Canine distemper virus in Lake Baikal seals (Phoca sibirica).

The virus epizootic which resulted in significant mortality in Siberian seals (Phoca sibirica) in Lake Baikal during 1987/88 was caused by canine distemper virus. Sequence analysis of the virus glycoprotein genes revealed that it was most closely related to recent European field isolates of canine distemper virus. This paper presents evidence that the same virus continued to circulate in seals in Lake Baikal after the initial epizootic. Three out of 45 brain tissue samples collected from seals culled in the spring of 1992 were positive for canine distemper virus-specific nucleic acid by the reverse transcription/polymerase chain reaction and the sequences were closely related to that of the original virus isolated in 1988.

Characterisation of morbilliviruses isolated from Lake Baikal seals (Phoca sibirica).

Sequence analysis of the haemagglutinin protein (H) gene of the morbillivirus (PDV-2) isolated from a Siberian seal (Phoca sibirica) during the 1987/1988 epizootic in Lake Baikal revealed that it was most closely related to two recent isolates of canine distemper virus (CDV) from Germany and different from CDV vaccines currently in use in that region. The virus continued to circulate in seals in Lake Baikal after the 1987/1988 epizootic since sera collected from culled seals in the spring of 1992 were positive in morbillivirus ELISA tests, reacting most strongly with the CDV antigen.

Relative immunocompetence of the newborn harbour seal, Phoca vitulina.

The immune system of many mammalian species is not fully developed at birth, with newborns obtaining temporary immunological protection from maternal antibodies. Little is known of the immune system of the harbour seal, and developmental aspects of its immune system have not been systematically studied. We collected blood and milk samples from nine free-ranging mother-pup pairs throughout the lactation period on Sable Island, Canada, in an effort to characterise developmental aspects of the immune system of this newborn pinniped. Pup lymphocytes responded stronger to the mitogens concanavalin A, phytohaemagglutinin, and pokeweed mitogen than the lymphocytes of their mothers. In contrast to newborn cats and dogs, newborn seal pups developed high specific antibody responses after immunisation with an inactivated rabies vaccine. Circulating levels of total IgG in newborn pups were low (3% of maternal levels), but increased rapidly after colostrum intake (to 65% of maternal levels after 15 days). A similar pattern of increase in pup serum was observed for phocine distemper virus specific antibodies which had been detected in the serum and milk of mothers, suggesting that the transfer of colostral antibodies is an important feature of temporary protection for the pup. We speculate that the relative immunocompetence of the harbour seal at birth reflects an adaptation to its relatively short nursing period and limited maternal care.

Comparative immunological characterization of type-specific and conserved B-cell epitopes of pinniped, felid and canid herpesviruses.

Murine monoclonal antibodies (MAbs) were generated against phocid herpesviruses (PhHV 2557/Han88 and 7848/Han90) isolated from European harbour seals (Phoca vitulina), and against strains of both felid (FHV strain FVR 605) and canid herpesviruses (CHV isolate 5105/Han89). MAbs were characterized with respect to certain biological properties and used to outline antigenicity profiles of isolates of PhHV (n = 8), FHV (n = 7) and CHV (n = 3) in enzyme immunoassays employing fixed infected cells. A close antigenic relationship between herpesviruses derived from pinnipeds and terrestrial carnivores became evident: The majority of the MAbs was directed against epitopes which were expressed by at least two of the viral species tested. A number of MAbs detected epitopes which were conserved between all isolates of PhHV, FHV and CHV. A few MAbs recognized type-specific B-cell epitopes and facilitated the identification of single viral species. Moreover, the PhHV isolate 7848/Han90 was antigenically distinguishable both from seven other phocid herpesvirus isolates and from FHV or CHV. PhHV 7848/Han90 proved to be antigenically distinct from all other viruses tested when examined by cross neutralization utilizing various reconvalescent and hyperimmune sera. Although more data are needed to ensure that PhHV 7848/Han90 indeed is a new genuine seal herpesvirus, the preliminary clustering of two groups of phocid herpesvirus isolates, tentatively designated PhHV-1 (type isolate 2557/Han88) and PhHV-2 (represented by 7848/Han90), seems to be justified. By using selected MAbs an unambiguous identification and typing of herpesvirus isolates derived from marine mammals and terrestrial carnivores is significantly facilitated.

Fusion protein gene nucleotide sequence similarities, shared antigenic sites and phylogenetic analysis suggest that phocid distemper virus type 2 and canine distemper virus belong to the same virus entity.

