We report biomolecular evidence of dolphin morbillivirus in 4 wild Eurasian otters (Lutra lutra) from southern Italy; 2 animals showed simultaneous immunohistochemical reactivity against morbilliviral antigen. These cases add further concern and support to the progressively expanding host range of dolphin morbillivirus in the western Mediterranean Sea

Cafiero, Maria Assunta

Carbone, Antonio

Caruso, Marta

Centelleghe, Cinzia

De Riso, Laura

Di Guardo, Giovanni

Galante, Domenico

Guarino, Laura

Mazzariol, Sandro

Padalino, Iolanda

Palazzo, Lucia

Parisi, Antonio

Petrella, Antonio

Troiano, Pasquale

Emerging Infectious Diseases

English

Archivio istituzionale della ricerca - Università di Padova

References  1. Powers AM, Logue CH. Changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus. J Gen Virol. 2007;88:2363–77. http://dx.doi.org/10.1099/vir.0.82858-0  2. Schuffenecker I, Iteman I, Michault A, Murri S, Frangeul L,  Vaney MC, et al. Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak. PLoS Med. 2006;3:e263.  http://dx.doi.org/10.1371/journal.pmed.0030263  3. Leparc-Goffart I, Nougairede A, Cassadou S, Prat C,  de Lamballerie X. Chikungunya in the Americas. Lancet. 2014; 383:514. http://dx.doi.org/10.1016/S0140-6736(14)60185-9  4. Chahar HS, Bharaj P, Dar L, Guleria R, Kabra SK, Broor S.  Co-infections with chikungunya virus and dengue virus in Delhi, India. Emerg Infect Dis. 2009;15:1077–80. http://dx.doi.org/ 10.3201/eid1507.080638  5. icddr,b. Seroprevalence of dengue virus infection in Dhaka,  Bangladesh, 2012. Health Sci Bull. 2014;2:1–6.  6. icddr,b. First identified outbreak of chikungunya in Bangladesh, 2008. Health Sci Bull. 2009;7:1–6.  7. Khatun S, Chakraborty A, Rahman M, Nasreen Banu N,  Rahman MM, Hasan SMM, et al. An outbreak of chikungunya in rural Bangladesh, 2011. PLoS Negl Trop Dis. 2015;9:e0003907. http://dx.doi.org/10.1371/journal.pntd.0003907  8. Kabir I, Dhimal M, Müller R, Banik S, Haque U. The 2017 Dhaka chikungunya outbreak. Lancet Infect Dis. 2017;17:1118.  http://dx.doi.org/10.1016/S1473-3099(17)30564-9  9. Sam IC, Loong SK, Michael JC, Chua CL, Wan Sulaiman WY, Vythilingam I, et al. Genotypic and phenotypic characterization  of chikungunya virus of different genotypes from Malaysia. PLoS One. 2012;7:e50476. http://dx.doi.org/10.1371/journal.pone.005047610. Huang JH, Yang CF, Su CL, Chang SF, Cheng CH, Yu SK, et al.  Imported chikungunya virus strains, Taiwan, 2006–2009. Emerg Infect Dis. 2009;15:1854–6. http://dx.doi.org/10.3201/eid1511.090398Address for correspondence: Mizanur Rahman, Molecular Diagnostics, Apollo Hospitals Dhaka, Plot-81, Block-E, Bashundhara, Dhaka-1229, Bangladesh; email: mizanur.rahman@apollodhaka.comDolphin Morbillivirus in  Eurasian Otters, ItalyIolanda Padalino, Giovanni Di Guardo,  Antonio Carbone, Pasquale Troiano,  Antonio Parisi, Domenico Galante,  Maria Assunta Cafiero, Marta Caruso,  Lucia Palazzo, Laura Guarino, Laura De Riso, Cinzia Centelleghe, Sandro Mazzariol,  Antonio PetrellaDOI: https://doi.org/10.3201/eid2502.180256Author affiliations: Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Foggia, Italy (I. Padalino, A. Carbone,  P. Troiano, A. Parisi, D. Galante, M.A. Cafiero, A. Petrella);  University of Teramo, Teramo, Italy (G. Di Guardo); Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Matera, Italy (M. Caruso); Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Potenza, Italy (L. Palazzo); Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata,  Taranto, Italy (L. Guarino); Ente Parco Nazionale del Cilento, Salerno, Italy (L. De Riso); University of Padua, Padua, Italy  (C. Centelleghe, S. Mazzariol)We report biomolecular evidence of dolphin morbillivirus in 4 wild Eurasian otters (Lutra lutra) from southern Italy; 2 ani-mals showed simultaneous immunohistochemical reactivity against morbilliviral antigen. These cases add further con-cern and support to the progressively expanding host range of dolphin morbillivirus in the western Mediterranean Sea.The genus Morbillivirus comprises several