Evolutionary evidence for multi-host transmission of cetacean morbillivirus

Figures & data

Cetacean morbillivirus detection in cetacean samples from the North Sea and the Mediterranean Sea

CeMV strain	Variant	Stranding year	Location	Host	Sample material	RT-PCR (Ct)	NGS coverage	GenBank accession no.	References
PMV	Ulster/88	1988	Northern Ireland, U.K.	Harbor porpoise	Vero cells	NI	5149×	MH430942	2
DMV	16A	1990	Spain	Striped dolphin	Lung	12	6023×	MH430934	28
DMV	16A-cc	1990	Spain	Striped dolphin	Vero cells	13	27×	MH430932	This study
DMV	16A-cc-vdol	1990	Spain	Striped dolphin	Vero-dolSLAM cells	13	1188×	MH430933	This study
DMV	muc	1990	Spain	Striped dolphin	Lung	13	1248×	MH430935	28
DMV	muc-cc	1990	Spain	Striped dolphin	Vero cells	13	20×	MH430936	This study
PMV	53	1990	Netherlands	Harbor porpoise	Vero cells	NI	9120×	MH430943	7
PMV	2990	1990	Netherlands	Harbor porpoise	Brain	32	26×	MH430945	7
PMV	2990-cc	1990	Netherlands	Harbor porpoise	Vero cells	NI	11,933×	MH430944	This study
DMV	DE/2007	2007	Germany	White-beaked dolphin	Brain	19	283×	MH430940	48
DMV	156	2010	Italy	Striped dolphin	Lung	19	50,169×	MH430937	This study
DMV	DMV/LA/NL/11.2	2011	Netherlands	White-beaked dolphin	Lung	23	21×	MH430941	24
DMV	DMV_Bph	2013	Italy	Fin whale	Brain	32	144×	MH430938	14, 49
DMV	DK/16^a	2016	Denmark	Fin whale	Lung	27	NI	MH430939	16

Ct value ≤ 40 = positive; Ct value > 40 = negative

RT-PCR reverse transcriptase PCR, Ct value threshold cycle, NGS next-generation sequencing, NI not investigated

^aGenome obtained by Sanger sequencing

Fig. 1 Recovery of full-length CeMV sequences by NGS and RACE.

a Genome coverage of wild-type CeMVs. The scale on the left indicates sequencing depth and genome regions are color-coded according to CeMV gene positions. b Sequencing chromatograms of leader and trailer regions of wild-type CeMV strains DMV-16A and PMV-2990 using RACE

Fig. 2 Bayesian phylogenetic analysis of full-length CeMVs excluding L sequences. Most common recent ancestor ages are presented at the nodes with posterior values > 0.7 shown in parenthesis. New CeMV genomes generated during this study are presented with circles at the tips. Branches were truncated for graphical reasons. The taxon names are presented as host-country/year of collection_variant_GenBank accession number. Branch colors represent virus strains. New CeMV variants and GenBank accession numbers Bph (MH430938), 156 (MH430937), DK/16 (MH430939), 16A (MH430934), muc (MH430935), DE/2007 (MH430940), NL/11.2 (MH430941), 2990 (MH430945), 53 (MH430943), and Ulster/88 (MH430942). Bp Balaenoptera physalus, La Lagenorhynchus albirostris, Pp Phocoena phocoena, Sc Stenella coeruleoalba, Sl Stenella longirostris, and Tt Tursiops truncatus

Fig. 3 Phylogeography of CeMV.

