Dolphin Morbillivirus Research Articles

A DNA vaccine against dolphin morbillivirus is immunogenic in bottlenose dolphins

The immunization of exotic species presents considerable challenges. Nevertheless, for facilities like zoos, animal parks, government facilities and non-profit conservation groups, the protection of valuable and endangered species from infectious disease is a growing concern. The rationale for immunization in these species parallels that for human and companion animals; to decrease the incidence of disease. The U.S. Navy Marine Mammal Program, in collaboration with industry and academic partners, has developed and evaluated a DNA vaccine targeting a marine viral pathogen – dolphin morbillivirus (DMV). The DMV vaccine consists of the fusion (F) and hemagglutinin (H) genes of DMV. Vaccine constructs (pVR-DMV-F and pVR-DMV-H) were evaluated for expression in vitro and then for immunogenicity in mice. Injection protocols were designed for application in Atlantic bottlenose dolphins ( Tursiops truncatus) to balance vaccine effectiveness with clinical utility. Six dolphins were inoculated, four animals received both pDMV-F and pDMV-H and two animals received a mock vaccine (vector alone). All animals received an inoculation week 0, followed by two booster injections weeks 8 and 14. Vaccine-specific immune responses were documented in all four vaccinated animals. To our knowledge, this is the first report of pathogen-specific immunogenicity to a DNA vaccine in an aquatic mammal species.

Involvement of morbilliviruses in the pathogenesis of demyelinating disease

Two members of the morbillivirus genus of the family Paramyxoviridae, canine distemper virus (CDV) and measles virus (MV), are well-known for their ability to cause a chronic demyelinating disease of the CNS in their natural hosts, dogs and humans, respectively. Both viruses have been studied for their potential involvement in the neuropathogenesis of the human demyelinating disease multiple sclerosis (MS). Recently, three new members of the morbillivirus genus, phocine distemper virus (PDV), porpoise morbillivirus (PMV) and dolphin morbillivirus (DMV), have been discovered. These viruses have also been shown to induce multifocal demyelinating disease in infected animals. This review focuses on morbillivirus-induced neuropathologies with emphasis on aetiopathogenesis of CNS demyelination. The possible involvement of a morbillivirus in the pathogenesis of multiple sclerosis is discussed.

Does Human Proximity Affect Antibody Prevalence in Marine-Foraging River Otters (Lontra canadensis)?

The investigation of diseases of free-ranging river otters (Lontra canadensis) is a primary conservation priority for this species; however, very little is known about diseases of river otters that forage in marine environments. To identify and better understand pathogens that could be important to marine-foraging river otters, other wildlife species, domestic animals, and humans and to determine if proximity to human population could be a factor in disease exposure, serum samples from 55 free-ranging marine-foraging river otters were tested for antibodies to selected pathogens. Thirty-five animals were captured in Prince William Sound, Alaska (USA), an area of low human density, and 20 were captured in the San Juan Islands, Washington State (USA), an area characterized by higher human density. Of 40 river otters tested by indirect immunofluorescent antibody test, 17.5% were seropositive (titer > or =320) for Toxoplasma gondii. All positive animals came from Washington. Of 35 river otters tested for antibodies to Leptospira interrogans using the microscopic agglutination test, 10 of 20 (50%) from Washington were seropositive (titer > or =200). None of the 15 tested animals from Alaska were positive. Antibodies to Neospora caninum (n=40), Sarcocystis neurona (n=40), Brucella abortus (n=55), avian influenza (n=40), canine distemper virus (n=55), phocine distemper virus (n=55), dolphin morbillivirus (n=55), porpoise morbillivirus (n=55), and Aleutian disease parvovirus (n=46) were not detected. Identifying exposure to T. gondii and L. interrogans in otters from Washington State but not in otters from Alaska suggests that living proximal to higher human density and its associated agricultural activities, domestic animals, and rodent populations could enhance river otter exposure to these pathogens.

