Dispatches

Two Morbilliviruses Implicated in Bottlenose Dolphin Epizootics

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Sequence analysis was performed on viral RNA isolated from bottlenose
dolphins (Tursiops truncatus) that died during two chronologically and
geographically separate epizootics in North America. Both dolphin
morbillivirus (DMV) and porpoise morbillivirus (PMV) were detected in
bottlenose dolphins that died during the 1987 U.S. Atlantic coast
epizootic. Our results indicate not only that these viruses are not species
specific, but also that both viruses were present in North America before
outbreaks in the Mediterranean and Irish Seas. Samples taken along the
Atlantic coast showed a statistically significant trend with DMV in the
north and an increasing incidence of PMV in samples isolated farther south.
In the 1993 Gulf of Mexico epizootic, only PMV was detected in bottlenose
dolphins that died. Thus, DMV and PMV are implicated as the causes of the
earliest known aquatic mammal morbilliviral outbreak, the U.S. Atlantic
coast epidemic; PMV is implicated in the Gulf of Mexico epidemic. The
presence of two pathogenic morbilliviruses that may circulate together or
separately complicates the epidemiology of cetacean morbilliviral diseases.

The only morbilliviruses known before 1989 were human measles virus, canine
distemper virus, rinderpest, and peste-des-petits-ruminants virus (1).
Recently, newly characterized morbilliviruses have been shown to be
epizootic-associated pathogens in pinnipeds and cetaceans. Phocine
distemper virus (2) was associated with a massive epizootic of harbor seals
(Phoca vitulina) in northwestern Europe (3) in 1988. During the harbor seal
phocine distemper virus outbreak, PMV was isolated from harbor porpoises
(Phocoena phocoena) that died along the Irish coast (4). DMV (5), was
isolated during an epizootic of striped dolphins (Stenella coeruleoalba) in
the Mediterranean Sea (6,7) in 1990-92.

Between June of 1987 and May of 1988, a morbillivirus epizootic caused a
tenfold increase in bottlenose dolphin stranding along the U.S. Atlantic
coast from New Jersey to Florida (8,9). More than half of the inshore
population of bottlenose dolphins in this area may have died.
Morbillivirus-related strandings of bottlenose dolphins along the Gulf of
Mexico coasts of Alabama, Mississippi, and Texas were also observed from
October 1993 through April 1994 (10,11).

Using reverse transcriptasepolymerase chain reaction (RT-PCR), we examined
tissue lysates made from formalin-fixed paraffin-embedded lung tissue from
stranded dolphins from the Atlantic epizootic and frozen unfixed lung
tissue from the Gulf of Mexico epizootic (11,12). RT-PCR-positive cases
were subsequently characterized by sequence analysis of segments of the
morbillivirus P gene in regions with the greatest diversity between DMV and
PMV.

We sequenced segments of the morbillivirus P gene from 29 dolphin specimens
(1 striped and 28 bottlenose dolphins) from the 1987-88 Atlantic epizootic;
sequence sufficient for viral identification was obtained in 25 (86%) of 29
cases. Sequence was also obtained from 7 (37%) of 19 bottlenose dolphin
specimens stranded in Texas during the Gulf of Mexico epizootic. Because of
the advanced state of postmortem decomposition of most of these specimens,
histologic and immunophenotypic analysis was not possible (13). Data
(stranding date, location, species, and morbillivirus sequence analysis
results) for these 32 cases are shown in Table 1. In addition, P gene
sequence was obtained from formalin-fixed paraffin-embedded lung tissue
from four of four striped dolphins from the 1990 Mediterranean Sea
epizootic and from one harbor porpoise recovered off the coast of Northern
Ireland in 1988 during the harbor seal epizootic (Table 1).

