# Genetic Relatedness of Dolphin Rhabdovirus with Fish Rhabdoviruses

Jurre Y. Siegers, Marco W.G. van de Bildt, Cornelis van Elk, Anita Schürch, Noël Tordo, Thijs Kuiken, Rogier Bodewes, Albert D.M.E. Osterhaus

► To cite this version:

Jurre Y. Siegers, Marco W.G. van de Bildt, Cornelis van Elk, Anita Schürch, Noël Tordo, et al. Genetic Relatedness of Dolphin Rhabdovirus with Fish Rhabdoviruses. Emerging Infectious Diseases, 2014, 20 (6), pp.1081-1082. 10.3201/eid2006.131880. pasteur-04840346

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Distributed under a Creative Commons Attribution 4.0 International License
9. Sumption KJ, Wright DJ, Cutler SJ, Dale BA. Human ehrlichiosis in the UK. Lancet. 1995;346:1487-8. http://dx.doi.org/10.1016/S0140-6736(95)92502-3

Address for correspondence: Peter Hagedorn, Robert Koch Institute, Nordufer 20, 13353, Berlin, Germany; email: hagedornP@rki.de

**Genetic Relatedness of Dolphin Rhabdovirus with Fish Rhabdoviruses**

**To the Editor:** Rhabdoviruses are enveloped, single-stranded, negative-sense RNA viruses that comprise a large and diverse family in the order *Mononegavirales* and infect arthropods, plants, fish, and mammals. There are 9 genera of rhabdoviruses: *Cytorhabdovirus*, *Ephemerovirus*, *Lys savirus*, *Novirhabdovirus*, *Nucleorhab-dovirus*, *Perhabdovirus*, *Sigmavirus*, *Tibrovirus*, and *Vesiculovirus*. In addi-tion, a substantial number of plant, ver-tebrate, and invertebrate rhabdoviruses have not been classified (1). Three gen- era (*Novirhabdovirus*, *Perhabdovirus*, and *Vesiculovirus*) comprise members that infect fresh water and marine fish (2). Fish rhabdoviruses pose a serious problem for aquaculture because of worldwide outbreaks of disease caused by novirhabdoviruses, perhabdovirus-es, and vesiculoviruses (3,4).

In 1992, a rhabdovirus-like virus was isolated from lung and kidney of a white-beaked dolphin (*Lagenorhyn-chus albirostris*) that had beached along the coast of the Netherlands (5). Although no macroscopic or mi-croscopic lesions were observed at necropsy, negative contrast electron microscopy showed typical rhabdo-virus-like, bullet-shaped particles in

Vero cell cultures that showed a fo-cal cytopathic effect (5). After this rhabdovirus-like virus was injected intracerebrally into brains of 1-day-old suckling mice, they died within 5 days (5). We report genetic and phylogenetic characterization of a dolphin rhabdovirus (DRV) and eval-uated the seroprevalence of DRV-neutralizing antibodies by using se-rum samples from various marine mammals collected during a 10-year period (2003-2012).

To characterize DRV, we per-formed random sequence amplifica-tion and deep sequencing with the 454 GS Junior Instrument (Roche, Basel, Switzerland) with DRV-infected Vero cell supernatants as described (6). From this analysis, we determined the complete coding sequence of DRV covered by 42,080 of 49,292 reads (minimum coverage 4 reads, average coverage 872 reads).

Genomic termini of DRV were determined by using a 3' and 5' rapid amplification of cDNA ends PCR and Sanger sequencing of obtained PCR amplicons. The complete ge-nome of DRV (GenBank accession no. KF958252) consists of 11,141 nt and has a typical rhabdovirus gene arrangement of 5 major open reading frames (ORFs) in the order 3'-nucleoprotein (N), phosphoprotein (P), matrix (M) protein, glycoprotein (G), and large (L) protein-5' (Figure, panel A, Appendix, wwwnc.cdc.gov/EID/article/20/6/13-1880-F1.htm). No additional ORFs ≥300 nt were detected. Between the major ORFs of DRV, intergenic sequences were present that ranged in size from 34 (P–M) to 83 (G–L) nucleotides. Pu-tative transcription initiation and transcription termination polyadenyl-ation sequences were AACA(G/U) and AUGA<sub>7</sub>, respectively.