Morbilliviral infection in a pygmy sperm whale ( Kogia breviceps) from Taiwanese waters

Morbilliviral infection was diagnosed in an adult male pygmy sperm whale ( Kogia breviceps) from southwestern Taiwan on the basis of pathological findings, immunohistochemical staining, and reverse transcription-polymerase chain reaction. The whale was found alive stranded on the beach and died after 5 days of medical care. It was thin and had dozens of nematode in the first stomach. The lungs were dark red and heavy. Histopathological examination revealed diffuse, moderate bronchointerstitial pneumonia. Intranuclear and intracytoplasmic inclusions with occasional syncytial cell formation were noted in the lungs, lymph nodes, and spleen. The RNA extracted from lung tissue was subjected to morbilliviral gene amplification. After priming with specific oligonucleotides, the cDNA covering the phosphoprotein (P) gene was copied and then amplified by PCR. The gene fragment amplified from the lung tissue was sequenced. Phylogenetic analysis of partial P gene revealed 97.6% sequence identity to the dolphin morbillivirus and 90.2% similarity to the pilot whale morbillivirus. Morbilliviral antigens were detected in the lungs, lymph nodes, and spleen by immunohistochemistry using polyclonal antibody against rinderpest virus. This is the first report of morbilliviral infection with genetic evidence in a pygmy sperm whale from the Western Pacific Ocean around Taiwan.

Full genome sequence of peste des petits ruminants virus, a member of the Morbillivirus genus

Peste des petits ruminants virus (PPRV) causes an acute febrile illness in small ruminant species, mostly sheep and goats. PPRV is a member of the Morbillivirus genus which includes measles, rinderpest (cattle plague), canine distemper, phocine distemper and the morbilliviruses found in whales, porpoises and dolphins. Full length genome sequences for these morbilliviruses are available and reverse genetic rescue systems have been developed for the viruses of terrestrial mammals, with the exception of PPRV. This paper presents the first published full length genome sequence for PPRV. The genome was found to be consistent with the rule-of-six and open reading frames (ORFs) were identified that encoded the eight proteins characteristic of morbilliviruses. At the nucleotide (nt) level, the full length genome of PPRV was most similar to that of rinderpest, the other ruminant morbillivirus. However, at the protein level five of the six structural proteins and the V protein showed a greater similarity to the dolphin morbillivirus (DMV) while only the C and L proteins showed a high relationship to rinderpest.

Morbillivirus Infections in Aquatic Mammals: A Brief Overview

Since 1987, at least eight morbillivirus infection (MI) epidemics have caused mass mortality of several free-living pinniped and cetacean populations around the world. The responsible agents, all belonging to the genus Morbillivirus (family Paramyxoviridae), have been characterized as either "canine distemper virus" strains, infecting pinnipeds, or as three new morbilliviruses, namely "phocid (phocine) distemper virus" , "porpoise morbillivirus" and "dolphin morbillivirus" . The last two agents are currently gathered under the common denomination of "cetacean morbillivirus". At post-mortem examination, a commonly occurring macroscopic lesion is represented by more or less severe bilateral pneumonia, with consolidation, congestion and oedema of both lungs, which fail to collapse. Histologically, a non-suppurative broncho-interstitial pneumonia, characterized by type II pneumocyte hyperplasia and by formation of endobronchial, endobronchiolar and endoalveolar "Warthin-Finkeldey type" syncytia, as well as a multifocal, non-suppurative encephalitis, associated with a severe and generalized lymphoid tissue depletion, are common pathological findings. Furthermore, eosinophilic viral inclusions are often detected, at both the intracytoplasmic and intranuclear level, within bronchial and bronchiolar epithelial, pulmonary syncytial, neuronal and other cell types. These inclusions, along with lymphoid and other cellular elements, are often found to be immunohistochemically positive for morbillivirus antigen. Among the still debated, or even controversial issues regarding MI in sea mammals, the one related to the origin of their causative agents is of particular concern. Another intriguing issue regards the synergistic effects, if any, associated with chronic exposure to a number of environmental pollutants, such as organochlorines and heavy metals. In fact, it is also unknown whether and how these chemicals contribute towards modulating the pathogenic and pathogenetic activity primarily displayed by sea mammal morbilliviruses.

