initial characterization of novel beaked whale ...cemv shares a common etiology with other morbilli...
TRANSCRIPT
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DISEASES OF AQUATIC ORGANISMSDis Aquat Org
Vol. 117: 215–227, 2016doi: 10.3354/dao02941
Published January 13
INTRODUCTION
Cetacean morbillivirus (CeMV; Family Paramyxo -viridae) is a member of the genus Morbillivirus,which includes 5 other virus species that affect mam-mals including the measles virus in humans, peste-des-petits-ruminants virus in ruminants, rinderpestvirus in cattle, phocine distemper virus in the trueseals, and canine distemper virus (CDV) in carni-vores (Barrett et al. 1993, van de Bildt et al. 2005).
CeMV shares a common etiology with other morbilli -viruses in that the immune system is the primary target, from which it can proliferate and spread toepithelial cells (reviewed in Van Bressem et al. 2014).Common secondary responses include pneumonia,meningoencephalitis, and hepatitis (Kennedy 1998,Reidarson et al. 1998, Taubenberger et al. 2000, DiGuardo et al. 2005). Classic pathological findings incases of clinical CeMV include lesions in the lungs,lymph nodes, and brain. Multinucleated cells (syn -
© Inter-Research 2016 · www.int-res.com*Corresponding author: [email protected]
Initial characterization of novel beaked whalemorbillivirus in Hawaiian cetaceans
Jessica M. Jacob1, Kristi L. West1, Gregg Levine2, Susan Sanchez3, Brenda A. Jensen1,*
1College of Natural and Computational Sciences, Hawai’i Pacific University, 45-045 Kamehameha Highway, Kaneohe, Hawai’i 96744, USA
2267 S. Kalaheo Avenue, Kailua, Hawai’i 96734, USA3Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA
ABSTRACT: Cetacean morbillivirus (CeMV) is a causative factor in epizootics that have resultedin thousands of deaths throughout the Atlantic and Mediterranean since 1987, but less is knownof its presence and significance in the Pacific. The first case of CeMV reported in Hawai’i was ina Longman’s beaked whale that stranded in 2010. The initial CeMV sequence from this individualindicated the possibility of a novel strain. To address this, archived samples from cetaceans thatstranded in Hawai’i between 1997 and 2014 were screened for CeMV. The beaked whale morbil-livirus (BWMV) was detected in 15 individuals representing 12 different species (24% of Code 1and 2 stranded cetaceans). The earliest detected case was a humpback whale that stranded in1998. Sequence comparisons of a 2.2 kb sequence spanning the phosphoprotein (P) and nucleo-capsid (N) genes strongly suggest that the BWMV represents a novel strain of CeMV present inHawai’i and the Central Pacific. In contrast to recently reported isolates from Brazil and Australiathat may represent a distinct clade, BWMV appears to be more closely related to known strains ofCeMV (dolphin morbillivirus; porpoise morbillivirus; and pilot whale morbillivirus). Detectionrates with repeat sampling of positive lymph nodes were between 2 and 61%, illustrating theextreme heterogeneity that can occur in affected tissues. Taken together, these results suggestthat BWMV may be common and established in Hawaiian cetacean populations. BWMV will beimportant for understanding CeMV and health threats in the relatively understudied cetaceans ofthe Pacific.
KEY WORDS: Cetacean · Morbillivirus · Pacific · Hawai’i
Resale or republication not permitted without written consent of the publisher
FREEREE ACCESSCCESS
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Dis Aquat Org 117: 215–227, 2016
cytia) and intranuclear inclusion bodies are oftennoted microscopically in these organ systems (Ken -ne dy 1998, Taubenberger et al. 2000, Di Guardo etal. 2005).
CeMV has caused many epizootics in the AtlanticOcean, Gulf of Mexico, Mediterranean Sea, and ad -jacent seas (Visser et al. 1993, Kennedy 1998, Bossartet al. 2010, Bellière et al. 2011a). The first knownCeMV epizootic occurred in 1987–1988 on the eastcoast of the USA and killed over 50% of the in-shoreAtlantic bottlenose dolphin Tursiops trun catus popu-lation (Lipscomb et al. 1994, Krafft et al. 1995, Tauben -berger et al. 1996). Another major event on the Span-ish coast of the Mediterranean Sea in 1990 killedthousands of striped dolphins Stenella coeruleoalba(Barrett et al. 1993, Blixenkrone-Møller et al. 1994,Bellière et al. 2011b). Between 2006 and 2007, CeMVwas detected in 9 long-finned pilot whales Globi-cephala melas that stranded along the southernSpanish Mediterranean coast and Balearic Islands(Fernández et al. 2008). More recently, in 2013CeMV was detected in 24 stranded cetaceans of var-ious species along the Tyrrhenian coast of Italy(Casalone et al. 2014), while another outbreak in2013 along the east coast of the USA killed hundredsof bottlenose dolphins (Brown et al. 2014).
While isolated cases of CeMV continue to be diag-nosed throughout the Pacific Ocean, it has notcaused any obvious epizootics in this region. The firstdocumented cases of CeMV in the Pacific were in 3common dolphins Delphinus delphis that stranded onthe California coast of the USA between 1995 and1997 (Reidarson et al. 1998). In 1998, a Pacific stripeddolphin Lagenorhynchus obliquidens stranded withCeMV on the coast of Japan (Uchida et al. 1999). Inthe Hawaiian Islands, the first diagnosed case ofCeMV was in a Longman’s beaked whale Indopace-tus pacificus that stranded in 2010 on the island ofMaui, followed soon after by a Blainville’s beakedwhale Mesoplodon densirostris (West et al. 2013),and a neonate sperm whale Physeter macrocephalusin 2011 (West et al. 2015).
