Perspectives of Morbillivirus Infections in Cetaceans: A Review Focused on South America

Paula A. Angel-Romero1, Dalia C. Barragán-Barrera1,2, Miguel H. Parra Ávila3

1 Laboratorio de Ecología Molecular de Vertebrados Acuáticos, Departamento de Ciencias Biológicas, Facultad de Ciencias, Universidad de los Andes, Bogotá, Colombia. 2 Fundación Macuáticos Colombia, Antioquia, Colombia. 3 Departamento de Ciencias Biológicas, Facultad de Ciencias, Universidad de los Andes, Bogotá, Colombia.

Correspondence author: Paula Andrea Angel Romero [email protected]

Advisor: Miguel Hernando Parra Ávila, PhD Chair Professor Biological Sciences Department Universidad de los Andes Bogotá, Colombia

Co-advisor: Dalia Carolina Barragán Barrera, PhD candidate Laboratorio de Ecología Molecular de Vertebrados Acuáticos - LEMVA Biological Sciences Department Universidad de los Andes Bogotá, Colombia

1 ABSTRACT

Cetacean Morbillivirus (CeMV) belongs to the Paramyxoviridae family and different strains have been discovered like porpoise morbillivirus (PMV), dolphin morbillivirus (DMV), pilot whale morbillivirus (PWMV), beaked whale morbillivirus (BWMV), and two recently unestablished lineages. CeMV is responsible for the death numerous individuals from a great variety of species through several outbreaks and mass strandings in several parts of the world. It is probably transmitted through the inhalation of aerosolized virus via the blowhole and it is favored by gregarious species and migratory behavior; besides the viral cellular entry is mediated by the SLAM receptor that provides an interface for morbillivirus H glycoprotein to attach, showing a coevolution process. The disease is categorized as a sub-acute, acute, chronic systemic disease or a chronic localized disease leading to a severe encephalitis and the diagnosis can be made by means of virus isolation, histology and immunohistochemistry (IHC), serology, or different Reverse Transcription Polymerase Chain Reaction (RT-PCR) variants. Most of CeMV reports have occurred in the USA and Europe, and that there is an information gap for South America; therefore, the aim of the current literature review is to present an overview of the CeMV mechanisms and characteristics, the pathology, pathogenesis, epidemiology, diagnosis and affected species of the disease, with a special emphasis on the lack of information and reports of this infectious agent in South America.

2 1. INTRODUCTION

Cetacean Morbillivirus (CeMV) belongs to the Order Mononegavirales, family Paramyxoviridae, subfamily Paramyxovirinae and Genus morbillivirus. There are currently seven members of this genus that cause severe diseases in their hosts, which include: measles virus (MV), canine distemper virus (CDV), rinderpest virus (RPV), peste-des-petits ruminants virus (PPRV) (Blixenkrone-Møller, Bolt, Jensen, Harder, & Svansson, 1996), phocine distemper virus (PDV), feline morbillivirus (FmoPV) and cetacean morbillivirus (CeMV) (Cassle et al., 2016; Jo, Osterhaus, & Ludlow, 2018). Regarding CeMV, it includes porpoise morbillivirus (PMV) isolated from harbor porpoises (Phocoena phocoena) from Northern Ireland (McCullough et al., 1991), dolphin morbillivirus (DMV) isolated from Mediterranean striped dolphins (Stenella coeruleoalba) (Van Bressem et al., 1991), pilot whale morbillivirus (PWMV) isolated from a long-finned pilot whale (Globicephala melas) from New Jersey, USA (Bellière, Esperón, & Sánchez-Vizcaíno, 2011; Taubenberger et al., 2000a), beaked whale morbillivirus (BWMV) isolated from a longman's beaked whale (Indopacetus pacificus) from Hawaii (West et al., 2013), and two recently discovered strains which are highly divergent and where identified from Indo-Pacific bottlenose dolphin (Tursiops aduncus) from the West Australian coast (Stephens et al., 2014) and from Guiana dolphin (Sotalia guianensis) from Brazil (Cassle et al., 2016; Groch et al., 2014).