Cetacean morbilliviruses are phylogenetically divergent

We performed a phylogenetic comparison of porpoise morbillivirus (PMV) and dolphin morbillivirus (DMV) isolates from porpoises and dolphins respectively according to criteria adopted by the World Health Organization for the phylogenetic comparison of measles viruses. PMV and DMV were more divergent than the most distantly related measles virus strains, thus challenging the classification of PMV and DMV as two strains of a single species, cetacean morbillivirus.

Sequence analysis of the phosphoprotein gene of peste des petits ruminants (PPR) virus: editing of the gene transcript

The gene encoding the phosphoprotein of the vaccine strain of Peste des petits ruminants (PPR) virus (Nigeria 75/1 vaccine strain) has been cloned and its nucleotide sequence been determined. This gene is 1655 nucleotides long and encodes two overlapping open reading frames (ORFs). Translation from the first AUG would produce a polypeptide of 509 amino acid residues with a predicted molecular mass of 54.9 kDa, the longest of the published morbillivirus P proteins. Translation from the second AUG would produce a protein of 177 amino acid residues with a predicted molecular mass of 20.3 kDa, analogous to the C proteins of other morbilliviruses. Evidence was found for the production of two types of P mRNA transcript, one a faithful transcript of the gene and the other with an extra G residue inserted at position 751. Translation from the first AUG of this second mRNA would produce a protein of 298 amino acids, with a predicted molecular mass 32.3 kDa, analogous to the V protein produced by other morbilliviruses. Sequences of the predicted P, C and V proteins were compared with those of the other morbillivirus sequences available to date. The P protein was found to be the most poorly conserved of the morbillivirus proteins, the amino acid identity ranging from 54% in case of Canine distemper virus (CDV) to 60% in the case of the Dolphin morbillivirus (DMV).

Changes in the receptorbinding haemagglutinin protein of wild-type morbilliviruses are not required for adaptation to Vero cells.

We examined the consequences of isolation and adaptation to Vero cells for the receptorbinding haemagglutinin (H) gene of four syncytia-forming isolates of canine distemper virus (CDV) and of a dolphin morbillivirus isolate. A Vero-adapted CDV isolate exhibited biased hypermutation, since 11 out of 12 nucleotide differences to other isolates from the same epidemic were U-C transitions. Most of these transitions appeared to have taken place during in vitro cultivation. Previously, biased hypermutation in morbilliviruses has almost exclusively been described for subacute sclerosing panencephalitis and measles inclusion body encephalitis, which are rare measles virus brain infections. Amino acid changes in the H proteins were not required for Vero cell adaptation, suggesting that Vero cells express receptors for wild-type morbilliviruses. This strongly indicate the existence of other morbillivirus receptors than CD46 and CDw150.

Rinderpest and peste des petits ruminants viruses exhibit neurovirulence in mice.

