported to Oʻahu for necropsy at the University of Hawaiʻi Health and Stranding Laboratory. Histopathological analysis revealed intranuclear inclusions within the bile duct epithelium, lymphoid depletion, rare syncytial cells in lymph nodes, and non-suppurative meningitis. Cerebellum tissue was processed for virus isolation using Vero.DogSLAMtag cells, as previously described [14]. After cytopathic effects were observed (i.e., plaques containing numerous multinucleated giant cells), viral RNA was extracted from the spent Vero.DogSLAMtag cell growth medium using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The extracted RNA then served as the template for construction of a cDNA sequencing library using a NEBNext Ultra II RNA Library Prep Kit (New England Biolabs).

Next-generation sequencing was performed on a MiSeq sequencer (Illumina) using a v3 chemistry 600-cycle kit, yielding 3,210,830 paired-end reads. These data were analyzed using default parameters in QIAGEN CLC Genomics Workbench version 24.0 (https://digitalinsights.qiagen.com), unless otherwise noted. Reads were trimmed, and the resulting 3,210,770 reads were de novo assembled, producing 40 contigs. BLASTN analysis of the contigs against the NCBI nr/nt database revealed that the largest contig (15,685 bp; %GC 41.8) displayed the highest nucleotide identity (73.83%) to Morbillivirus ceti isolate MQ989 (GenBank accession number PV298625). The integrity of this contig, representing most of the genomic sequence, including the coding-complete sequence of Fraser’s dolphin morbillivirus (FDMV), was verified by visual inspection of the alignment of mapped reads to the contig, resulting in an average coverage depth of 43 reads/nt. Annotation of the FDMV open reading frames was performed using the Genome Annotation Transfer Utility (GATU; https://4virology.net/virology-ca-tools/gatu/) with the M. ceti isolate DK/16 genome used as the reference sequence. The FDMV genome sequence is available in GenBank (accession number PX396315), and the sequence reads are available under BioProject accession number PRJNA1427737. The expected six open reading frames were predicted, encoding the nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H), and RNA-dependent RNA polymerase (L) (Fig. 1A). Figure 2A illustrates the genomic features of FDMV, including nucleotide (nt) positions and basic characteristics of the deduced proteins.

Fig. 1.

(A) Genome organization (3′-to-5′) of FDMV (GenBank accession number PX396315). The genes are shown as colored arrows that are drawn to scale. Abbreviations: N = nucleocapsid; P = phosphoprotein; M = matrix; F = fusion; H = hemagglutinin; L = RNA-dependent RNA polymerase. For the P gene, the smaller green arrows represent the region of C protein (leaky scanning) and V protein (RNA editing). (B) Phylogenetic analysis comparison of the complete L gene amino acid sequences of the Fraser’s dolphin morbillivirus (bold) from Fraser’s dolphin (Lagenodelphis hosei) to representatives of the genus Morbillivirus. The branch lengths represent the number of inferred substitutions as indicated by the scale. Abbreviations: DMV = dolphin morbillivirus; PMV = porpoise morbillivirus; PWMV = pilot whale morbillivirus; GDMV = Guiana dolphin morbillivirus; FDMV = Fraser’s dolphin morbillivirus; MV = measles virus; RP = rinderpest virus; PPRV = peste-des-petits-ruminants virus; PDV = phocine distemper virus; CDV = canine distemper virus; MM = Myotis bat morbillivirus; MS = porcine morbillivirus; MP = Phyllostomus bat morbillivirus; FMV = feline morbillivirus

Fig. 2.

(A) Genomic features and characteristics of the deduced proteins of FDMV (GenBank accession number PX396315). (B) Genetic comparison of the complete RNA-dependent RNA polymerase (L) gene of the novel FDMV from Fraser’s dolphin (Lagenodelphis hosei) to representatives of the genus Morbillivirus. Values are expressed as a percentage of nucleotide identity. Abbreviations: DMV = dolphin morbillivirus; PMV = porpoise morbillivirus; PWMV = pilot whale morbillivirus; GDMV = Guiana dolphin morbillivirus; FDMV = Fraser’s dolphin morbillivirus; MV = measles virus; RP = rinderpest virus; PPRV = peste-des-petits-ruminants virus; PDV = phocine distemper virus; CDV = canine distemper virus; MM = Myotis bat morbillivirus; MS = porcine morbillivirus; MP = Phyllostomus bat morbillivirus; FMV = feline morbillivirus

Phylogenetic analysis included 41 complete L gene amino acid sequences from members of the genus Morbillivirus selected from the National Center for Biotechnology Information (NCBI) GenBank database. Sequences were aligned using ClustalW, with gap opening and extension penalties set to 5 and 1, respectively. Phylogenetic relationships were inferred using the maximum-likelihood method with the best-fit amino acid substitution model (JTT + F+G + I) implemented in MEGA12 [15]. The same sequences were used to generate a sequence identity matrix from their nucleotide (nt) sequences using the Sequence Demarcation Tool v1.3 [16]. Currently, the International Committee on Taxonomy of Viruses (ICTV) defines species demarcation within the family Paramyxoviridae based on evolutionary distances derived from complete L protein amino acid sequences in phylogenetic trees, with distinct species characterized by branch lengths > 0.03.