Nucleotide sequencing of the fusion protein (F) gene of phocid distemper virus-2 (PDV-2), recently isolated from Baikal seals (Phoca sibirica), revealed an open reading frame (nucleotides 84 to 2075) with two potential in-frame ATG translation initiation codons. We suggest that the second in-frame ATG triplet at positions 264 to 266 initiates the translation, resulting in a protein of 537 amino acid residues with a calculated M(r) of 63,035. The putative F1/F2 cleavage site, located approximately 100 amino acid residues from the N terminus, is identical to those of the F proteins of phocid distemper virus-1 (PDV-1) isolated from European harbour seals (Phoca vitulina) and of canine distemper virus (CDV). A full scale comparison of morbillivirus F genes reveals that the conserved F0 extracellular protein-encoding region contains a large number of non-expressed mutations, suggesting that this part of the protein is under strong functional constraints. Phylogenetic analysis of morbillivirus F gene nucleotide sequences revealed a closer evolutionary relationship between PDV-2 and CDV than between PDV-1 and PDV-2. These data were supported by cross-reactivity patterns of PDV-2 and CDV obtained with monoclonal antibodies to structural proteins of PDV-1 and CDV, and suggest that PDV-2 is a strain of CDV, resulting from a trans-species infection.

Mitogen and antigen induced B and T cell responses of peripheral blood mononuclear cells from the harbour seal (Phoca vitulina).

In vitro assays were developed for studies concerning the functioning of the immune system of the harbour seal (Phoca vitulina). Proliferative responses of peripheral blood mononuclear cells (PBMC) were measured after stimulation with different concentrations of the mitogens concanavalin A (Con A), pokeweed mitogen (PWM), phytohaemagglutinin (PHA) or lipopolysaccharide from Salmonella typhimurium (LPS). Con A and PWM induced strong proliferative responses, while PHA and LPS induced comparatively low proliferative responses. Responses of mitogen stimulated PBMC to recombinant human interleukin-2 (rhIL-2) and in vitro immunoglobulin production by mitogen stimulated PBMC were measured to discriminate between stimulation of T cells and B cells. It was found that Con A and PHA stimulate phocine T cells, PWM stimulates both T cells and B cells and LPS predominantly stimulates phocine B cells. Antigen-specific immune responses were measured after immunization of seals with an inactivated rabies vaccine and/or with tetanus toxoid. Antigen-specific proliferation of PBMC and the presence of antigen-specific antibody forming cells were demonstrated for both antigens in the PBMC of immunized animals. The responses measured in vitro correlated well with the development of specific serum antibody titers to these antigens.

Dolphin and porpoise morbilliviruses are genetically distinct from phocine distemper virus.

The morbilliviruses recently isolated from two cetacean species in the North and Mediterranean Seas have been shown to differ from phocine distemper virus isolated from European seals using monoclonal antibodies. We have identified a "universal" morbillivirus primer set, based on highly conserved regions of the morbillivirus phosphoprotein (P) gene and used this to amplify a region surrounding the RNA editing site from all known members of the group. Sequence analysis of this region of the gene shows that the dolphin and porpoise viruses are related but quite different from all other members of the group, forming a distinct lineage more closely related to the ruminant morbilliviruses than to the carnivore viruses.

Characterization of morbilliviruses isolated from dolphins and porpoises in Europe.

A previously unidentified morbillivirus was isolated from two harbour porpoises (Phocoena phocoena) that had died in the Dutch Waddensea (North Sea) in 1990. This porpoise morbillivirus (PMV) and a dolphin morbillivirus (DMV), which had recently caused a heavy mortality in Mediterranean striped dolphins (Stenella coeruleoalba), were compared antigenically with other members of the genus Morbillivirus, including the newly recognized phocine distemper virus type 1. DMV and PMV proved to be similar but distinct morbillivurses, closely related to rinderpest virus and peste-des-petitsruminants virus. Cell cultures of cetacean, pinniped, ruminant and canine origin showed a different pattern of susceptibility to DMV and PMV infection. Ruminants and dogs proved to be susceptible to experimental infection with DMV and PMV, which both caused a transient leukopenia most pronounced in the ruminants. Pre-exposure of dogs to DMV and PMV protected them from developing CDV viraemia and clinical signs upon challenge infection with virulent CDV. A serological survey among stranded animals of different cetacean species in Europe indicated that infections with DMV- and PMV-like morbilliviruses are not uncommon among these aquatic mammals.

Prevalence of morbilliviruses among pinniped and cetacean species.