lympho-ep-ithelio-neurotropic, highly pathogenic RNA viruses of domestic and wild vertebrates, including aquatic mammals. Among them, cetacean morbillivirus (CeMV) has been re-sponsible since the 1980s for dramatic epidemics in free-ranging cetaceans worldwide (1). Specifically, the CeMV strain termed dolphin morbillivirus (DMV) has caused at least 4 unusual mortality events (UMEs) among striped dolphins (Stenella coeruleoalba) in the western Mediter-ranean Sea and, to a lesser extent, long-finned pilot whales (Globicephala melas) and other wild cetaceans from the same region (1–3).We report evidence of DMV infection in 4 wild Eur-asian otters (Lutra lutra) from southern Italy (Apulia and Basilicata regions). The animals, all adult females, be-longed to a group of 7 individuals found dead at Parco Na-zionale del Cilento, a large national park that extends to the coastline of southwestern Italy. The animals underwent necropsy at Istituto Zooprofilattico Sperimentale della Pug-lia e della Basilicata (Foggia, Italy) during 2016 and 2017, according to an official agreement between the institute and the park aimed at assessing the health and conservation sta-tus of the otter population.Within a multidisciplinary approach framework, we conducted in-depth histopathologic, microbiologic, par-asitologic, and ecotoxicologic analyses on the 7 otters, along with biomolecular (reverse transcription PCR [RT-PCR]) and immunohistochemical (IHC) investigations for Morbillivirus spp. After using a technique amplifying a highly conserved fragment of the Morbillivirus nucleo-protein (NP) gene (4), we applied 2 additional methods aimed at detecting DMV-specific hemagglutinin (HA) (5) and NP gene sequences (6) for more detailed analy-sis. To increase the biomolecular results’ reliability, we performed all the extraction, amplification, and sequenc-ing steps in 3 different laboratories. We also conducted the histopathological and IHC analyses in 3 different 372 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 2, February 2019RESEARCH LETTERSlaboratories. For IHC analysis, we used a commercial monoclonal antibody (VMRD, https://www.vmrd.com) against the NP antigen of canine distemper virus (CDV), including adequate morbillivirus-positive and -negative control tissues in each run.At necropsy, we found multiple traumatic injuries, probably caused by motor vehicles and deemed the pu-tative cause of death, in all 7 otters. Microscopically, we observed a bilateral, subacute-to-chronic, broncho- or bronchiolo-interstitial pneumonia, showing endobronchial, endobronchiolar, or endoalveolar macrophage infiltration, thickening of alveolar septa, and lympho-histiocytic septal infiltration, in 2 otters; 1 also showed a multifocal, portal and lobular, nonsuppurative hepatitis. Lungs and kidneys from these 2 animals were molecularly positive for DMV (Figure, panel A). Their pulmonary tissue showed positive morbilliviral antigen immunostaining in vascular walls and endothelial cells, as well as in alveolar epithelial cells (con-sistent, or not, with hyperplastic type II pneumocytes); we also found positive immunostaining in thickened alveolar septa and endoalveolar macrophages (Figure, panel B). Al-though the advanced postmortem autolysis did not enable us to obtain reliable histopathological information from the 5 remaining otters, biomolecular analyses yielded positive results from several tissues (submandibular lymph node, parotidal gland, lung, brain, heart, kidney, urinary bladder, and liver) in 2 of them (Figure, panel A).Sequencing of all the 200-bp NP gene viral amplicons obtained (GenBank accession nos.  