a Bayesian phylogenetic analysis of partial P genes (400 bp). Most common recent ancestor ages are presented at the nodes in cursive with 95% highest posterior density interval values in brackets and as gray horizontal bars. Posterior values > 0.5 are shown in parenthesis. CeMV genomes in this study are presented with black circles at the tips. Each branch is color-coded according to the ocean/sea in which the cetaceans were found. The taxon names are presented as virus_host/Country-year of collection/variant_GenBank accession number_ID for b. Sequences 17* and 24* were extracted from a published paper²². Sequence 21* was kindly provided by Dr. Stone and Jianning Wang. Host abbreviations: Bp Balaenoptera physalus, Dd Delphinus delphis, Gg Grampus griseus, Gm Globicephala melas, Ip Indopacetus pacificus, Kb Kogia breviceps, La Lagenorhynchus albirostris, Mm Monachus monachus, Mn Megaptera novaeangliae, Pp Phocoena phocoena, Sb Steno bredanensis, Sc Stenella coeruleoalba, Sg Sotalia guianensis, Sl Stenella longirostris, Tt Tursiops truncatus, and Zc Ziphius cavirostris. b A world map of CeMV migration using sequences from a. Locations of viruses in the map are relative and were obtained from the publications in which the sequences were published. The hashtag (#) indicates no sequence availability (No. 32: CeMV-5 from AU/2010 and No. 33: CeMV-1_JP/1999). Proposed virus migratory routes (dashed lines) were based on sequence clades from the phylogenetic tree in a

Sequence variation between wild-type and isolated CeMV strains

Strain-variant	Gene	Position (ORF)	Codon	Wild-type	Vero	V. dolSLAM	Mutation
DMV-16A	Leader	26	—	a	a/t	a	g.26A>T
	P	693	231	c	g	c	dS
	P	1375	459	Ser^1	Pro	Pro	S459P
	M	310	104	Thr	Ala	Thr	T104A
	M	502	168	Asn	His	Asn	N168H
	F	1339	447	Arg	Gly	Arg	R447G
	L	2618	873	Ile^2	Thr	Thr	I873T
DMV-muc	Leader	11	—	g	a	—	g.11G>A
	M	523	175	Arg	Gly	—	R175G
	M	681	227	Lys	Asn	—	K227N
PMV-2990	M	265	89	Glu	Lys	—	E89K
	H	224	75	Asn	Asn	—	S75N
	L	406	126	Trp	Trp	—	G126W
	L	4125	1375	t	c	—	dS

dS Synonymous substitution

Fig. 4 Amino acid changes between DMV variants. Defined molecular signatures of each monophyletic group are color-colored. Single changes not representing the group are shown in red. The asterisk denotes sequences obtained from outbreaks. The taxon names are presented as host-Country/year of collection_variant (first three characters). a comparison of amino acid changes in the nucleoprotein, phosphoprotein, matrix, fusion and hemagglutinin in the complete set of DMV genomes. b Comparison of amino acid changes in the L protein. DMV variants and GenBank accession numbers: Bph (MH430938), 156 (MH430937), Sc-ES/2007 (HQ829973), Gm-ES/2007(HQ829972) IZSPLV (MF589987), 631MMS031711 (KU720625); BCF20110815-LA001 (KU720624), GW2010007A (KU720623), DK/2016 (MH430939), 16A (MH430934), muc (MH430935), ref (AJ608288), DE/2007 (MH430940), and 11.2 (MH430941). Host abbreviations: Bp Balaenoptera physalus, La Lagenorhynchus albirostris, Pp Phocoena phocoena, Sc Stenella coeruleoalba, Sl Stenella longirostris, and Tt Tursiops truncatus

Fig. 5 CeMV use of the seal SLAM receptor.

a Characterization of SLAM expression on Vero cells. Vero-dolSLAM cells (teal colored), Vero-phocaSLAM cells (purple colored) and untransfected Vero cells (gray line) were stained with a HA-tag antibody and analyzed by flow cytometry. b Cell-to-cell fusion activity of DMV, CDV-dog/2016, and CDV-phoca/1988 glycoproteins in Vero-dolSLAM cells (left) and Vero-phocaSLAM cells (right) as measured by β-galactosidase activity. Relative luminescence unit (RLU) values on the Y-axis were calculated by subtracting the RLU values obtained from Vero-dolSLAM or Vero-phocaSLAM cells with values generated from Vero cells (control). Experiments were performed in quadruplicates (n = 3). c Multistep growth curve analyses of DMV-16A inoculated at an moi of 0.001 in Vero cells (control), Vero-dolSLAM and Vero-phocaSLAM cells. Virus titers were determined in triplicates

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