Complete Genome Sequencing of the Divergent Guiana Dolphin Morbillivirus (GDMV), Brazil

Abstract

Cetacean morbillivirus (CeMV) is a major threat to cetaceans worldwide, causing individual deaths and outbreaks of mass mortality. Based on partial sequences of the viral phosphoprotein, CeMV is subclassified into seven strains and two distinct lineages. To date, only CeMV-1 strains, including the dolphin morbillivirus (DMV), have been completely sequenced. The CeMV-2 lineage was first reported in Guiana dolphins (Sotalia guianensis) in Brazil and was associated with an unusual mortality event in 2017-2018. Here we provide the nearly complete Guiana dolphin morbillivirus (GDMV) genome sequence, representing the first within the CeMV-2 lineage. GDMV was isolated using Vero.DogSLAMtag cells, the viral RNA was extracted, and deep sequencing analysis was performed. Gaps in the viral genome were completed by Sanger sequencing. The final genome length was 15,607 nucleotides covering 99.3% of the DMV reference genome, including full sequences of the six structural proteins encoded by morbillivirus. The sequence similarity was 74-77.9% to other CeMV strains, with highest identity to the DMV. The complete L protein amino acid sequence comparison-based taxonomy indicates that GDMV is a distinct morbillivirus species; however, as GDMV and CeMV-1 strains infect a similar host spectrum, our findings support that GDMV represents a new CeMV-2 lineage.

Keywords: Sotalia guianensis; cell culture; cetacean morbillivirus; epizootics; marine mammal stranding; mortality outbreak; virus isolation.

Figure 1

(A) Vero/dog SLAM, mock infected cells, 10×, 40 h; (B) Vero/dog SLAM, syncytium. 10×, 40 h post infection (p.i.); (C) Vero/dog SLAM, syncytium and central area of cell loss surrounded by syncytia; 4×, 96 h p.i.; (D) Immunohistochemistry for morbillivirus using an antibody from canine distemper virus nucleoprotein. There is strong positive nuclear and cytoplasmic immunolabeling of Vero/dog SLAM cells syncytia. 4×; 96 h p.i.

Figure 2

Unrooted phylogenetic tree showing complete genome nucleotide sequences of the cetacean morbillivirus (CeMV) isolate found in a stranded Guiana dolphin (Sotalia guianensis) from Rio de Janeiro, Brazil, 2017, and those of other previously described morbilliviruses. The phylogram was generated by the neighbor-joining method, bootstrap of 1000 replicates, with a total of 15,020 bp. The tree is drawn to scale, with branch lengths and the evolutionary distances computed using the p-distance method. For comparison, recognized CeMV strains were included when available. CeMV-1 and CeMV-2 represent the two distinct CeMV lineages. The sequence names include GenBank accession number, species of cetacean, year of stranding, and location. The scale bar indicates nucleotide substitutions per site. Abbreviations for each part of the sequence name are as follows: Bp, Balaenoptera physalus; Gma, Globicephala macrorhynchus; La, Lagenorhynchus albirostris; Pp, Phocoena phocoena; Sc, Stenella coeruleoalba; Sg, Sotalia guianensis. Abbreviations for each CeMV strain or morbillivirus species are as follows: CDV, canine distemper virus; DMV, dolphin morbillivirus; FeMV, feline morbillivirus; GDMV, Guiana dolphin morbillivirus; MeV, measles virus; MBaMV, Myotis bat morbillivirus, PDV, phocine distemper virus; PMV, porpoise morbillivirus; PoMV, porcine morbillivirus; PPRV, peste des petits ruminants virus; PWMV, pilot whale morbillivirus; RPV, rinderpest virus.

Figure 3

Estimates of evolutionary divergence based on amino acid p-distances per gene between GDMV_SG_2017 and available CeMV sequences representative of DMV (MH430933.1), PWMV (ON492026), and PMV (MH430945.1) strains.

Figure 4

Phylogenetic analysis of complete L protein amino acid sequences of cetacean morbillivirus (CeMV, Morbillivirus ceti) isolate found in a stranded Guiana dolphin (Sotalia guianensis) from Rio de Janeiro, Brazil, 2017, and those of other previously described morbilliviruses. Complete L protein amino acid sequences were aligned by Clustal W with gap generation penalties of 5 and extension penalties of 1 in both multiple and pairwise alignments. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 23 amino acid sequences with a total of 2201 positions. The sequence names include GenBank accession number, species of cetacean, year of stranding, and location. The scale bar indicates nucleotide substitutions per site. DMV, dolphin morbillivirus; GDMV, Guiana dolphin morbillivirus; PMV, porpoise morbillivirus; PWMV, pilot whale morbillivirus.