The branch length of FDMV (0.11) is greater than that observed for other cetacean morbilliviruses (CeMVs), which range from 0.01 to 0.03 (Fig. 1B). Notably, Guiana dolphin morbillivirus (GDMV) exhibits a branch length of 0.07 and has been suggested by Groch et al. [17] to be a distinct species from M. ceti. Genetic analysis indicated that FDMV shares 87–89.6% nucleotide sequence identity with other CeMV strains in comparison to the L gene (Fig. 2B). Based on these findings, FDMV may also represent a new species in the genus Morbillivirus. Phylogenetic analysis further supports FDMV as the most basal member of the CeMV clade (Fig. 1B), consistent with a unique evolutionary lineage.

The phylogenetic placement of FDMV as the most basal member of the CeMV clade provides important insight into the evolutionary history and expanding diversity of morbilliviruses infecting cetaceans. Given the wide geographic range of many CeMV strains, resulting from the migratory behavior of infected cetaceans, future work is needed to determine whether FDMV is restricted to Hawaiian Fraser’s dolphins or occurs throughout the species’ range. While many CeMV strains can infect a variety of marine mammal species, the host range of FDMV remains unknown. Interestingly, cell-to-cell fusion and growth kinetics assays have demonstrated that CeMV strains can utilize either dolphin or seal CD150 as cellular receptors [18]. This suggests that spillover events into Hawaiʻi’s unique marine mammals, such as endangered Hawaiian monk seals or insular false killer whales (Pseudorca crassidens), recently estimated at a population size of 139 individuals, may be possible [19].

Two studies [20, 21] on Mediterranean cetaceans described DMV dynamics in Italian waters, reporting the introduction of a new strain (NE Atlantic DMV) into the population since 2012. The emergence of this strain in immunologically naive cetaceans likely triggered epidemic activity, facilitated by the high population density and gregarious behavior of striped dolphins (Stenella coeruleoalba) in the region. This illustrates how movement of infected hosts between regions can introduce novel viral variants and shape local CeMV diversity. A similar mechanism may explain the presence and diversification of CeMV strains detected in Hawaiʻi, where oceanic connectivity and movements of pelagic cetaceans could allow periodic viral introductions followed by local circulation within susceptible populations. In addition to FDMV, the BWMV strain is known to circulate among Hawaiian cetacean species [3] and may represent a comparable scenario of regional introduction and persistence, analogous to the Mediterranean. However, because a complete genome sequence for BWMV is not yet available, its evolutionary relationships, origin, and degree of regional adaptation remain uncertain. Such processes could contribute to the emergence of genetically distinct regional strains, consistent with the pronounced divergence and basal phylogenetic placement observed for FDMV.

Groch et al. [17] classified CeMV strains, based on partial P gene sequences, into two lineages: CeMV-1, which includes DMV, PMV, PWMV, and BWMV, and CeMV-2, which includes GDMV and the Indo-Pacific dolphin morbillivirus (IPDMV). Considering its phylogenetic placement and divergence, FDMV could potentially be considered a candidate for a new lineage, tentatively referred to as CeMV-3. However, formal classification will require further genomic and biological characterization, including the availability of complete genome sequences for IPDMV and BWMV. These findings underscore the distinctness of FDMV within the CeMV clade, suggesting that it may represent a novel Morbillivirus species.

Author contributions

Thomas B. Waltzek, Kristi West and John A. Lednicky contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nelmarie Landrau-Giovannetti. The first draft of the manuscript was written by Nelmarie Landrau-Giovannetti and all authors commented on previous versions of the manuscript. All authors read and approved of the final manuscript.

Funding

This project was funded by the NSF PRFB Award 2209115, the US Commander Pacific Fleet Environmental Readiness Division, and funding for stranding response and necropsy operational support was received from the NOAA John H. Prescott Marine Mammal Rescue Assistance Program.

Declarations

Competing interests

All of the authors declare that there is no conflict of interest.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.