Members of the morbillivirus genus, canine distemper (CDV), phocine distemper virus (PDV), and the cetacean viruses of dolphins and porpoises exhibit high levels of CNS infection in their natural hosts. CNS complications are rare for measles virus (MV) and are not associated with rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) infection. However, it is possible that all morbilliviruses infect the CNS but in some hosts are rapidly cleared by the immune response. In this study, we assessed whether RPV and PPRV have the potential to be neurovirulent. We describe the outcome of infection, of selected mouse strains, with isolates of RPV, PPRV, PDV, porpoise morbillivirus (PMV), dolphin morbillivirus (DMV), and a wild-type strain of MV. In the case of RPV virus, strains with different passage histories have been examined. The results of experiments with these viruses were compared with those using neuroadapted and vaccine strains of MV, which acted as positive and negative controls respectively. Intracerebral inoculation with RPV (Saudi/81) and PPRV (Nigeria75/1) strains produced infection in Balb/C and Cd1, but not C57 suckling mice, whereas the CAM/RB rodent-adapted strain of MV infected all three strains of mice. Weanling mice were only infected by CAM/RB. Intranasal and intraperitoneal inoculation failed to produce infection with any virus strains. We have shown that, both RPV and PPRV, in common with other morbilliviruses are neurovirulent in a permissive system. Transient infection of the CNS of cattle and goats with RPV and PPRV, respectively, remains a possibility, which could provide relevant models for the initial stages of MV infection in humans.

Molecular Evolution of Viral Fusion and Matrix Protein Genes and Phylogenetic Relationships among the Paramyxoviridae

Phylogenetic relationships among the Paramyxoviridae, a broad family of viruses whose members cause devastating diseases of wildlife, livestock, and humans, were examined with both fusion (F) and matrix (M) protein-coding sequences. Neighbor-joining trees of F and M protein sequences showed that the Paramyxoviridae was divided into the two traditionally recognized subfamilies, the Paramyxovirinae and the Pneumovirinae. Within the Paramyxovirinae, the results also showed groups corresponding to three currently recognized genera: Respirovirus, Morbillivirus, and Rubulavirus. The relationships among the three genera of the Paramyxovirinae were resolved with M protein sequences and there was significant bootstrap support (100%) showing that members of the genus Respirovirus and the genus Morbillivirus were more closely related to each other than to members of the genus Rubulavirus. Both F and M phylogenies showed that Newcastle disease virus (NDV) was more closely related to the genus Rubulavirus than to the other two genera but were consistent with the proposal (B. S. Seal et al., 2000, Virus Res. 66, 1–11) that NDV be classified as a separate genus within the Paramyxovirinae. Both F and M phylogenies were also consistent with the proposal (L. Wang et al., 2000, J. Virol 74, 9972–9979) that Hendra virus be classified as a new genus closely related and basal to the genus Morbillivirus. Rinderpest was most closely related to measles and a more derived virus than to canine distemper virus, phocine distemper virus, or dolphin morbillivirus.

An insight into the epidemiology of dolphin morbillivirus worldwide

Serum samples from 288 cetaceans representing 25 species and originating from 11 different countries were collected between 1995 and 1999 and examined for the presence of dolphin morbillivirus (DMV)-specific antibodies by an indirect ELISA (iELISA) ( N=267) or a plaque reduction assay ( N=21). A total of 35 odontocetes were seropositive: three harbour porpoises ( Phocoena phocoena) and a common dolphin ( Delphinus delphis) from the Northeastern (NE) Atlantic, a bottlenose dolphin ( Tursiops truncatus) from Kent (England), three striped dolphins ( Stenella coeruleoalba), two Risso’s dolphins ( Grampus griseus) and a bottlenose dolphin from the Mediterranean Sea, one common dolphin from the Southwest (SW) Indian Ocean, three Fraser’s dolphins ( Lagenodelphis hosei) from the SW Atlantic, 18 long-finned pilot whales ( Globicephala melas) and a bottlenose dolphin from the SW Pacific as well as a captive bottlenose dolphin ( Tursiops aduncus) originally from Taiwan. The presence of morbillivirus antibodies in 17 of these animals was further examined in other iELISAs and virus neutralization tests. Our results indicate that DMV infects cetaceans worldwide. This is the first report of DMV-seropositive animals from the SW Indian, SW Atlantic and West Pacific Oceans. Prevalence of DMV-seropositives was 85.7% in 21 pilot whales from the SW Pacific and both sexually mature and immature individuals were infected. This indicates that DMV is endemic in these animals. The same situation may occur among Fraser’s dolphins from the SW Atlantic. The prevalence of DMV-seropositives was 5.26% and 5.36% in 19 common dolphins and 56 harbour porpoise from the NE Atlantic, respectively, and 18.75% in 16 striped dolphins from the Mediterranean. Prevalence varied significantly with sexual maturity in harbour porpoises and striped dolphins; all DMV-seropositives being mature animals. The prevalence of seropositive harbour porpoise and striped dolphins appeared to have decreased since previous studies. These data suggest that DMV is not endemic within these populations, that they are losing their humoral immunity against the virus and that they may be vulnerable to new epidemics.