Morbilliviruses are known to cause a severe disease due to immunosuppression as they are lymphotropic and replication process starts in lymphoid tissue before infection other tissues and epithelial cells takes place (Barrett et al., 1995; Delpeut, Noyce, & Richardson, 2014; Osterhaus et al., 1995; Shimizu et al., 2013; Van Bressem et al., 2014). CeMV is responsible for the death numerous individuals from a great variety of species through several epidemics and outbreaks, since it was first isolated from S. coeruleoalba in 1990 in Spain as DMV strain, which is one of the highest mortalities reported (Aguilar & Raga, 1993).

3 Here we provide an overview of the CeMV mechanisms and characteristics, the pathology, pathogenesis, epidemiology, diagnosis and affected species of the disease, with a special emphasis on the lack of information and reports of this infectious agent in South America.

2. CETACEAN MORBILLIVIRUS CHARACTERISTICS

2.1. Antigenic and molecular characteristics

Morbilliviruses are characterized by a lipid envelope that contains the helical nucleocapsid with the linear non-segmented, negative-sense, single-stranded RNA (Barrett, 1999). RNA viruses have a very high mutation rate (i.e. from ~10−2 to ~10−5 mutations/site/replication) which leads them to genotypic and phenotypic variations causing the appearance of variants or strains that have differences regarding the immunological properties, the virulence, the host rage and tropism and the epidemiology (Beffagna, Centelleghe, Franzo, Di Guardo, & Mazzariol, 2017).

The DMV is 15,702-bp long and is composed of six different structural proteins each of them codified by a transcription unit or gene and two virulence factor proteins. The principal protein is the nucleocapsid protein (N) which contains the viral genetic material in a ribonucleoprotein complex (RNP), that protects the RNA from enzymes present in the cytoplasm of the host cell that can cause its degradation. N protein also acts as an acceptor molecule for the RNA-dependent-RNA polymerase allowing it to attach to mediate replication and transcription processes (Jo et al., 2018). It has 523 amino acids and presents high variability at the C-terminus. On the contrary, the N protein is highly conserved at the amino terminus but displays a region of high variability at the carboxyl terminus (Banyard, Tiwari, & Barrett, 2011). The phosphoprotein (P) and the large protein (L) are also contained in the RNP complex (Jo et al., 2018).

The proteins associated to the viral membrane are the matrix protein (M), the fusion protein (F) and hemagglutinin glycoprotein (H). M protein is found in the inside of the viral envelope, which is hydrophobic, enriched positively charged residues that are believed to interact with the RNP and it does not seem to be modified post-translationally (Barrett,

4 1999) and it allows the assembly of the virus and the exit of the cell (Jo et al., 2018); F protein is a conserved acylated protein and H glycoprotein is more variable, both proteins are responsible for the attachment and fusion with the host cell (Barrett, 1999). The H protein is a crucial structure to perform the cell entry, and associated with the morbillivirus F protein, cause cell-to-cell fusion and therefore a cytopathic effect (CPE) (Banyard et al., 2011). Regarding the virulence factors, non-structural V and C proteins are derived from the P gene, by editing of the mRNA by insertion of a G residue and by translation of an overlapping reading frame (ORF) (Bellière et al., 2011).

Advances in molecular biology techniques have allowed the diagnosis of CeMV infections, in order to stablish phylogenetic relationships between different morbillivirus strains. It is of high importance to clarify the differences between strains as it was proven that the amino acids which differentiated DMV isolated from S. coeruleoalba and fin whale (Balaenoptera physalus) from different epidemics outbreaks where under diversifying selection, which can lead eventually to a potential host switch (Beffagna et al., 2017). Likewise, these techniques provide valuable information about the virus epidemiology and are important to develop candidate vaccines (Barrett, 1999).