Molecular characterization has revealed informa-tion about CeMV structure and the 3 commonly rec-ognized strains. The CeMV genome is composed of6 different genes: 5’-N-P-M-F-H-L-3’. The nucleo-capsid (N) gene and the phosphoprotein (P) genehave been particularly important for characterizingCeMV. The P-gene is a highly conserved structuralprotein that can be used to identify morbillivirus spe-cies in various mammals (Barrett et al. 1993, Visser etal. 1993, Bolt et al. 1995), while the N-gene thatencodes the N protein is less conserved across mor-
billivirus species, and can be used to characterizeand distinguish between different morbillivirus spe-cies and strains (Diallo et al. 1994). The porpoise mor-billivirus (PMV) strain was associated with severalepizootics in the Atlantic Ocean in the 1980s and1990s (Kennedy et al. 1988), while dolphin morbil-livirus (DMV) has caused outbreaks in the AtlanticOcean and the Mediterranean Sea from the 1980s tothe present day (Blixenkrone-Møller et al. 1994). Incontrast, pilot whale morbillivirus (PWMV) has notbeen associated with any major outbreaks (Tauben-berger et al. 2000).
The discovery of CeMV in the Longman’s beakedwhale demonstrated the need to more closely exam-ine CeMV in Hawaiian cetaceans, as well as toaddress the hypothesis that the beaked whale mor-billivirus (BWMV) represents a novel strain of CeMVin the Pacific. The purpose of this research was three-fold: (1) to survey the archive of stranded Hawaiiancetacean tissues for CeMV using reverse transcrip-tion polymerase chain reaction (RT-PCR) in order toamplify the P-gene region, (2) to characterize theHawaiian CeMV isolates by sequencing the P- andN-gene regions using direct sequencing, and (3) toexamine the heterogeneous distribution of CeMV intissues. This retroactive survey is the first compre-hensive examination of CeMV occurrence in Hawai-ian cetaceans.
MATERIALS AND METHODS
Sample collection
Since 2006, the Hawai’i Pacific University (HPU)Marine Mammal Stranding Team has partnered withthe National Oceanographic and Atmospheric Ad -ministration (NOAA) to conduct cause of deathinvestigations in cetaceans that strand in the Hawai-ian Islands and the Pacific Islands Region (PIR).These investigations include gross pathology, histo -pathology, and other diagnostics as indicated and asfeasible. This study examined 62 cetaceans thatstranded between 1997 and 2014; 58 in the mainHawaiian Islands and 4 in the PIR (see Fig. 1). Indi-viduals were characterized as either Code 1 (firstobserved alive and sampled soon after death), orCode 2 (freshly dead with little decomposition oforgans) as described by Geraci & Lounsbury (1993).Pending availability, between 3 and 25 tissues wereexamined, including brain (cerebrum and cerebel-lum), spleen, liver, various lymph nodes, lung (leftand right), kidney (left and right), and blood. Beaked
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Jacob et al.: Novel beaked whale morbillivirus in Hawaiian cetaceans
whale muscle, blubber, and skin were screened toassess the feasibility of identifying CeMV in skin andblubber biopsies. Amniotic fluid, uterus, and ovariesfrom a pregnant striped dolphin were also examined.
RNA extraction, quantification, and quality control
Tissues designated for RNA isolation were main-tained between −60 and −80°C. Beginning in 2011,samples were also preserved in RNA Later (#67106,Qiagen) when dry ice was not available. Sub-sam-ples (10 mm3) were processed using the SV TotalRNA Isolation System Kit (#Z3100, Promega) fol-lowing the manufacturer’s instructions and recom-mendations for maximum extraction efficiency. AllRNA and PCR procedures occurred under PCRsterile conditions.
RNA was quantified by UV spectroscopy in aSmartSpec Plus UV Spectrophotometer (BioRad).Total RNA quality was assessed by RNA gels: 1.2%sterile agarose with 1× 3-(N-morpholino) propane-sulfonic acid buffer, 2.88 ml of 37% formaldehyde,and 8 µl of ethidium bromide (Masek et al. 2005).RNA samples were prepared by combining 10 µl ofRNA with 10 µl of formaldehyde sample buffer(Lonza), incubated at 65°C for 5 min, and cooled onice for 1 min prior to loading. The prominence of ribo-somal RNA subunits and the relative distribution ofRNA relative to the subunits were used to qualita-tively assess the RNA. Well-defined RNA subunitsindicate high quality RNA that is not severelydegraded.
Characterization of CeMV isolates usingP- and N-genes
Approximately 100 to 1000 ng total RNA was usedas a template for cDNA synthesis with MoloneyMurine Leukemia Virus Reverse Transcriptase (MMLV-RT) (#M1701, Promega) and random primers (#C1181,Promega) according to the manufacturer’s instruc-tions. PCR was used to amplify a 429 bp P-gene frag-ment from cDNA. A total of 1 µM of the primers P429-forward and P429-reverse (Table 1) (Barrett et al.1993) and 2 µl cDNA were used with Go Taq Hot StartGreen PCR master mix (#M5123, Promega). PCRproducts were confirmed using standard gel electro-phoresis in a 1% agarose gel containing ethidiumbromide. A positive CeMV test generated a distinctband around 429 bp in length on the gel. The absenceof a band around 429 bp indicated that CeMV was notdetected. Amplification of a 322 bp fragment of thehousekeeping gene β-actin was used as a positivecontrol for RNA extraction and reverse transcription(Table 1) (Jensen 2000). The positive PCR control con-sisted of template cDNA from one of several tissuesfrom the Longman’s beaked whale that was confirmedpositive by the Athens Diagnostic Laboratory (Col-lege of Veterinary Medicine, University of Georgia,Athens, GA) (West et al. 2013). Replacement of thecDNA template with nuclease-free water confirmedthat cross-contamination did not occur during samplepreparation. Each positive tissue was confirmed by anadditional independent analysis of the cDNA that in-cluded a random selection of experimental tissuesplus the positive and negative controls.