The deduced amino acid se-quence of genes of DRV and several other rhabdoviruses were aligned by using MUSCLE in MEGA5 version 5.2) (7). Ambiguous aligned regions

were removed by using the Gblocks program (8). Phylogenetic analysis of the L and G genes was performed by using the neighbor-joining method in MEGA5 (7). This analysis showed that DRV is most closely related to fish rhabdoviruses of the genera *Per-habdovirus* and *Vesiculovirus* and unassigned fish rhabdoviruses with strong bootstrap support (Figure, panels B–D, Appendix).

Deduced amino acid sequences of the 5 major genes had the high-est, although weak, homology with those of various fish rhabdoviruses by pairwise identity analyses: N (48%) with hybrid snakehead virus (HSHV), *Monopterus albus* rhabdovirus (MARV), and *Siniperca chautsi* rhab-dovirus (SCRV); P (18%–20%) with eel virus European X (EVEX), HSHV, MARV, and SCRV; M (27%–33%) with lake trout rhabdovirus, Swedish sea trout rhabdovirus, and EVEX; G (30%–32%) with perch rhabdovirus, lake trout rhabdovirus, Swedish sea trout rhabdovirus, HSHV, MARV, SCRV, and EVEX; and L (54%–56%) with perch rhabdovirus, HSHV, and EVEX. This close relationship with fish rhabdoviruses is surprising be cause DRV was isolated from tissues of a mammal and propagated in mam-malian cell lines at 37°C, which does not occur with related viruses isolated from fish.

To evaluate whether DRV or re-lated viruses circulate among species of cetaceans, we performed serologic screening by using a virus neutraliza-tion assay as described (5). The speci-ficity of this assay was tested by using a panel of rhabdovirus-specific anti-sera obtained from cetaceans of vari-ous species (5). The serum samples had been collected for diagnostic pur-poses from mainly juvenile cetaceans stranded along the coast of the Neth-erlands during 2003–2012. These spe-cies included 2 Atlantic white-sided dolphins (*Lagenorhynchus acutus*), 79 harbor porpoises (*Phocoena pho-coena*), 9 striped dolphins (*Stenella*
coeruleoabla), and 6 white-beaked dolphins (*Lagenorhynchus albirostris*). Serum samples from 145 bottlenose dolphins (*Tursiops truncates*) from the collection of the Dolphinarium Harderwijk (Harderwijk, the Netherlands) were also tested. DRV-neu-tralizing antibodies were detected in serum samples from 1 bottlenose dolphin (7%), 5 striped dolphins (55%), 1 white-beaked dolphin (17%), and 3 harbor porpoises (4%). These results suggested that DRV or closely related viruses continue to infect members of cetacean species (6).

Although rhabdovirus evolution-ary pathways are complicated (9), our analysis suggests that DRV is a possible derivative of fish rhabdoviruses. DRV might have originated from an uniden-tified fish rhabdovirus and might cycle between fish and marine mammals, similar to that suggested for cycling of vesicular stomatitis virus between arthropods and terrestrial mammals (10). Future analyses of sequences from other marine mammal rhabdovirus se-quences might support the validity of our phylogenetic analysis and result in creation of a new group containing ma-rine mammal rhabdoviruses.

This study was supported by the Eu-
ropean Community Seventh Framework
Program (FP7/2007–2013) under the
project European Management Platform
for Emerging and Re-emerging Infectious
Disease Entities, European Community
grant agreement no. 223498, and by the
Virgo Consortium.