2.2. Mechanisms of Cellular Entry and Receptors

The general cellular entry process starts with the H glycoprotein, which is responsible for the attachment to the host cell membrane to initiate the cell entry process (Banyard et al., 2011), and together with the F protein, the process of cell-to-cell fusion is induced causing the fusion of the cell membrane and M protein (Banyard et al., 2011; Barrett et al., 1995; Delpeut et al., 2014). Morbilliviruses are characterized by the ability to infect immune cells like B and T-lymphocytes, macrophages, activated monocytes and dendritic cells. the last two cells mentioned, express the cellular receptor signaling lymphocytic activated molecule (SLAM or CD150) which is the receptor of the morbilliviruses H protein and the infection with CeMV usually leads to an acute disease, lymphopenia or low lymphocyte count in the blood and immunosuppression, leaving organisms vulnerable to secondary infections and also affecting the central nervous system (CNS) causing a severe encephalitis (Jo et al., 2018;

5 Ludlow, Rennick, Nambulli, De Swart, & Paul Duprex, 2014; Ohishi et al., 2010; Ohishi, Suzuki, & Maruyama, 2012; Tatsuo, Ono, & Yanagi, 2001).

The SLAM receptors family share similar domain structures, in which the ectodomain has a distal membrane immunoglobulin variable (IgV) domain and a proximal membrane immunoglobulin constant-2 (IgC2) domain that provides a target for morbillivirus H glycoprotein (Ohishi et al., 2010; Ohishi et al., 2012). The genes that codify for these receptors, are located in chromosome 1 in humans and mice, which indicates that probably this receptor family had a common origin and it is an ancestral gene (Ohishi et al., 2010). Furthermore, it has been reported that the phylogenetic tree of the H viral gene overlaps the tree of the host SLAM gene with few exceptions, indicating a coevolution process (Beffagna et al., 2017). The SLAMs of close related species show great homology, for example between killer whale (Orcinus orca) and Pacific white-sided dolphin (Lagenorhynchus obliquidens) there is a 99% amino acid identity; and between them and artiodactyla order (cow and sheep) 84–85%; between spotted seal (Phoca largha) and walrus (Odobenus rosmarus) the amino acid identity is 99%, and compared between them and canine SLAM 84% of identity; and the American manatee (Trichechus manatus) SLAM presented high homology with that of the African elephant (Loxodonta africana), 86% (Ohishi et al., 2010). Nevertheless, changes either in the SLAM or in the H protein, affect the host susceptibility, the host tropism, the viral infectivity and pathogenesis (Beffagna et al., 2017; Delpeut et al., 2014; Jo et al., 2018).

The infected dendritic cells and macrophages migrate to lymph nodes affecting a great amount of activated T and B cells too, allowing the dissemination of the virus through the whole lymphatic system. Due to the viral tropism, CeMV can infect epithelial tissues using the poliovirus-receptor-like 4 (PVRL4 or Nectin-4) as a receptor, because this protein is expressed at polarized epithelial cells, allowing the viral exit process and transmission to other hosts by shedding the viruses in respiratory secretions, urine and feces (Delpeut et al., 2014; Jo et al., 2018; Shimizu et al., 2013). Besides, CD147 is a transmembrane glycoprotein receptor and it has been proposed to function as a viral entry receptor for MV

6 and PDV. CD147 that belongs to the immunoglobulin family is expressed in several endothelial and neuronal cells (Van Bressem et al., 2014; Watanabe et al., 2010).

2.3. Cetacean Morbillivirus transmission and excretion

Morbillivirus transmission and circulation in cetacean wild population is still unclear (Banyard et al., 2011); however, it is believed that it occurs through the inhalation of aerosolized virus via the blowhole released by infected individuals as reported in other morbillivirus (Jo et al., 2018; Van Bressem, Van Waerebeek, & Raga, 1999). This horizontal transmission of the virus is likely to occur in cetaceans that present a gregarious behavior, that travel in big groups and possibly favored by the synchronic breathing (Raga et al., 2008). It has been proposed that all body orifices and skin are a potential route for viral excretion including dermal, urinary and fecal secretions; nevertheless, the virus is diluted and inactivated as it is much less likely to result in lateral transmission (Kennedy, 1998).