MG836265–8) showed remarkable differences from the available CDV sequences but high homology (99%) with 2 DMV sequences (Gen-Bank accession nos. EF469546.1, KU720625.1). We com-pared an additional 456-bp HA gene fragment (GenBank provisional accession no. MG905831), obtained from the kidney of 1 of the otters we tested, with 2 DMV isolates re-covered from Mediterranean striped dolphins in 2007; this fragment displayed 100% homology with 1 DMV isolate (GenBank accession no. HQ829973) and 99.56% homol-ogy with the other (accession no. AJ608288).Susceptibility to morbilliviruses is not new to L. lutra otters; CDV infection has been documented, with subse-quent risk for viral spillover to coastal pinnipeds (7). In this respect, the endemic behavior and the progressively expanding host range of DMV in the western Mediter-ranean Sea are a matter of concern (2,3,8), as exemplified by a peculiar case of infection in a captive harbor seal (Phoca vitulina), a species with a mixed marine–terres-trial ecology (9).How the otters in this study may have acquired DMV infection is unknown. Of note, 8 cases of DMV infection were detected during 2016 and 2017 in dolphins strand-ed along the Ionian Sea coast, in an area geographically consistent with that in which the 4 DMV-infected otters were found. It is possible that >1 dolphins with DMV-associated brain lesions could have entered the rivers or lagoons inhabited by the otters and transmitted the virus to them. Alternatively, the otters’ movement toward and placement close to the sea, clearly documented for 1 of them (A. Petrella, unpub. data), could underlie a subse-quent encounter with DMV-infected dolphins. As an addi-tional or complementary option, susceptible invertebrate hosts might have acted as DMV reservoirs, similarly to what has been reported for Baicalia carinata and Limnaea auricularia mollusks, which probably were a source of infection during the CDV epidemic among Baikal seals (Pusa sibirica) in 1987 (10).In conclusion, we identified DMV infection among Eurasian otters in southwestern Italy, along the coast of the  Emerging Infectious Diseases • www.cdc.gov/eid •Vol. 25, No. 2, February 2019 373RESEARCH LETTERSFigure. Evidence of dolphin morbillivirus infection in Eurasian otters (Lutra lutra), southwestern Italy. A) Comparison of nucleoprotein gene amplification products from infected otters, obtained by reverse transcription PCR. A specific band at the expected molecular weight of 287 bp is shown. Lane 1, molecular weight marker (Tracklt 100bp DNA Ladder; Invitrogen,  http://www.thermofisher.com); lane 2, positive control (lung tissue from an infected striped dolphin, Stenella coeruleoalba); lane 3, negative control (distilled water); lanes 4–7, samples from morbillivirus-positive Eurasian otters: LL-290, lung (lane 4); LL-291, kidney (lane 5); LL-3380, lung (lane 6); LL-7318, lung (lane 7). B) Mayer’s hematoxylin counterstain of lung tissue shows marked and widespread immunohistochemical labeling for morbillivirus antigen (dark areas), particularly evident at the level of vascular walls and endothelial cells and, to a lesser extent, of alveolar epithelial cells (morphologically consistent, or not, with hyperplastic type II pneumocytes) as well as of thickened alveolar septa. Scale bar indicates 100 μm.western Mediterranean Sea. The effect of DMV infection on the health and conservation of the threatened Eurasian otter populations warrants further investigation.AcknowledgmentsWe thank Antonio Lo Polito, Maria Luigia Prencipe, and  Fabio Gisoldo for their valuable technical support. We also  thank Gabriella Di Francesco, Alessio Lorusso, Daniela  Malatesta, and Guendalina Zaccaria for their valuable support in performing histopathological, immunohistochemical, and  biomolecular analyses as well as for their greatly appreciated contribution to fruitful and insightful discussions on the data reported in the manuscript.About the AuthorDr. Padalino works at the Virology Department of Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Foggia, Italy, where she is in charge of the Molecular Biology and Cell Culture Laboratory. Her research is mainly focused on the molecular diagnosis and epidemiologic surveillance of viral infectious diseases in domestic and wild animals.References  1. Van Bressem MF, Duignan PJ, Banyard A, Barbieri M,  Colegrove KM, De Guise S, et al. Cetacean morbillivirus: current knowledge and future directions. Viruses. 2014;6:5145–81.  