217
Target Primer pair Sequence (5’-3’) Product length (bp)
P-gene P429F ATG TTT ATG ATC ACA GCG GT 429 P429R ATT GGG TTG CAC CAC TTG TC
N- and P-genes MVJJP429R GGG TTG CAC CAC CTG TCA A 1949 MVJJ630RRC GCA TCT ATT CTT GCA CAA ATT T
N- and P-genes MVJJ391F CAG ATG TCA GCA TCA GAT TAG TTG 240 MVJJ630R AAA TTT GTG CAA GAA TAG ATG C
N- and P-genes MVJJ749F ATG GTT GGA TGC TGT GAG AAA TC 770 MVJJ1518R GAG TGT CTT TGC TGA GAG TCG T
N- and P-genes MVJJ391F CAG ATG TCA GCA TCA GAT TAG TTG 646 MVJJ1036R GAC GTC TCG CCC ATC TGT T
N- and P-genes MVJJ749F ATG GTT GGA TGC TGT GAG AAA TC 288 MVJJ1036R GAC GTC TCG CCC ATC TGT T
β-actin gene BelActin_D1 AGA GCA AGA GAG GCA TCC TG 322 BelActin_D1 TAG ATA GGC ACG TGT GGG
Table 1. Primers used to screen and characterize cetacean morbillivirus (CeMV) P- and N-genes in stranded Hawaiian cetaceans. Primer pairs are listed, and the target nucleotide annealing location is included in the name
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Dis Aquat Org 117: 215–227, 2016
Five additional primer pairs were designed usingPrimer 3 and MacVector software to target the N-gene and additional P-gene regions based on DMVse quence (GenBank acc. no. AJ608288) (Table 1). Allbands of expected size were excised and purifiedusing the Wizard SV Gel and PCR Clean-Up System(#A9282, Promega) and prepared in duplicate for for-ward and reverse direct sequencing at the Universityof Hawai’i at Manoa’s Advanced Studies of Geno -mics, Proteomics, and Bioinformatics (ASGPB) labo-ratory. The number of independent sequence readscontributing to the consensus sequence for individ-ual isolates ranged from 5 to 33, depending on thenumber of tissues in which CeMV was detected.
Percent similarity tables and phylogenetic treeswere generated using MacVector Multiple SequenceAnalysis ClustalW software. Partial P-gene se quencesare available for 2 other CeMV strains (DMV andPWMV) and 2 regional isolates (Southwest AtlanticBrazilian [SWAt] and Swan River from Australia). Se-quence similarities to DMV, PWMV, SWAt, and SwanRiver isolates were calculated in 2 ways. First, nucleo-tide similarities were calculated based on the 216 bpP-gene sequence common to all individuals. Second,each animal’s consensus sequence (of varying lengths)was compared to the full length DMV sequence usingthe nucleotide basic local alignment search tool(BLAST N) algorithm. N-gene nucleotide similaritiesamong the Hawaiian CeMV, DMV, PWMV, PMV, andSwan River isolates were based on 188 bp, the lengthof the Swan River consensus sequence. In order toavoid bias from longer sequences, percent nucleotidesimilarities among CeMV sequences were based onthe longest continuous sequence common to all. TheN- and P-gene sequence similarities were examinedusing the neighbor-joining tree method to compareHawaiian CeMV, DMV, PMV, PWMV, SWAt, andSwan River, with CDV as an outgroup. Nucleotide dif-ferences were calculated by Tamura-Nei, with gapsdistributed proportionally and no gamma correction.
Heterogeneity of CeMV
CeMV distribution throughout tissues was exam-ined in lymph nodes from KW2010012 (Blainville’sbeaked whale Mesoplodon densirostris) and KW2011021 (rough-toothed dolphin Steno bredanensis).Each lymph node was sub-sampled into 100 mm3 tis-sue blocks (n = 24, 6, and 9 for M. densirostris medi-astinal lymph, M. densirostris anal lymph, and S.bredanensis anal lymph, respectively). Two 10 mm3
sub-samples of each tissue block were screened for
morbillivirus by RT-PCR as described above. Thedetection rate, defined as the ability to detect CeMVacross lymph node sub-sections, was used to reportthe ability to amplify CeMV (Katayama et al. 1998,Shin et al. 2004). The detection rate was calculatedfor each individual lymph node, for interior sub-sam-ples (edge and middle cuts from sub-samples that didnot touch the end of the lymph node), and for exteriorsub-samples (edge and middle cuts from sub-sam-ples that included the end of the lymph node).
RESULTS
RT-PCR amplification of the CeMV P-gene
Suites of tissues (3 to 25 ind.−1) collected from Code1 and Code 2 cetaceans stranded in Hawai’i werescreened using PCR targeting the CeMV P-gene.CeMV was detected in 15 animals out of a total of 58stranded Hawaiian cetaceans examined. CeMV wasnot de tected in the 4 animals from outside Hawai’i inthe PIR: 2 Cuvier’s beaked whales Ziphius cavirostrisfrom Saipan, 1 spotted dolphin Stenella attenuatafrom Guam, and 1 melon-headed whale Pepono-cephala electra from Kosrae, Micronesia.
In addition to the Longman’s beaked whale,Blainville’s beaked whale (West et al. 2013) andsperm whale (West et al. 2015) that had been re -ported previously, CeMV was detected in 12 addi-tional Hawaiian cetaceans representing 9 additionalspecies: Cuvier’s beaked whale (1/1), humpbackwhale Megaptera novaeangliae (1/7), pygmy spermwhale Kogia breviceps (1/1), rough-toothed dolphin(1/2), spinner dolphin Stenella longirostris (3/16),spotted dolphin (1/2), striped dolphin (2/11), bottle-nose dolphin (1/3), and Risso’s dolphin Grampusgriseus (1/1) (Table 2, Fig. 1). The 6 Hawaiian speciesin which CeMV was not detected included the dwarfsperm whale Kogia sima (0/3), false killer whalePseudorca crassidens (0/2), killer whale Orcinus orca(0/1), melon-headed whale (0/3), pygmy killer whaleFeresa attenuata (0/1), and short-finned pilot whaleGlobicephala macrorhynchus (0/1).