Furthermore, there is evidence for vertical and transplacental transmission as in the case of a G. melas fetus (Fernandez et al., 2008), a B. physalus newborn positive for DMV (Mazzariol et al., 2016), a calf of S. ceoruleoalba with a CNS infection (Di Guardo et al., 2011) and morbillivirus antigen in lactating mammary gland which could transfer morbillivirus through milk secretion to the calf (Domingo et al., 1992) and in a sperm whale (Physeter macrocephalus) neonate (West et al., 2015), which supports maternal transfer of the virus (Beffagna et al., 2017; Jo et al., 2018). Viral antigen has been detected in other tissues as the male reproductive tract of P. phocoena (Kennedy et al., 1992) and in S. ceoruleoalba; and in the mammary glands of bottlenose dolphins (Tursiops truncatus) (Domingo et al., 1992; Kennedy, 1998; Schulman et al., 1997), suggesting the possible venereal and vertical transmission of the virus through lactation to the offspring.

The dissemination of the virus is poorly understood; however, big cetaceans such as P. macrocephalus and B. physalus are organisms that migrate and travel great distances carrying the virus and can be acting as vectors, but these animals have a solitary behavior or live and travel in small pods (Beffagna et al., 2017; Jo et al., 2018). Besides, G. melas is

7 considered to be the endemic source of the virus as it has a high seroprevalence observed in different tissue samples obtained in several stranding events (Bossart et al., 2010). This species live in large groups, they move great distances and have a wide pelagic distribution that overlaps with several other cetacean species, suiting the requirements of the virus to be maintained and transmitted (Banyard et al., 2011; Sierra et al., 2016). The distribution across large areas and the movement patterns lead to the infection of naïve populations which can result in epidemics, mortality and mass stranding events (Banyard et al., 2011). For this reason, it is important to assess species that perform annual migrations such as big mysticetes in which humpback whales (Megaptera novaeangliae), blue whales (Balaenoptera musculus), sei whales (Balaenoptera borealis), right whales (Eubalaena australis) among others, which live in the South Atlantic and Pacific and no information is available.

2.4. Cetacean Morbillivirus origin evolution

Closely related species of cetaceans like the hippopotamus (Hippopotamus amphibius) that belong to the Cetartiodactyla clade (Milinkovitch & Thewissen, 1997; Nikaido, Rooney, & Okada, 1999) are commonly affected by RPV and PPRV (Barrett et al., 1995; Kumar et al., 2014), making possible a host jump between a member of the clade and cetacean species and the ecological isolation due to the colonization of the ocean led the virus differentiate as Cetacean Morbillivirus (Ohishi et al., 2012; Shimizu et al., 2013; Van Bressem et al., 2014), as DMV and PMV are more closely related to ruminant morbilliviruses (RPV, PPRV) (Barrett et al., 1995; Di Guardo, Marruchella, Agrimi, & Kennedy, 2005; Kennedy, 1998; Visser et al., 1993) than to distemper viruses (Barrett et al., 1993; Blixenkrone-Moller, Bolt, Gottschalck, & Kenter, 1994; Blixenkrone-Møller et al., 1996; Bolt & Blixenkrone-Møller, 1994; Visser et al., 1993). The ecological isolation, a great variety of possible hosts for the virus, their migratory and gregarious behavior favors this hypothesis (Van Bressem et al., 1999).

8 3. DISEASE

Morbillivirus infections are lymphotropic in the first stages and subsequently the epitheliotropic phase takes place as the virus disseminates throughout the body by infected immune cells (Delpeut et al., 2014; Jo et al., 2018). The pathology and pathogenesis of the disease is classified in acute, sub-acute, chronic systemic and chronic localized as in some cases it could cause encephalitis. There is poor evidence that support subclinical infections, however in a few cases it was possible to detect antigens with no evident clinical signs or histological lesions (Bossart et al., 2010; Reidarson et al., 1998).

3.1. External Clinical Signs

Clinical signs of morbillivirus infection are observed in stranded animals that present neurological or behavioral changes, like apathy and disorientation (Elk et al., 2014; Stone et al., 2011). Besides, it is common to observe a poor body condition, highly parasitized on the skin, abnormal respiratory rates or dyspnea, muscle tremors, trembling and seizures (Elk et al., 2014; Jauniaux et al., 2000).