http://dx.doi.org/10.3390/v6125145  2. Mazzariol S, Centelleghe C, Beffagna G, Povinelli M, Terracciano G, Cocumelli C, et al. Mediterranean fin whales (Balaenoptera physalus) threatened by dolphin morbillivirus. Emerg Infect Dis. 2016;22:302–5. http://dx.doi.org/10.3201/eid2202.150882  3. Mazzariol S, Centelleghe C, Di Provvido A, Di Renzo L, Cardeti G,  Cersini A, et al. Dolphin morbillivirus associated with a mass stranding of sperm whales, Italy. Emerg Infect Dis. 2017;23: 144–6. http://dx.doi.org/10.3201/eid2301.160239  4. Frisk AL, König M, Moritz A, Baumgärtner W. Detection of canine distemper virus nucleoprotein RNA by reverse transcription-PCR using serum, whole blood, and cerebrospinal fluid from dogs with distemper. J Clin Microbiol. 1999;37:3634–43.  5. Centelleghe C, Beffagna G, Zanetti R, Zappulli V, Di Guardo G,  Mazzariol S. Molecular analysis of dolphin morbillivirus: a new sensitive detection method based on nested RT-PCR. J Virol  Methods. 2016;235:85–91. http://dx.doi.org/10.1016/ j.jviromet.2016.05.005  6. Verna F, Giorda F, Miceli I, Rizzo G, Pautasso A, Romano A,  et al. Detection of morbillivirus infection by RT-PCR RFLP  analysis in cetaceans and carnivores. J Virol Methods. 2017; 247:22–7. http://dx.doi.org/10.1016/j.jviromet.2017.05.009  7. Mos L, Ross PS, McIntosh D, Raverty S. Canine distemper virus in river otters in British Columbia as an emergent risk for coastal pinnipeds. Vet Rec. 2003;152:237–9. http://dx.doi.org/10.1136/vr.152.8.237  8. Jo WK, Osterhaus AD, Ludlow M. Transmission of morbilliviruses within and among marine mammal species. Curr Opin Virol. 2018;28:133–41. http://dx.doi.org/10.1016/j.coviro.2017.12.005  9. Mazzariol S, Peletto S, Mondin A, Centelleghe C, Di Guardo G, Di Francesco CE, et al. Dolphin morbillivirus infection in a captive harbor seal (Phoca vitulina). J Clin Microbiol. 2013;51:708–11. http://dx.doi.org/10.1128/JCM.02710-1210. Kondratov IG, Denikina NN, Belikov SI, Durymanova AA,  Ustinova EN, Shestopalov AM. Mollusks as a natural reservoir  of morbilliviruses. Dokl Biol Sci. 2003;389:154–6.  http://dx.doi.org/10.1023/A:1023435312334Address for correspondence: Giovanni Di Guardo, University of Teramo, Faculty of Veterinary Medicine, Località Piano d’Accio, 64100, Teramo, Italy; email: gdiguardo@unite.itLittle Evidence of Zika  Virus Infection in Wild  Long-Tailed Macaques,  Peninsular MalaysiaChong Long Chua, Yoke Fun Chan,  Eva S.G. Soh Andu, Jeffrine J. Rovie-Ryan, Frankie Thomas Sitam, Khebir Verasahib,  I-Ching SamAuthor affiliations: University of Malaya, Kuala Lumpur, Malaysia (C.L. Chua, Y.F. Chan, E.S.G. Soh Andu, I-C. Sam); Department of Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur (J.J. Rovie-Ryan, F.T. Sitam); National Public Health Laboratory, Sungai Buloh, Malaysia (K. Verasahib)DOI: https://doi.org/10.3201/eid2502.180258We tested a sample of 234 wild long-tailed macaques (Ma-caca fascicularis) trapped in Peninsular Malaysia in 2009, 2010, and 2016 for Zika virus RNA and antibodies. None were positive for RNA, and only 1.3% were seropositive for neutralizing antibodies. Long-tailed macaques are unlikely to be reservoirs for Zika virus in Malaysia.Zika virus, first isolated from a rhesus macaque (Macaca mulatta) in the Zika Forest in Uganda, reemerged in the Pacific Islands and Americas in 2015 and caused un-precedented outbreaks associated with serious congenital syndromes (1). The role of animal reservoirs for Zika virus is unclear, although in Africa, nonhuman primates (NHPs) are suspected to be involved in maintaining a sylvatic cycle, as they are for 2 other flaviviruses (yellow fever and dengue viruses) also transmitted by Aedes mosquitoes. The pres-ence of a sylvatic cycle for Zika virus in Africa is supported by a seroprevalence of 0%–16% in African green monkeys (Chlorocebus sabaeus) and vervet monkeys (Chlorocebus 374 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 2, February 2019RESEARCH LETTERS

Dolphin Morbillivirus in Eurasian Otters, Italy

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