Since 1987, morbillivirus infections have caused serious disease outbreaks with high mortality among aquatic mammals. Baikal seals (Phoca sibirica) in Siberia were involved in an outbreak caused by a virus closely related to canine distemper virus (CDV) in 1987. Harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) in north-western Europe were struck by a newly recognised morbillivirus of seals (PDV1). A serological survey has indicated that these morbillivirus infections frequently occur among several pinniped species. Besides pinnipeds, the presence of morbillivirus infections among cetaceans, including whales, dolphins and porpoises, has been demonstrated since the outbreak of PDV among seals in north-western Europe. Morbillivirus was isolated from several stranded harbour porpoises (Phocoena phocoena). This virus proved to be very similar to a virus which was isolated during a disease outbreak with high mortality among striped dolphins (Stenella coeruleoalba) in the Mediterranean area. The viruses isolated from these cetacean species were quite different from the viruses isolated from the seals. They proved more related to the ruminant morbilliviruses, peste des petits ruminants virus and rinderpest virus. The potential transmission of the dolphin morbillivirus to the endangered population of the Mediterranean monk seal (Monachus monachus) has been considered. Studies are presently being conducted into the possibility of inducing protection against morbillivirus infection in this species by vaccination with an immune stimulating complex (ISCOM) preparation based on CDV.

Isolation of a virus with rhabdovirus morphology from a white-beaked dolphin (Lagenorhynchus albirostris).

A virus with rhabdovirus morphology which proved to be antigenically distinct from rabies virus and vesicular stomatitis virus was isolated from a dolphin that had beached on the Dutch coast. Neutralizing antibodies to this virus were found in several European marine mammal species.

Morbillivirus infections in aquatic mammals.

Infections with morbilliviruses have caused heavy losses among different populations of aquatic mammals during the last 5 years. Two different morbilliviruses were isolated from disease outbreaks among seals in Europe and Siberia: phocid distemper virus-1 (PDV-1) and phocid distemper virus-2 (PDV-2) respectively. PDV-1 was characterized as a newly identified morbillivirus, most related to canine distemper virus (CDV), whereas PDV-2 most probably is a strain of CDV. Morbilliviruses were also isolated from porpoises--porpoise morbillivirus (PMV)--and dolphins--dolphin morbillivirus (DMV)--which had stranded on the coasts of Europe. PMV and DMV proved to be closely related to, but distinct from 2 ruminant morbilliviruses, rinderpest virus (RPV) and peste-des-petits-ruminants virus (PPRV). Serological surveys carried out among pinniped and cetacean species in the seas of Europe and North America indicated that infections with these newly discovered morbilliviruses or closely related viruses commonly occur among aquatic mammal species.

Dolphin morbillivirus infection in different parts of the Mediterranean Sea.

Morbillivirus were isolated from Mediterranean striped dolphins (Stenella coeruleoalba) dying along the coasts of Italy and Greece in 1991. They were antigenically identical to the morbilliviruses isolated from striped dolphins in Spain in 1990.

Molecular and serological studies on the recent seal virus epizootics in Europe and Siberia.

The virus epizootics which occurred in seals in both Europe and Siberia during 1987/1988 were caused by two different morbilliviruses, referred to as phocid distemper virus (PDV) 1 and 2, respectively. Molecular and serological studies have shown that the European virus is quite distinct from canine distemper virus (CDV), its closest relative in the morbillivirus group. Analysis of tissues obtained from infected seals from a wide geographical distribution over Northern Europe showed that the infectious agent (PDV 1) was identical in all cases. Nucleotide sequence analysis of one of the virus genes suggested that this virus has evolved away from CDV over a long time period and is most probably an enzootic virus of marine mammals. In contrast, the virus (PDV 2) which caused the deaths of many Siberian seals was indistinguishable, both serologically and at the molecular level, from CDV and must have originated from a land source.

Canine distemper virus ISCOMs induce protection in harbour seals (Phoca vitulina) against phocid distemper but still allow subsequent infection with phocid distemper virus-1.

A candidate canine distemper virus (CDV) ISCOM vaccine has been shown to be effective in protecting harbour seals (Phoca vitulina) from phocid distemper in 1988. However, of the 35 harbour seals receiving this vaccine upon admission to a seal rehabilitation and research centre (Pieterburen, The Netherlands) in 1989, six developed mild inflammatory symptoms of the respiratory tract. Phocid distemper virus-1 (PDV-1) could be isolated from three of these animals. This indicates that the vaccine affords protection from phocid distemper, but may still allow PDV-1 infection of the respiratory tract. Contacts with non-vaccinated seals should then be prevented until no more virus is excreted. It is speculated that this PDV-1 infection of the respiratory tract in CDV-ISCOM vaccinated seals is followed by a lifelong immunity.