3.2. Acute systemic disease

Morbilliviral infection that results in an acute fatal disease is associated to several organs and tissues, the lungs are one of the principal organs affected, showing severe inflammation and several sings of the infection. It is characterized by interstitial broncho-pneumonia with multinucleate syncytia, type II pneumocyte hyperplasia and exudation of mononuclear cells in the alveolar and the bronchiolar lumina, as well as necrosis in both mononuclear cells and syncytia (Barrett et al., 1995; Domingo et al., 1992). Severe depletion of lymph nodes and lymph node hypoplasia is frequently seen and intranuclear inclusion bodies have been detected in respiratory epithelia and in syncytia (Jauniaux et al., 2000). There is evidence of viral replication in the brain as non-suppurative encephalitis has been reported (Domingo et al., 1992; Duignan, Geraci, Raga, & Calzada, 1992; Kennedy et al., 1991; Schulman et al., 1997; Stephens et al., 2014; Stone et al., 2011; Stone, Blyde, Saliki, & Morton, 2012).

9 3.3. Sub-Acute systemic disease

As a consequence of the severe immunosuppression caused by the acute stage of the infection, animals that survive are prone to opportunistic secondary infections that can infect even the brain as reported mycotic pathogens as Aspergillus sp. Many of the clinical sings and lesions from the acute disease may be no longer observable due to the inflammatory response to the secondary pathogens; however, non-suppurative demyelinating meningoencephalitis is characteristic of sub-acute infection (Di Guardo et al., 2005; Di Guardo et al., 1995; Domingo et al., 1992; Duignan et al., 1992; Elk et al., 2014; Fernandez et al., 2008; Groch et al., 2014; Stephens et al., 2014; Stone et al., 2011).

3.4. Chronic systemic disease

This stage of the disease is common in animals that survived to acute and sub-acute infections. Severe secondary infections in several organs due to the advanced immunosuppression and complications from the central nervous system viral infection. A profound lymphoid depletion, lesions or antigens detected in the hepatic sinusoid blood vessels, mesenteric lymph nodes or it can be presented as a no signs of the disease observable, but viral RNA is persistent in the blood and lymphoid organs (Domingo et al., 1992; Mariano Domingo et al., 1995; Elk et al., 2014; Stephens et al., 2014; Taubenberger et al., 1996).

3.5. Chronic Localized disease – Encephalitis

This disease category refers to a form of the disease associated to DMV that develop only in the CNS causing lesions in the brain, localized in the cerebral cortex, subcortical white matter, thalamus and almost none in the cerebellum (Domingo et al., 1995), sharing characteristics with a subacute sclerosing panencephalitis (SSPE) (Garg, 2008) and occasionally multinucleate syncytia (Domingo et al., 1992). The encephalitis may cause the death of the infected individuals as it affects vital areas of the brain such as the dorsal motor nucleus of the vagus nerve, responsible for cardio-respiratory systems (Di Guardo et al., 2011, 2013). Morbilliviral antigen has been detected in great amounts by patches and not

10 in a continuous form, suggesting that the spread and infections occurs cell-to-cell rather than blood-borne infections that would affect larger and continuous areas as well as inclusion bodies in neurons (Di Guardo et al., 2011). The mechanism of infection remain unclear, but it may be related with CD147 and other cellular receptors associated with the immuno-privileged condition of the brain; however, the role of this receptors need further investigation (Di Guardo et al., 2005; Fernandez et al., 2008; Sierra et al., 2016; Stone et al., 2012; Van Bressem et al., 2014; Watanabe et al., 2010), as well as evaluate if the death and transmission of the virus are related with the CNS form of the disease.