Morbillivirus P gene sequencing allowed viral identification of 37 stranded
cetaceans from four geographically and chronologically separate epizootics.
In the U.S. Atlantic Coast 1987-88 epizootic, mixed infection was present
involving both recognized cetacean morbilliviruses, DMV and PMV (Table 1).
Twelve animals (including the striped dolphin) were infected with DMV, nine
with PMV, and four with both. From the later Gulf of Mexico
morbillivirus-associated epizootic, only PMV was identified. The
morbillivirus infecting striped dolphins in the Mediterranean Sea epizootic
was identified as DMV (6). All four striped dolphin specimens examined in
this study were typed as DMV infected. A harbor porpoise stranded on the
coast of Northern Ireland during the harbor seal epizootic in northwestern
Europe was typed as PMV-infected, consistent with the published analysis of
these animals (14).

                          Table 1. Case summary
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  Date found         Stranding               Dolphin       Morbillivirus
                     by state                species         sequence(a)
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 4 Aug. 1987 New Jersey               Tursiops truncatus   DMV
 5 Aug. 1987 New Jersey               T. truncatus         DMV
 9 Aug. 1987 New Jersey               T. truncatus         DMV
 11 Aug. 1987New Jersey               T. truncatus         DMV
 23 Aug. 1987New Jersey               T. truncatus         DMV + PMV
 3 Sep. 1987 New Jersey               Stenella coeruloalba DMV
 5 Sep. 1987 New Jersey               T. truncatus         DMV + PMV
 6 Sep. 1987 New Jersey               T. truncatus         PMV
 14 Aug. 1987Virginia                 T. truncatus         PMV
 14 Aug. 1987Virginia                 T. truncatus         DMV + PMV
 15 Aug. 1987Virginia                 T. truncatus         PMV
 26 Aug. 1987Virginia                 T. truncatus         DMV + PMV
 29 Aug. 1987Virginia                 T. truncatus         PMV
 29 Aug. 1987Virginia                 T. truncatus         DMV
 4 Sep. 1987 Virginia                 T. truncatus         DMV
 5 Oct. 1987 Virginia                 T. truncatus         DMV
 6 Oct. 1987 Virginia                 T. truncatus         DMV
 7 Oct. 1987 Virginia                 T. truncatus         DMV
 21 Dec. 1987Florida (Atlantic Coast) T. truncatus         PMV
 21 Dec. 1987Florida (Atlantic Coast) T. truncatus         PMV
 1 Jan. 1988 Florida (Atlantic Coast) T. truncatus         PMV
 18 Jan. 1988Florida (Atlantic Coast) T. truncatus         PMV
 9 Feb. 1988 Florida (Atlantic Coast) T. truncatus         DMV
 9 Feb. 1988 Florida (Atlantic Coast) T. truncatus         PMV
 10 Feb. 1988Florida (Atlantic Coast) T. truncatus         DMV
 4 Apr. 1993 Texas                    T. truncatus         PMV
 4 Apr. 1993 Texas                    T. truncatus         PMV
 4 Apr. 1993 Texas                    T. truncatus         PMV
 11 Apr. 1993Texas                    T. truncatus         PMV
 15 Apr. 1993Texas                    T. truncatus         PMV
 19 Apr. 1993Texas                    T. truncatus         PMV
 21 Apr. 1993Texas                    T. truncatus         PMV
 1991        Mediterranean Sea        S. coeruloalba       DMV
 1991        Mediterranean Sea        S. coeruloalba       DMV
 1991        Mediterranean Sea        S. coeruloalba       DMV
 1991        Mediterranean Sea        S. coeruloalba       DMV
 1988        Northern Ireland         Phocoena phocoena    PMV

 a RNA isolation, RT-PCR, and cycle sequencing were performed (11,20).
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Partial P gene nucleotide sequence data are presented with published DMV
and PMV P gene sequences (Figure). Sequence data from the PMV-infected
cases (Atlantic epizootic cases, Gulf of Mexico epizootic cases, Irish
harbor porpoise) showed no within-group variation. Data from all cases
showed two single nucleotide changes from the published PMV sequence (12),
at positions 206 and 209 (aligned with the measles virus P gene sense
mRNA). Likewise, no sequence variation was noted for the DMV-infected
animals (Atlantic and Mediterranean epizootics). However, a two-nucleotide
inversion, at coding positions 206-207, was consistently noted when
compared to the published DMV sequence (12) (Figure). In four cases from
the Atlantic epizootic cycle sequencing indicated simultaneous infection
with DMV and PMV. To confirm that the two different sequences were present
simultaneously, PCR product was cloned, and plasmid DNA from individual
colonies was sequenced. In each of the four doubly infected cases,
approximately half the clones contained the DMV sequence, and half
contained the PMV sequence. No clones containing hybrid sequences were
found. Since PMV and DMV cause non-species-specific infections, more
appropriate designations for these viruses might be cetacean morbillivirus
1 for PMV (the first detected cetacean morbilivirus [4]) and cetacean
morbillivirus 2 for DMV.