Jurre Y. Siegers,
Marco W.G. van de Bildt,
Cornelis E. van Elk,
Anita C. Schürch, Noël Tordo,
Thijs Kuiken, Rogier Bodewes,
and Albert D.M.E. Osterhaus

Author affiliations: Erasmus Medical Centre, Rotterdam, the Netherlands (J.Y. Siegers, M.W.G van de Bildt, C.E. van Elk, A.C. Schürch, T. Kuiken, R. Bodewes, A.D.M.E. Osterhaus); Dolphinarium Harderwijk, Harderwijk, the Netherlands (C.E. van Elk);

SOS Dolphin Foundation, Harderwijk, the Netherlands (C.E. van Elk); Institut Pasteur, Paris, France (N. Tordo); and Viroclinics Biosciences, Rotterdam, the Netherlands (A.D.M.E. Osterhaus)

DOI: http://dx.doi.org/10.3201/eid2006.131880

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2. Hoffmann B, Beer M, Schütze H, Mettenleiter TC. Fish rhabdoviruses: molecular epidemiology and evolution. Curr Top Microbiol Immunol. 2005;292:81–117. http://dx.doi.org/10.1007/3-540-27485-5_5

3. Crane M, Hyatt A. Viruses of fish: an overview of significant pathogens. Viruses. 2011;3:2025–46. http://dx.doi.org/10.3390/v3112025

4. van Beurden SJ, Engelsma MY, Roozenburg I, Voorbergen-Laarman MA, van Tulden PW, Kerkhoff S, et al. Viral diseases of wild and farmed European eel *Anguilla anguilla* with particular reference to the Netherlands. Dis Aquat Organ. 2012;101:69–86. http://dx.doi.org/10.3354/dao02501

5. Osterhaus AD, Broeders HW, Teppema JS, Kuiken T, House JA, Vos HW, et al. Isolation of a virus with rhabdovirus morphology from a white-beaked dolphin (*Lagenorhynchus albirostris*). Arch Virol. 1993;133:189–93. http://dx.doi.org/10.1007/BF01309754

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7. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9. http://dx.doi.org/10.1093/molbev/msr121

8. Talavera G, Castresana J. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol. 2007;56:564–77. http://dx.doi.org/10.1080/10635150701472164

9. Kuzmin IV, Novella IS, Dietzgen RG, Padhi A, Rupprecht CE. The rhabdoviruses: biodiversity, phylogenetics, and evolution. Infect Genet Evol. 2009;9:541–53. http://dx.doi.org/10.1016/j.meegid.2009.02.005

10 Novella IS, Ebendick-Corpus BE, Zárate S, Miller EL. Emergence of mammalian cell-adapted vesicular stomatitis virus from persistent infections of insect vector cells. J Virol. 2007;81:6664–8. http://dx.doi.org/10.1128/JVI.02365-06

Address for correspondence: Rogier Bodewes, Department of Viroscience, Erasmus Medical Centre, Dr. Molewaterplein 50, 3015GE Rotterdam, the Netherlands; email: r.bodewes@erasmusmc.nl

Genetic and
Ecologic Variability
among Anaplasma
phagocytophilum
Strains,
Northern Italy

To the Editor: The tick-borne pathogen *Anaplasma phagocytophilum* is an increasing potential public health threat across Europe. Its intra-specific genetic variability is associated with different reservoir host and vector tick species (1–4); however, the roles of various vertebrates as competent reservoirs of *A. phagocytophilum* in Europe need clarification (1). During March 2011–June 2013, we studied the prevalence and genetic variability of *A. phagocytophilum* in 821 questing *Ixodes ricinus* ticks (155 adults [A], 666 nymphs [N] collected by standard blanket dragging) and 284 engorged ixodid ticks (61A, 191N, 21 larvae [L]) collected from humans, dogs, sheep, hunted wild ungulates, live-trapped birds, and rodents. Blood samples from 1,295 rodents (yellow-necked mice [*Apodemus flavicollis*]), bank voles [*Myodes glareolus*], and harvest mice [*Moscardinus avellanarius*]) were also analyzed. All animal-handling procedures and ethical issues