Morbillivirus ecology in polar bears ( Ursus maritimus )

Polar bear (Ursus maritimus) morbillivirus infection was initially reported by Follmann and co-workers in 1996, based upon serologic results using canine distemper virus (CDV). The impetus for the evaluation of polar bear populations for morbillivirus infections was prompted by epidemics of canine distemper-like disease in seal populations in the north Atlantic regions of Greenland, Europe, and Russia. Since marine morbilliviruses have been further characterized into three major species, phocine distemper virus (PDV), dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV), it was of value to determine the origin of the polar bear infection. One hundred serum samples were selected from a group of sera collected from regions of Alaska and Russia and tested by differential serum neutralization assay against the three major marine morbilliviruses and CDV, to determine the predominant virus infecting the polar bear. Polar bears had higher serum antibody titers to CDV than they did to PDV, DMV, and PMV. These data suggest that polar bears are being infected with a morbillivirus of terrestrial origin. Furthermore, based on the high serum antibody prevalence in the population, the virus may be indigenous to the polar bear and not necessarily the result of interspecies transmission from other arctic mammals susceptible to CDV and/or marine morbilliviruses.

Identification of morbilliviruses of probable cetacean origin in carcases of Mediterranean monk seals (Monachus monachus)

Two morbilliviruses were isolated from carcases of Mediterranean monk seals (Monachus monachus) which had died in coastal areas of Greece and Mauritania. They were characterised as being closely related to...

Molecular genetic evidence of a novel morbillivirus in a long-finned pilot whale (Globicephalus melas).

A long-finned pilot whale with morbilliviral disease was stranded in New Jersey. An immunohistochemical stain demonstrated morbilliviral antigen. Reverse transcriptase-polymerase chain reaction for morbillivirus P and N genes was positive. Novel sequences most closely related to, but distinct from, those of dolphin and porpoise morbilliviruses suggest that this virus may represent a third member of the cetacean morbillivirus group.

The matrix protein gene sequence analysis reveals close relationship between peste des petits ruminants virus (PPRV) and dolphin morbillivirus

The gene encoding the matrix protein of peste des petits ruminants virus (PPRV) has been cloned and its nucleotide sequence determined. This gene is 1466 nucleotides long and contains an open reading frame (ORF) capable of encoding a basic protein of 335 amino acid residues with a predicted molecular weight of 38 057 Da. This ORF starts at position 33–35 and ends with the codon TAA at position 1038–1040 thus leaving a long untranslated region (426 nucleotides) at the 3′ end of the messenger RNA. This fragment is very G/C rich (68.5%) and in contrast to the ORF region appears to be least conserved in the M gene sequence of the morbilliviruses. A comparison of the PPRV M protein with those of other viruses in the group confirms the previously noted high degree of conservation for this protein sequence. The percent of identity within the group ranges from 76.7 to 86.9%, the highest being with the dolphin morbillivirus matrix protein.

Morbillivirus downregulation of CD46.