CeMV was most frequently detected in lymphnode (13/337), followed by brain (cerebrum 6/48,cerebellum 2/49, unknown brain 0/18), lung (5/130),liver (4/69), spleen (3/68), kidney (1/116), and blood(1/5) (Table 3). CeMV was not detected in muscle(0/3), blubber (0/3), skin (0/3), uterus (0/1), ovary(0/2), or amniotic fluid (0/1) (Table 3). CeMV wasdetected in more females (9/24) than males (6/38)(Table 2). CeMV was most often detected in adults
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Jacob et al.: Novel beaked whale morbillivirus in Hawaiian cetaceans 219
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023
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Tab
le 2
. C
etac
ean
mor
bil
livi
rus
(CeM
V)
det
ecti
on i
n s
tran
ded
Haw
aiia
n c
etac
ean
s. B
oth
pos
itiv
e an
d n
egat
ive
resu
lts
are
ind
icat
ed s
ince
th
e n
um
ber
an
d t
ype
of a
vail
able
tiss
ues
var
ied
by
case
. Th
e n
um
ber
aft
er t
he
tiss
ue
abb
revi
atio
n in
dic
ates
how
man
y ti
mes
RN
A w
as in
dep
end
entl
y ex
trac
ted
fro
m t
issu
e su
b-s
amp
les.
Cod
e 1:
ob
serv
ed a
live
, sa
mp
led
soo
n a
fter
dea
th; C
ode
2: r
ecen
tly
dea
d w
ith
min
imal
org
an d
ecom
pos
itio
n
-
Dis Aquat Org 117: 215–227, 2016
(5/26) and sub-adults (4/12), followed by calves(3/17), juveniles (2/2), and neonates (1/3), but CeMVwas never detected in yearlings (0/2) (Table 2).CeMV was detected in more Code 2 (fresh dead) ani-mals (8/33), than Code 1 (stranded alive) animals(7/29) (Table 2).
Characterization of CeMV isolates usingP- and N-genes
The P-gene sequence was obtained for all ani-mals except the spotted dolphin (KW2009015),
despite re peated attempts. The nu -cleotide similarity be tween P-geneconsensus sequen ces from individualstranded cetaceans ranged from 97.2to 100% (Table 4). The 216 bp P-gene sequence common to all indi-viduals was between 80.6 and 83.8%similar to PWMV and DMV, respec-tively (Table 4). Using the BLAST Nalgorithm to compare the full DMVsequence, Hawaiian isolates of the P-gene were 83 to 88% similar toDMV.
Multiple overlapping sequencesacross the N- and P-genes werealigned to generate a 2180 bp con-sensus segment of the HawaiianCeMV genome. This se quence spansthe majority of the CeMV N- and P-genes. The P-gene region consensuswas generated using the 14 individu-
als in which CeMV was detected and sequenced.Ultimately, 3 individual cases and associatedorgans were the most useful for characterizationof the N-gene and additional P-gene regions:KW2010005 (Longman’s beaked whale) mediastinallymph, ce rebrum, and right lung; 15320 (pygmysperm whale) lung (GenBank acc. no. KM975650);and KW2010012 (Blainville’s beaked whale) cere-brum (GenBank acc. no. KM975651). Be cause theLongman’s beaked whale sequence is 98 to 100%identical to each Hawaiian isolate at each compa-rable locus (Table 4), and is identical to theHawaiian consensus, the Longman’s BWMV (Gen-
220
Fig. 1. Stranding locations of cetaceans with beaked whale cetacean morbilli -virus (BWMV) in Hawai’i. Each black dot represents the location of a strandedcetacean in which BWMV was detected. The figure was created using
ArcMap v.10.2.1 (ESRI)
Tissue type No. of ind. CeMV-positive ind. (n = 15) No. of tissue No. of CeMV Positivewith archived With archived With positive samples screened detections diagnostictissue (n = 62) tissue tissue (n = 853) (n = 35) cases (%)a
Lymph node 47 13 5 337 13 33Brain 55 13 6 115 8 40Lung 58 15 4 130 5 27Liver 59 15 4 69 4 27Spleen 55 13 2 68 3 13Kidney 58 14 1 116 1 7Skin 3 3 0 3 0 0Blubber 3 3 0 3 0 0Muscle 2 1 0 3 0 0Uterus 1 1 0 1 0 0Ovary 1 1 0 2 0 0Blood 5 2 1 5 1 7Amniotic fluid 1 1 0 1 0 0aCalculated as: [CeMV-positive individuals with this positive tissue / total no. of positive animals (= 15)] × 100
Table 3. Cetacean morbillivirus (CeMV) detection in various tissues from stranded cetaceans. A total of 853 archived tissue samples were screened; CeMV was detected in 35 tissue samples from 15 animals
-
Jacob et al.: Novel beaked whale morbillivirus in Hawaiian cetaceans
Bank acc. no. KM4600 45) was used for all furthercomparisons to known strains/isolates (Fig. 2).
Using the BLAST N GenBank algorithm andMac Vector 12.5 ClustalW sequence comparisonalgorithm, we determined that BWMV was 85.6%similar to DMV. Because of limited sequence in -formation for PMV and PWMV, the full length ofthis sequence could only be compared to DMV. Inthe 378 bp region of comparable P-gene sequence,the BWMV consensus sequence was 87.6 and85.7% similar to DMV and PWMV, respectively(Figs. 2 & 3). BWMV was also compared to the 2new regional isolates from Brazil (SWAt; Groch etal. 2014) and Australia (Swan River; Stephens etal. 2014). Nucleotide similarities based on 216 bpof P-gene sequence ranged from 70.8 to 72.7% forboth regional isolates (Figs. 2 & 3). The 1139 bp ofthe BWMV N-gene sequence was then comparedto the previously known CeMV strains DMV,PWMV, and PMV (Figs. 2 & 3). Using a 230 bpsequence common to all, the BWMV N-genenucleotide sequence was 87.8, 88.7, and 83.9%similar to DMV, PMV, and PWMV, respectively.The BWMV N-gene was also 72.7% identical to a188 bp partial sequence from the Swan River iso-late. Phylogenetic analysis of both N- and P-genesusing CDV as an outgroup indicated that BWMVclustered with the known strains DMV, PMV, andPWMV while the Swan River and SWAt isolateswere distinct (Fig. 3).
Heterogeneity of CeMV
The overall detection rate for all 3 lymph nodes thatwere tested was 27%. However, the detection ratevaried substantially between lymph nodes: 25% forKW2010012 (Blainville’s beaked whale) anal lymph;2% for KW2010012 (Blainville’s beaked whale)mediastinal lymph; and 61% for KW2011021 (rough-toothed dolphin) anal lymph.