3.6. Co-infections

Several coinfections have been reported in morbillivirus infected animals, due to the profound immune suppression they suffer, allowing the colonization of secondary opportunistic bacterial, fungal and/or parasitic infections (Stone et al., 2011). Infection with several agents is believed to have a synergistic effect with the progress of the disease (Cassle et al., 2016). Regarding parasitic infections Toxoplasma gondii has been reported in adult B. physalus (Di Guardo et al., 2013; Mazzariol et al., 2012), S. coeruleoalba (Di Guardo et al., 2013; Domingo et al., 1992) and T. truncatus (Casalone et al., 2014; Di Guardo et al., 2013); nematodes at different developmental stages belonging to the genus Crassicauda where present in lung tissue (Jauniaux et al., 2000), nematodes such as Halocercus lagenorhynchi were also found in the second stomach chamber of G. melas (Taubenberger et al., 2000). Ectoparasitic copepods has been reported in the blubber layer of B. physalus (Jauniaux et al., 2000).

For bacterial infections, Escherichia coli, Enterococcus sp. and Staphylococcus sp. were commonly reported in the lung and the pulmonary lymph node in White-Beaked dolphins (Lagenorhynchus albirostris) (Elk et al., 2014) and Salmonella sp. infection in B. physalus (Jauniaux et al., 2000). Vibrio alginolyticus was isolated from the brain, lung, trachea and sinus of a juvenile T. truncatus; however, there were no lesions associated (Cassle et al., 2016). Streptococcus phocae was found in Short-beaked Common dolphin (Delphinus delphis) and it is associated to different pathologies and tissues, such as

11 bronchopneumonia, metritis and sepsis (Díaz-Delgado et al., 2017), and Brucella sp. was reported as a coinfection in a neonatal P. macrocephalus (West et al., 2015).

Fungal infections have been reported in the CNS and in the respiratory system as Aspergillus sp. hyphae in the lung parenchyma of a S. ceoruleoalba (Domingo et al., 1992), in G. melas, and in short-finned pilot whales (Globicephala macrorrhynchus) in Spain (Fernandez et al., 2008). Aspergillus fumigatus was present in the lung, trachea, and brain of a juvenile T. truncatus (Cassle et al., 2016).

Several lesions in the oral cavity are caused by secondary opportunistic agents (Domingo et al., 1992). Besides, the immunosuppression caused by the Morbilliviral infection also allows secondary viral infections like Papillomavirus and Herpes Virus (HV) in cetaceans (Bellière et al., 2010; Casalone, Mazzariol, Pautasso, Guardo, et al., 2014; Díaz-Delgado et al., 2017; Soto et al., 2012). Two morbillivirus strains involved in a Morbilliviral infection (DMV and PMV) were detected in Bottlenose dolphins in the U.S. Atlantic coast in 1987 (Taubenberger et al., 2000).

3.7. Immunology and Epidemiology

Morbillivirus infections have a severe effect in the immune system of the host, as it causes immunosuppression and leukopenia (Beineke, Siebert, Wohlsein, & Baumgärtner, 2010; Heaney, Barrett, & Cosby, 2002; Schlender et al., 1996). It has been reported T cell proliferation, increases in lysozyme concentrations and monocyte phagocytosis; however the immune cells present the cellular receptor that allows the entry of the virus leading to depletion of responding cells and lymphoid organs (Jo et al., 2018; Van Bressem et al., 2014). Antibodies titers can be detected in individuals but the timing of the Morbilliviral infection (active/inactive) and the stage (acute, subacute, chronic, subclinical) cannot be determined (Beineke et al., 2010; Van Bressem et al., 2014). Newborns could present acquired immunity through maternal transmission of antibodies through milk or placenta if the mother was previously exposed to the viral entity; however, it would only last for some

12 months leaving the individuals vulnerable to the infection (Griffin & Bellini, 1996; Van Bressem et al., 2014).

The epidemiology of the morbillivirus infections is unclear and it is hard to assess (Morris et al., 2015); however, morbillivirus is very infectious and biology and behavior of the species facilitate the transmission. Morbillivirus require great population numbers of vulnerable or naïve individuals to persist endemically (Black, 1991; Van Bressem et al., 2014). Many affected cetacean species are gregarious, however the persistence of CeMV is a mystery in the ecology of the disease as population numbers are not that big (Almberg, Cross, & Smith, 2010; Van Bressem et al., 2014); nevertheless, herd immunity plays an important role in the epidemiology of the disease and it is believed that the decrease on it could be responsible for the outbreaks.