                          176                                        215
Published DMV             ATCTGCTCCC  AGGATTAAGG  TCGAGAGATC  TGCTGACGTT
Atlantic/Mediter. DMV     ----------  ----------  ----------  GT--------
Atlantic/Gulf/Irish PMV   ----------  ----------  ----------  AT-C--T---
Published PMV             ----------  ----------  ----------  -T----T---

                          216                                      255
Published DMV             GAGACTATAA  GCAGTGAAGA  GCTACAAGGA  CTGATTAGAT
Atlantic/Mediter. DMV     ----------  ----------  ----------  ----------
Atlantic/Gulf/Irish PMV   ----------  -------G--  A--T------  -----C----
Published PMV             ----------  -------G--  A--T------  -----C----

                          256                                        295
Published DMV             CTCAGAGTCA  AAAACATAAT  GGATTTGGAG  TAGACAGATT
Atlantic/Mediter. DMV     ----------  ----------  ----------  ----------
Atlantic/Gulf/Irish PMV   ------AC--  C--G--G---  -----C----  G-----A--C
Published PMV             ------AC--  C--G--G---  -----C----  G-----A--C

                          296         313
Published DMV             CCTAAAGGTC  CCACCAAT
Atlantic/Mediter. DMV     ----------  --------
Atlantic/Gulf/Irish PMV   TT--------  --------
Published PMV             TT--------  --------

Figure. Partial nucleotide sequence of DMV and PMV morbillivirus P gene
compared with published DMV and PMV sequences (12). Primer sequences used
were 5'-ATC TGC TCC CAG GAT TAA GGT CGA-3' (forward) and 5'-CGG GAT TGG TGG
GAC CTT TA-3' (reverse). RT- PCR was performed (11). PCR products were
cycle sequenced (20) or cloned into PDK101 (21) and sequenced by using T7
Sequenase (Amersham Corporation, Arlington Heights, IL) according to the
manufacturers instructions.

Bottlenose dolphins of the U.S. Atlantic coast migrate seasonally (15). The
1987-88 epizootic moved south from New Jersey to the east coast of Florida
with the southerly fall migration. In-shore bottlenose dolphins rarely
travel around the southern tip of the Florida peninsula (Randall Wells,
pers. comm.), which explains the end of the 1987 epizootic on the east
coast of Florida in May of 1988. Sequences were obtained from dolphins
stranded during this epizootic in three U.S. states, New Jersey, Virginia,
and Florida. From the Gulf of Mexico epizootic, which occurred 5 years
later, sequence data from seven dolphins stranded in Texas were obtained. A
significant trend (chi square for trends [16], p = 0.00023) was noted among
the specimens analyzed from the New Jersey, Virginia, and Florida Atlantic
coasts in which the percentage of animals infected with DMV decreased,
while PMV infection increased (Table 2). In the 1993 die-off, 100% of the
animals from which sequence information was obtained were PMV-infected. The
epidemiologic significance of this finding is unknown. It is possible that
dolphins of the southern Atlantic coast and those in the Gulf of Mexico
were more susceptible to infection with (or had an increased death rate
from) PMV than with DMV.