The overall detection rate was higher in the innersub-samples (32%) compared to the outer sub- samples (26%). The outer sample detection rateswere 10% for Blainville’s beaked whale anallymph, 0% for Blainville’s beaked whale mediastinallymph, and 56% for rough-toothed dolphin anallymph. The inner sample detection rates were 100%for Blainville’s beaked whale anal lymph, 6% forBlainville’s beaked whale mediastinal lymph, and100% for rough-toothed dolphin anal lymph.
DISCUSSION
Since the initial report of a potentially unique iso-late of CeMV in the Longman’s beaked whale (Westet al. 2013), our detailed analysis of the P- and N-gene regions of the Hawaiian CeMV isolates stronglysuggest that the isolates from this study are from anew, previously uncharacterized strain of CeMV. Asof this study, this BWMV was isolated from 12 differ-
221
GenBank acc. no. DMV PWMV SWAt Swan River BWMV
DMV P-gene AJ608288 −PWMV P-gene AF200817 88.4 −SWAt (Brazilian) CeMV P-gene KF711855 75.9 75.9 −Swan River (Australian) CeMV P-gene N/A 75.9 75.9 100.0 −15063-001 (Humpback whale) KM460046 83.8 82.4 72.7 72.7 100.015320-001 (Pygmy sperm whale) KM460047 83.8 82.4 72.7 72.7 100.015487-001 (Spinner dolphin) KM460048 83.3 81.9 72.2 72.2 99.5KW2008008 (Cuvier’s beaked whale) KM460049 83.8 82.4 72.7 72.7 100.0KW2009004 (Spinner dolphin) KM460050 82.9 81.9 72.2 72.2 99.1KW2010005 (Longman’s beaked whale) KM460045 83.8 82.4 72.7 72.7 −KW2010008 (Striped dolphin) KM460051 83.8 82.4 72.7 72.7 100.0KW2010012 (Blainville’s beaked whale) KM460052 83.8 82.4 72.7 72.7 100.0KW2011001 (Bottlenose dolphin) KM975649 81.9 80.6 70.8 70.8 98.1KW2011008 (Sperm whale) KJ482570 83.3 81.9 72.2 72.2 99.5KW2011021 (Rough-toothed dolphin) KM460053 83.8 82.4 72.7 72.7 100.0KW2013015 (Spinner dolphin) KM975648 83.8 82.4 72.7 72.7 100.0KW2013023 (Risso’s dolphin) KM460054 83.8 82.4 72.7 72.7 100.0KW2014006 (Striped dolphin) KM460055 83.8 82.4 72.7 72.7 100.0
Table 4. P-gene percent nucleotide identities among the Hawaiian cetacean morbillivirus (CeMV) isolates and other distinctCeMV strains/isolates. DMV: dolphin morbillivirus; PWMV: pilot whale morbillivirus; SWAt: Southwest Atlantic Brazilian
regional isolate; Swan River: Swan River, Australia regional isolate; BWMV: beaked whale morbillivirus
-
Dis Aquat Org 117: 215–227, 2016222
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
1020
3040
5060
7080
9010
011
0C
AG
AT
GT
CA
GC
AT
CA
GA
TT
AG
TT
GA
AG
TG
GGT
CC
AA
AG
TG
AA
AA
GT
CC
TTT
AT
CA
GG
TCC
TC
AC
TT
TT
GC
AT
CC
AG
AG
GT
GGC
CA
AC
AAT
GG
AA
GGA
CGG
AA
GGC
AGG
AC
GGA
CT
AT
TT
CT
CT
AT
CA
GA
TG
TC
AG
CA
TC
AG
AT
TA
GT
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AA
GT
AA
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CA
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GA
GAA
AG
TC
CC
TA
TC
AG
GG
CT
CA
CT
TT
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CA
TC
CA
GA
GG
CGG
CT
AAA
TA
TG
GA
GG
AT
GA
GG
CG
GA
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AC
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TC
TC
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GC
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CA
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CC
AA
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AA
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CCT
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CA
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--
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-
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
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MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
120
130
140
150
160
170
180
190
200
210
220
230
TC
AG
ACC
TGG
CA
GA
GG
AG
GGG
AGG
AT
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CA
GA
GG
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CC
AA
CT
GG
TT
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AA
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GA
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AG
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CA
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AT
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CC
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GA
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AA
TT
CA
AC
AT
AC
TC