DMV is considered the most virulent common strand and it is associated to the chronic encephalitis Morbilliviral disease, responsible for several massive marine mammals stranding (Jo et al., 2018). Studies suggest that Pilot whale (Globicephala spp.), Dusky dolphin (Lagenorhynchus obscurus), Fraser’s dolphin (Lagenodelphis hosei), melon-headed whale (Peponocephala electra) may act as CeMV vectors and reservoirs (Jo et al., 2018) as well as subclinical infected animals pose a risk for other naïve cetaceans with which they are associate from the same or different species (Stone et al., 2011, 2012).

3.8. Aggravating Factors

It has been proposed that there are few other factors that affect cetaceans and their susceptibility to morbillivirus and other infections which are environmental pollution and climate change. Several environmental pollutants are immunotoxic are playing a role in causing an even more serious immunosuppression making the host susceptible to morbillivirus or to secondary opportunistic infections (Beineke et al., 2010; Di Guardo et al., 2013; Fernandez et al., 2008). Studies have demonstrated that pesticides and persistent polychlorinated biphenyls (PCBs) are related to immunosuppression and affect reproduction. These pesticides could be related too with pneumonias caused by parasites,

13 bacteria, as the pollutant burdens in the individuals have a correlation with morbillivirus infections (Jepson et al., 2016).

Synergistic effects of these possible aggravating factors are still debated as they can increase the severity of the infection as cetaceans are exposed to numerous pollutants such as organochlorines (PCBs, DDTs, dioxins, etc.), heavy metals (Hg, Pb, Cd, etc.) and emerging pollutants such as flame retardants (PBDEs) and microplastic polymers among others. It is necessary to evaluate the role that these compounds have in modulating the infections (Di Guardo et al., 2005;2015; Gaydos, Balcomb, Osborne, & Dierauf, 2004). Besides, other anthropogenic forces could have an effect on cetaceans’ health and susceptibility to the disease such as negative interactions with fisheries coupled with rise in sea surface level temperature could affect prey availability, reduced population sizes leading to inbreeding and climate change could have an impact in migration and species distribution (Aguilar & Raga, 1993; Di Guardo, Mazzariol, & Fernández, 2011; Echeverri-zuluaga, Duque-garcía, & Ruiz-saenz, 2015; Swart, Harder, Ross, Vos, & Osterhaus, 1995).

4. DIAGNOSIS

Several techniques have been developed to detect the virus and diagnose a morbilliviral infection with virus isolation and serological techniques, differentiate the strains with specialized genome amplification techniques such as reverse transcription polymerase chain reaction (RT-PCR), detect the stage of the disease, the immune status of the population among other factors by means of histology, immunohistochemistry and serological studies (Barrett & Rossiter, 1999; Duignan et al., 1995; Duignan et al., 1995; Fauquier et al., 2017; Van Bressem et al., 1993). For this, several tissue samples must be collected including different brain areas, cerebellum, lung, spleen, mesenteric and pulmonary lymph nodes heart and skeletal muscle (Di Guardo et al., 2013; Müller et al., 2002) to assess the degree of infection, the infected organs and the tropism of the virus.

14 4.1. Virus isolation

Virus isolation is considered the gold standard CeMV diagnosis; however, this technique presents many difficulties associated with the decomposition of stranded carcasses tissues. Both DMV and PMV have been isolated by using lung tissue homogenates, which is inoculated in monolayers of African green monkey kidney (Vero) and it takes several weeks of repeated passages to obtain viral growth (Blixenkrone-Moller et al., 1994; Visser et al., 1993); however, Vero cells that express the canine SLAM (Vero.DogSLAMtag cells) allow a shorter incubation time in the order of four to nine days, improving virus replication (Banyard et al., 2011; Grant, Banyard, Barrett, Saliki, & Romero, 2009; Nielsen, Smith, Weingartl, Lair, & Measures, 2008; Peletto et al., 2018). This technique offers other advantages such as providing the antigen for serological studies and it allowing the extraction and sequencing of the genetic material to carry out phylogenetic analysis and to determine the strain (Bolt et al., 1994).