 Table 2. Percentage of DM-V and PMV-infected animals by location
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    Stranding location       No.        No. DMV        No. PMV
                           dolphins  infected (%)   infected (%)
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 Atlantic, New Jersey         8         7 (70)a        3 (30)a

 Atlantic, Virginia           10        7 (58)a        5 (42)a

 Atlantic, Florida            7         2 (29)         5 (71)
 Gulf of Mexico, Texas        7            0           7 (100)

 a Including the four cases of simultaneous DMV and PMV infection.
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To test whether enzootic morbillivirus infection could be detected in
bottlenose dolphins, RT-PCR was performed on formalin-fixed
paraffin-embedded tissue samples from 11 dolphins stranded along the U.S.
Atlantic coast from 1974 to 1985 (before the 1987 epizootic). None of the
dolphins had histologic or immunohistochemical evidence of morbillivirus
infection (9). Amplifiable RNA was obtained from samples from eight
animals, and all were negative for morbillivirus. Tissue specimens from
seven bottlenose dolphins stranded on the Florida coast of the Gulf of
Mexico in the interval between the Atlantic and Gulf of Mexico epizootics
were also examined by RT-PCR for morbillivirus. None of these animals had
histologic or immunohistochemical evidence of morbillivirus infection (data
not shown). However, one of these samples was weakly positive for the
morbillivirus P gene 78-bp product (11). Sequence information, however,
could not be obtained from this sample. This result suggests the
possibility of an enzootic infection in dolphins along the U.S. coast after
the initial 1987 morbillivirus epizootic.

Serum antibodies to canine distemper virus were detected in specimens of 6
of 13 free-ranging bottlenose dolphins during the U.S. Atlantic epizootic
in 1987 (17), indicating exposure to morbillivirus. A recent study by
Duignan, et al. (18) presented serologic evidence of morbillivirus
infection in 11 of 15 cetacean species in the western Atlantic since 1986.
Virus neutralizing titers were higher against DMV and PMV than against
peste-des-petits ruminants virus, phocine distemper virus, or canine
distemper virus. Enzootic morbillivirus infection of two species of pilot
whale (Globicephala melas and G. macrorhynchus) was recently demonstrated
in animals in the western Atlantic (19), with the earliest titer from a
pilot whale stranded in 1982. Neutralizing antibody titers in these species
were also higher against DMV and PMV, and were observed in 108 of 125
animals tested. However, confirmation of active infection by viral sequence
analysis was not performed in either of these studies. It is, therefore,
possible that the pilot whale mass strandings reported in 1982 and 1986
were morbillivirus induced, but confirmation of this hypothesis would
require histologic and/or viral sequence confirmation.

Geographically and temporally distinct morbillivirus die-offs, as well as
the evidence presented in this study that the two previously described
cetacean morbilliviruses are not species-specific, raise many questions
about the epidemiology of this family of viruses. The viruses implicated in
the 1988 European porpoise deaths and 1990 Mediterranean dolphin epizootic
were present in bottlenose dolphins of the 1987 U.S. die-off. Duignan, et
al. hypothesized that pilot whales enzootically infected with
morbilliviruses could act as long-distance vectors between America and
Europe (19). Confirmation would require sequence analysis of pilot whale
samples collected in both regions. The distribution of DMV and PMV
infection in the U.S. epizootics suggests that the later epizootic may have
been initiated by rare contact between PMV-infected Atlantic dolphins and
immunologically naive Gulf dolphins. Further investigation of serum
antibody titers, PCR evidence of enzootic infection, and sequence
identification of morbillivirus species are required for the better
understanding of these epizootics.

Acknowledgments

This study was partially supported by a grant from the Center for Marine
and Estuarine Disease Research, U.S. Environmental Protection Agency. We
thank S. Kennedy, M. Domingo, the U.S. National Marine Fisheries Service,
and the U.S. Marine Mammal Stranding Networks for providing samples.

Jeffery K. Taubenberger, M.D., Ph.D.,* Mark Tsai, M.S.,* Amy E. Krafft,
Ph.D.,* Jack H. Lichy, M.D., Ph.D.,* Ann H. Reid, M.A.,* F. Yvonne
Schulman, D.V.M., and Thomas P. Lipscomb, D.V.M.

*Division of Molecular Pathology, Department of Cellular Pathology, and
Department of Veterinary Pathology, Armed Forces Institute of
Pathology,Washington, DC 20306-6000 USA

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