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GG
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TC
TA
TC
CT
C-
--
--
--
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--
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BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
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MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
240
250
260
270
280
290
300
310
320
330
340
GC
AC
AA
AT
TT
GG
AT
CC
TC
CCT
CGG
CC
AA
AGG
CA
GT
CA
CT
GC
TC
CG
GGA
CAA
CT
GC
GGG
CC
GGA
TTT
CG
GGA
GA
TG
AG
GC
GG
TG
GA
TT
AA
GT
AT
AC
CC
AG
CA
AC
GC
CG
TG
TA
GT
GG
GT
GA
GT
TT
CG
CA
CA
AA
TT
TG
GA
TC
CT
AT
TTA
GC
CA
AG
GC
AG
TC
AC
TG
CT
CC
AG
AT
AC
TG
CA
GC
TG
AC
TC
CGG
AG
AC
GGA
GG
CG
GT
GG
AT
TA
AA
TTA
TA
CT
CCA
GC
AA
CG
CC
GT
GT
AG
TG
GG
TG
AG
TT
TC
GC
AC
AA
AT
CTT
GG
AT
TT
TTA
CT
AG
CC
AA
GG
CA
GT
CA
CT
GC
TC
CA
GA
TA
CT
GC
AG
CT
GA
CT
CT
GGA
GA
TG
AG
GC
GG
TG
GA
TT
AA
GT
AT
AC
CC
AG
CA
AC
GC
CG
TG
TG
GGT
AGG
GT
GA
GT
TT
C-
--
--
--
--
--
--
--
--
--
--
--
--
--
--
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BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
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MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
350
360
370
380
390
400
410
420
430
440
450
460
GG
CT
TG
AT
AAA
AG
GA
TG
GT
TA
GA
TG
CT
GT
GA
GA
AA
TC
GG
AT
TG
CA
GGA
AGG
AC
TTT
GTT
CA
TTT
GA
GG
AG
AT
TC
AT
GG
TG
GC
AT
TA
AT
TT
TG
GGA
TA
TC
AG
AAA
GA
AC
AC
CA
GG
AA
AC
AA
AC
CG
GC
TT
GA
CA
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AT
GG
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AT
GC
TG
TG
AG
AA
AT
CG
GA
TT
GC
GG
AG
GA
CC
TA
TC
GT
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AG
GA
GA
TT
CA
TG
GT
GG
CA
TT
AA
TT
TT
AG
AT
AT
CA
AG
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AA
CA
CC
AG
GG
AAA
CA
AA
CC
GG
CT
CGG
AC
AA
AG
GA
TG
GT
TA
GA
TG
CC
GGT
GA
GA
AA
TC
GG
AT
TG
CG
GA
GG
AT
CCT
AT
CG
TT
GA
GA
AAG
AT
TC
AT
GG
TA
GGC
AT
TA
AT
TT
TA
GA
TA
TC
AA
AA
GG
AAC
AC
CA
GG
AA
AC
AA
AC
C-
--
--
--
--
--
--
--
--
--
--
--
--
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--
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--
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--
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--
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--
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--
--
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--
--
--
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--
--
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--
--
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--
--
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--
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CCC
--
--
--
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--
--
--
--
--
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--
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--
--
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--
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--
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--
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--
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--
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BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
470
480
490
500
510
520
530
540
550
560
570
CA
GG
AT
TG
CT
GA
GAA
TG
AT
AT
GC
GA
TA
TA
GA
CA
CA
TTA
CAA
TC
GT
CG
AG
GC
AG
GG
CCT
TG
CC
AAG
TTT
TC
AT
CC
TA
AC
TA
TT
AAA
GTT
TC
GG
GAA
TT
GA
AA
CA
AT
GT
AC
CC
AGG
CT
TTT
AG
GG
TT
GC
AG
GA
TT
GC
CGG
AG
AAT
GA
TA
TG
CG
AC
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AG
AC
AC
CTT
AT
AT
CG
TC
GA
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CA
GG
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TA
GC
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TC
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AA
AC
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CTT
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GG
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CA
GG
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GA
AAA
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GA
TA
TA
GA
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CC
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TA
TC
GT
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AG