There is evidence that CD46 (membrane cofactor protein) is a cellular receptor for vaccine and laboratory-passaged strains of measles virus (MV). Following infection with these MV strains, CD46 is downregulated from the cell surface, and consequent complement-mediated lysis has been shown to occur upon infection of a human monocytic cell line. The MV hemagglutinin (H) protein alone is capable of inducing this downregulation. Some wild-type strains of MV fail to downregulate CD46, despite infection being prevented by anti-CD46 antibodies. In this study we show that CD46 is also downregulated to the same extent by wild-type, vaccine, and laboratory-passaged strains of rinderpest virus (RPV), although CD46 did not appear to be the receptor for RPV. Expression of the RPV H protein by a nonreplicating adenovirus vector was also found to cause this downregulation. A vaccine strain of peste des petits ruminants virus caused slight downregulation of CD46 in infected Vero cells, while wild-type and vaccine strains of canine distemper virus and a wild-type strain of dolphin morbillivirus failed to downregulate CD46. Downregulation of CD46 can, therefore, be a function independent of the use of this protein as a virus receptor.

Morbillivirus infection in stranded common dolphins from the Pacific Ocean.

From August 1995 to August 1997, six of 18 common dolphins (Delphinus delphis) that stranded along beaches of southern California (USA) tested antibody positive for dolphin morbillivirus (DMV). Titers ascertained by virus neutralization ranged from 1:50 to 1:910 while those determined by ELISA ranged from 1:80 to 1:195. The first individual to strand survived and was released back into the Pacific Ocean 14 mo later. Histopathologic examination of tissues from the other five dolphins did not reveal lesions characteristic of morbilliviral disease; however, morbilliviral RNA was detected in three of the five by reverse transcriptase-polymerase chain reaction testing. This is the first report of morbilliviral infection in any marine mammal species in the northern hemisphere of the Pacific Ocean. These data indicate that DMV, or a closely related morbillivirus, is present in the Pacific Ocean and infection of common dolphins may not be associated with morbillivirus disease.

Monk seal mortality: virus or toxin?

During the past few months, more than half of the total population of about 300 highly endangered Mediterranean monk seals ( Monachus monachus) on the western Saharan coast of Africa, died in a mysterious disease outbreak 1. Epizootiological and postmortem findings were reminiscent of similar outbreaks amongst pinniped and cetacean species in recent years, which were caused by an infection with newly discovered morbilliviruses (for review see Osterhaus et al. 2). Virological, as well as toxicological, analysis performed on tissue samples collected from relatively fresh carcasses during the outbreak indicate that infection with a virus closely related to dolphin morbillivirus (DMV), possibly originating from affected dolphins in the same area, was the primary cause of the outbreak. Therefore it is concluded that vaccination with a safe and effective non-replicating vaccine should be considered as a management tool in the conservation of Mediterranean monk seals.

Serological evidence of morbillivirus infection in small cetaceans from the Southeast Pacific

The presence of morbillivirus-specific serum antibodies was examined by an indirect enzyme linked immunosorbent assay (iELISA) and virus neutralization tests in serum samples from 30 dusky dolphins ( Lagenorhynchus obscurus), 8 long-snouted common dolphins ( Delphinus capensis), 2 inshore and 6 offshore bottlenose dolphins ( Tursiops truncatus) and 20 Burmeister's porpoises ( Phocoena spinipinnis) taken in fisheries off central Peru in 1993–1995. The sera from six dusky dolphins, one common dolphin and three offshore bottlenose dolphins were positive on a coat of dolphin morbillivirus (DMV) antigen in the iELISA. Several of these sera were also positive when tested against peste des petits ruminants and rinderpest virus antigen. Porpoise morbillivirus and/or DMV neutralizing antibodies were detected in the sera of two bottlenose and three dusky dolphins that reacted positively with DMV antigen in iELISA and also in the sera of one common, one dusky and one bottlenose dolphin that were negative in the iELISA. These results strongly suggest that viruses closely related, or identical, to the cetacean morbillivirus present in the North Atlantic and the Mediterranean Sea infect several species of Delphinidae of the Southeastern Pacific. No convincing morbillivirus-specific antibody positive reactions were detected in the sera from either the Burmeister's porpoises or the inshore bottlenose dolphins.