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AG
GC
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CT
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CC
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TT
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TC
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AA
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TT
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GC
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CA
TC
CT
AA
CT
AT
CA
AA
TT
CG
GA
AT
TG
AA
AC
GAA
TG
TA
CC
CA
GGC
AC
TTT
GGG
AC
TTG
--
--
--
--
--
--
--
--
--
-TT
GT
GGA
TA
TA
GA
TAA
CA
TTA
TA
TT
GGT
TGG
AG
GC
AG
GC
CCT
CGG
CC
AAG
TTT
TC
AT
TCC
TA
AC
CAA
TC
AA
AT
TC
GG
AA
TT
GA
GAA
CA
AT
GT
AT
CCC
TGG
CT
TTT
AG
GC
CTT
T-
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
580
590
600
610
620
630
640
650
660
670
680
690
CA
TG
AA
TT
CT
CA
GG
TG
AG
TTT
AA
CT
AC
AA
TT
GA
GT
CC
TT
GA
TG
AA
TC
TC
TA
TC
AA
CA
AAA
TG
GG
CG
AG
AC
TG
CA
CC
GT
AC
AT
GG
TG
AAT
TC
TT
GA
AA
AC
TC
GAA
TT
CA
GA
AT
AAA
ATT
TC
AC
AT
GA
AT
TC
TC
GGG
GT
GA
AT
TA
AC
TA
CA
GTT
TG
AG
TC
TC
TTA
AAT
GA
AC
CCT
CT
AC
CCA
AC
AG
AT
GG
GC
GA
GA
CT
GC
AC
CG
TA
CA
TG
GT
AA
TT
CT
TG
AA
AA
CT
CA
AAT
TC
AG
AA
CA
AG
TT
CA
CA
TG
AA
TT
CT
CA
GG
TG
AA
TT
AA
CC
AAC
GAA
TC
GGA
GT
CC
TT
GA
TG
AA
TC
TC
TA
TC
AA
CA
GA
TG
GG
CG
AG
AC
TG
CA
CC
GT
AC
AT
GG
TG
AAT
CCC
TC
GGA
AA
AC
TC
TAA
TT
CA
GA
AC
AA
GT
TC
AC
AT
GA
AT
TT
GCC
CGG
GA
GGA
AT
TA
AC
TA
CA
AT
TG
AG
TC
CT
TG
AT
GA
AC
CCT
CT
AT
CA
AC
AG
AT
GG
GC
GA
GA
CT
GC
TCC
CG
TA
CA
TG
GT
AA
TT
CT
CGG
AA
AA
CT
CA
AAT
CCC
AG
AA
CA
AG
TT
--
CA
TG
AG
TTT
CT
CA
GG
TG
AG
TTT
GAA
CC
AAC
CAA
TT
GA
GT
CA
CTT
TAA
TG
AA
TT
TTG
TTA
TC
AA
CA
GA
TG
GG
AGG
AG
AC
TG
CA
CC
GT
AC
AT
GG
TA
AT
TC
TT
G-
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
700
710
720
730
740
750
760
770
780
790
800
GT
GC
GGG
GT
TC
AT
AC
CC
CCC
TA
TT
GT
GG
AG
CT
AT
GC
AA
TG
GG
AG
TT
GG
GGG
TT
GA
AC
TT
GA
GAA
AT
TC
AA
TG
GG
TG
GA
CT
TA
AT
TT
CGG
GT
CG
TT
CT
TA
TTT
TT
GA
CC
CT
GC
GTT
AT
TT
CA
GG
TG
CA
GG
TT
CA
TA
CC
CG
CCT
AT
TA
TTG
GA
GC
TA
TG
CA
AT
GG
GA
GT
TG
GA
GT
TG
AA
CT
TG
AA
AA
TT
CA
AT
GG
GT
GG
AC
TT
AA
TT
TT
GG
TC
GT
TC
TT
AC
TT
CGG
AC
CC
TG
CA
TA
TT
TC
AG
GT
GC
AG
GT
TC
AT
AC
CC
AT
TTA
CTT
GT
GG
AG
CT
AT
GC
AA
TG
GG
AG
TT
GG
AG
TT
GA
GCC
TT
GA
AA
AC
TTC
AA
TG
GG
TG
GA
CT
TA
AT
TT
TG
GT
CG
TT
CT
TA
CT
TT
GA
CC
CT
GC
AT
AT
TT
CA
G-
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
810
820
830
840
850
860
870
880
890
900
910
920
AT
TA
GG
CCC
AA
GA
GA
TG
GT
CA
GG
AG
AT
CA
GC
AG
GT
AA
GG
TA
AAG
CT
CA
TC
AC
TC
GGC
TG
CA
GA
AC
TG
GGG
GA
TC
AC
AG
CC
GA
AGG
AC
GC
GAA
AA
CT
CGG
TT
TC
TGG
AG
AT
TG
CT
GC
ACC
AG
GC
CA
CTT
GGG
GT
CA
AG
AG
AT
GG
TC
AG
GA
GA
TC
AG
CA
GG
CAA
AG
GT
GA
GC
TC
AT
CG
CCT
AG
CC
GGC
AG
AA
CT
AG
GG
AT
CA
CA
GC
CG
AG
GA
CG
CC
AA
AC
TT
GT
CTT
CC
GA
GA
TT
GC
TG
CG
CA
GG
CT
GTT
TA
GG
TC
AA
GA
GA
TG
GT
CA
GG
AG
AT
CA
GC
AG
GT
AA
GG
TG
AG
CT
CA
TC
AC
TA
GC
TG
CA
GA
AT
TTA
GG
GA
TC
AC
AG
CT
GGA
GG
AT
GGC
CA
AA
CT
TG
TT
TC
CG
AG
AT
TG
CT
GC
GC
AG
GC
T-
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
930
940
950
960
970
980
990
1000
1010
1020
1030
AA
CG
AT
GGA
CA
GA
GC
AG
GC
AAG
AG
CA
GTT
GGG
GT
CC
CA
AG
CCA
GAA
AC
CA
GA
TA
TC
AT
TC
CCT
TC
AT
CC
TG
AT
AAG
AG
GC
GA
ATT
CC
AA
TTG
TTT
CC
AG
GG
AA
TA
AT
TCC
TC
GT
GT
AAA
AT
GA
GA
ATT
GA
AT
GGA
CG
AC
AG
AG
CT
AA
TA
GA
GC
AA
TA
GG
TC
CC
AA
AC
AA
AA
CC
AG
AT
AT
CG
TTT
TC
TT
CA
TC
CT
GA
CA
GA
GG
CG
AT
GCC
CA
GT
ACC
TC
CA
GG
GA
AC
AAT
CC
TTT
CG
CGG
CA
AA
CGG
AG
GG
TG
AA
CG
AC
GA
CA
GA
GC
TA
AT
AG
GGG
CA
AT
AG
GT
CC
GAA
GAA
CA
AA
AC
CA
GG
TTA
TC
AT
TT
CT
TC
AT
CC
TG
AC
AG
AG
GA
GGA
GTT
CT
AAG
TG
CT
CC
GGG
GG
AA
TA
TC
CC
TA
GGT
GC
AA
AT
GA
AGG
GC
GG-
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
Fig
. 2.
Seq
uen
ce a
lig
nm
ent
of b
eak
ed w
hal
e ce
tace
an m
orb
illi
viru
s (B
WM
V)
wit
h t
he
5 d
isti
nct
cet
acea
n m
orb
illi
viru
s (C
eMV
) st
rain
s/is
olat
es.
Th
e 21
80 b
p B
WM
Vco
nse
nsu
s se
qu
ence
sp
ann
ing
mos
t of
th
e N
- an
d P
-gen
es (
Gen
Ban
k a
cc. n
o. K
M46
0045
) is
ali
gn
ed w
ith
dol
ph
in m
orb
illi
viru
s (D
MV
; Gen
Ban
k a
cc. n
o. A
J608
288)
, th
ep
arti
al N
-gen
e se
qu
ence
of
the
por
poi
se m
orb
illi
viru
s (P
MV
; G
enB
ank
acc
. n
o. A
Y94
9833
), t
he
par
tial
P-
and
N-g
ene
seq
uen
ces
of t
he
pil
ot w
hal
e m
orb
illi
viru
s(P
WM
V;
Gen
Ban
k a
cc.
no.
AF
200 8
17 a
nd
AF
2008
18),
th
e p
arti
al P
-gen
e se
qu
ence
of
the
Sou
thw
est
Atl
anti
c (B
razi
lian
) is
olat
e (S
WA
t; G
enB
ank
acc
. n
o. K
F71
1855
),an
d th
e p
arti
al P
- an
d N
-gen
e se
qu
ence
s of
the
Au
stra
lian
(Sw
an R
iver
) iso
late
. Bas
ed o
n D
MV
gen
ome
anal
ysis
, th
e N
-gen
e op
en r
ead
ing
fram
e (O
RF
) en
ds
at 1
139
bp
(*),
a 2
80 b
p n
on-c
odin
g r
egio
n b
etw
een
the
2 g
enes
exi
sts
from
114
0 to
141
9 b
p, a
nd
the
P-g
ene
OR
F s
tart
s at
142
0 b
p (*
*). D
ash
es (−
) in
dic
ate
no
seq
uen
ce is
ava
ilab
le
Fig
. 2 c
onti
nu
ed o
n n
ext
pag
e
-
Jacob et al.: Novel beaked whale morbillivirus in Hawaiian cetaceans 223
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
1160
1170
1180
1190
1200
1210
1220
1230
1240
1250
1260
GA
CA
GT
AAC
TG
AC
GA
CC
CG
AAT
TA
GC
AAC
CC
AA
AA
GT
CA
ACC
CG
AG
GC
CTT
TG
GC
CA
AG
GTT
GA
AAA
AG
CCA
AT
GGG
CCG
TA
AA
GG
TTT
GC
TA
GAA
AG
ACC
CAA
GAA
ACC
CC
CG
CG
TG
AT
GCC
CA
CCA
GC
GC
AG
AC
AA
CA
CT
GA
CG
AC
CC
AA
TT
AG
TA
TCC
CA
AA
AA
TTC
AG
CT
GGA
GG
CA
TT
AGG
CC
AA
GA
TG
AG
AG
CC
AT
GG
CC
--
-AA
AG
CT
AT
TTG
GGA
AA
AC
CA
-AA
GGG
CC
CG
CG
TTG
AT
GT
CCA
CT
GGC
GC
AC
GA
CA
AC
GCC
TG
AT
GGA
TCC
CA
GTT
TG
GGT
AC
CC
AA
AA
GT
CA
GC
CG
AG
GC
AT
TG
GC
CA
GGG
AT
GA
GA
GC
CA
TG
GC
C-
--
AAA
GC
TA
CTT
AGG
AA
AA
CC
A-
AAA
GC
TCC
GC
ATT
GA
CA
CCC
AC
CG
CG
CA
T-
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
1270
1280
1290
1300
1310
1320
1330
1340
1350
1360
1370
1380
TG
TTC
TA
TAA
AT
GA
TAA
AA
GA
CCC
TA
CTT
TGG
GC
TG
AAA
TA
CA
AA
AG
TT
TTC
TG
CCC
TC
AAA
GA
AAG
CA
CCC
AG
AT
TTT
CA
TT
TTT
TT
CA
TC
AG
TA
TT
AC
AA
AA
AA
CT
TA
GG
AC
CA
AA
GT
CC
AA
AC
TAA
TG
TT
TA
TA
AT
GGA
TAA
AA
GA
TCC
TA
CT
TG
GC
TG
AAA
--
-CC
AA
AA
GG
AT
CC
AC
TC
TG
AG
CA
GC
GT
CA
GA
CT
TT
GTT
AT
TT
TC
AT
CA
ATT
AT
TA
CA
AA
AA
AC
TT
AG
GA
CC
AA
AG
TC
CA
AG
GAA
AT
GT
TT
AT
AA
CGG
AC
AAA
AG
AC
CCT
AC
TC
GGG
CT
AAA
A-
--
CCA
AA
AG
GA
CCC
CA
TTT
CT
GA
GC
AG
CG
TT
AAT
AAC
TT
CA
CAA
CTT
TT
CA
TC
AG
TA
TT
AC
AA
AA
AA
CT
TA
GG
AC
CA
AG
GGT
CC
CAA
AA
AAA
T-
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
1390
1400
1410
1420**
*
1430
1440
1450
1460
1470
1480
1490
CG
AA
CT
CC
AC
AC
CCA
TC
TTC
CTT
CC
AC
CA
AAC
AC
CA
TC
CC
CA
AA
TG
GC
AG
AA
GGA
GC
AG
GC
CC
AAT
CA
TG
TC
AG
TTA
AG
GG
AC
TC
GGA
GT
GC
CCT
CA
GG
TTC
TC
TC
AG
AG
AG
AAA
TC
CG
CC
CG
AT
GT
GGA
CCC
TC
CA
CA
TC
AT
TT
CA
TTC
CA
CC
GA
CA
CC
AT
CC
CC
AA
AT
GG
CA
GA
GGG
AG
CA
GG
CC
TAA
TC
AT
GT
CA
ATT
AA
GG
GA
CT
TGG
AG
TG
TCC
TC
AA
ATT
CT
CT
CA
GA
GA
AAA
AT
CC
GC
CC
GA
TG
CG
AA
CT
CC
AC
AT
CA
TT
TC
TTT
CC
AC
CG
AC
AC
TG
TTC
TCC
CA
A-
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
1500
1510
1520
1530
1540
1550
1560
1570
1580
1590
1600
1610
CT
GT
TGG
AG
AAT
TA
AA
GGA
GGG
CT
CCA
AA
TC
AAT
CC
GA
GGG
CA
AA
ACC
AGG
CC
GA
TGG
AG
GGA
AT
CA
AG
CG
AG
AG
TC
AC
CCA
CC
AG
GA
CA
AC
TC
AA
AAA
AC
GGG
AC
AC
TC
TT
GA
TT
TC
GA
CG
AA
TC
AT
GC
TG
TC
GGA
AAA
TT
AA
GGG
AA
GGC
CCC
AA
AT
TAA
TC
CG
AA
GGC
AA
GG
CCC
GGC
CT
GC
GGA
AGG
AA
TC
AA
GC
GA
GA
GT
CA
TCC
AC
CA
GG
AC
AA
CT
CC
GAA
AA
AA
GGA
CA
CT
CT
TG
AT
TT
CG
AC
GA
AT
CA
TG
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
BW
MV
N a
nd P
-gen
esD
MV
N a
nd P
-gen
esP
MV
N-g
ene
PW
MV
N a
nd P
-gen
esS
wan
Riv
er N
and
P-g
enes
SW
At P
-gen
e
1620
1630
1640
1650
1660
1670
1680
1690
1700
1710
1720
CT
CT
TC
AG
CA
AT
TG
GGA
CC
AG
AA
GCC
GT
AC
CG
CA
TG
CTT
AC
TT
GG
TG
AT
GA
TA
CA
GG
AC
TTT
GGG
AA
AAA
CC
AA
GGT
TA
CA
TC
CC
TA
CTT
GA
TGG
GA
GA
GC
TCC
AAA
GC
CA
GG
AG
AC
TTT
GGG
GA
AA
AC
TC
TT
CA
GC
AA
TT
AGG
AC
CA
GA
AA
CCG
TA
CC
GC
AT
GT
TTA
CT
TG
GT
GA
TG
AT
AC
AG
GA
TTT
TA
GGA
GC
AAC
CA
GGG
TT
AC
AT
CC
CT
AA
TTG
AA
GGG
AG
AG
CC
CCG
AAG
CC
AG
GA
GA
CA
TTC
GGG
AA
AG
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--