4.2. Histology and immunohistochemistry (IHC)

Histological techniques have been used to detect morbilliviral infection in tissues sampled from stranded organisms and coupled with immunohistochemistry (IHC) CeMV antigen can be detected even if the preservation of the tissue or the carcass is poor or in cases in which opportunistic pathogens obstruct the virus-caused lesions (Di Guardo et al., 2013; Groch et al., 2014; West et al., 2013). IHC is performed in formalin-fixed tissues obtained from the brain, lung, spleen and lymph nodes (Müller et al., 2002) using commercially available monoclonal antibodies (MoAb) to CDV N protein (Bossart et al., 2010; Elk et al., 2014) and to PDV hemagglutinin protein which detect CeMV as the primary antibody as they recognize the same epitope from the Morbillivirus genus (Di Guardo et al., 2011; Stephens et al., 2014; West et al., 2013) and biotinylated Goat Anti-Mouse IgG antibody as a secondary antibody is commonly used (Di Guardo et al., 2013) followed by a Avidin-Biotin peroxidase complex to produce a colored label (Müller et al., 2002; Müller, Siebert, Wünschmann, Artelt, & Baumgärtner, 2000).

15 4.3. Serology

Different serology techniques have been used to detect antibodies against CeMV infection such as virus neutralization (VN) tests, plaque reduction (PR) assays and indirect enzyme- linked immunosorbent assays (iELISAs). VN it is a sensible technique that allow the detection of antibodies in serum samples by neutralizing the virus previously grown in Vero cells (Bossart et al., 2010) and preventing the infection of susceptible cells which results in a Cytopathic effect (CPE). The exposition to CeMV is consider positive when the result of the test are titers of 1:16 or higher (Elk et al., 2014; Müller et al., 2000; Van Bressem et al., 2001; Van Bressem, Van Waerebeek, Fleming, & Barrett, 1998). PR assay allow the detection of antibodies by the dilution of the serum and exposure to the virus, by the reduction of plaques of infected Vero cells (Nielsen et al., 2008; Nielsen, Stewart, Measures, Duignan, & House, 2000). Both VN and PR can only recognize the surface glycoproteins H and F of the virus (Barrett et al., 1993).

Indirect ELISA is useful for the detection of antibodies for N, P, F and H CeMV proteins (Orvell, Blixenkrone-Moller, Svansson, & Have, 1990). The presence of the virus-specific antibodies was detected by means of a horseradish-peroxidase conjugated Protein A (Sigma) obtained from the cell wall of Staphylococcus aureus that binds to several species’ immunoglobulins and it is revealed with a chromogen substrate and optical density (OD) is measured to determine the result of the assay (Van Bressem et al., 1998).

4.4. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

There are several ways to perform this molecular technique in which the most simple and common is a RT-PCR with universal morbillivirus primer set targeting the conserved regions of the P gene (Barrett et al., 1993), primers amplify a fragment of 287 base pairs from a conserved terminal region of N gene (Di Guardo et al., 2011; Di Guardo et al., 2013; Raga et al., 2008) or universal primers for a highly conserved region in the F gene (Rubio-Guerri et al., 2013). Specialized PCRs have been developed like protocols to amplify degraded RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples (Reidarson et al., 1998),

16 reverse transcription-quantitative PCR (qRT-PCR) using a set of primers to amplify DMV N gene (de Medeiros Bento et al., 2016), nested RT-PCR targeting conserved regions of the DMV H gene to increase sensitivity and specificity to the detection and diagnosis of the virus (C Centelleghe et al., 2016), RT-PCR RFLP using degenerate primers for a conserved N terminus of the N gene of 287 bp followed by MseI RFLP, to distinguish between DMV and CDV (Di Guardo et al., 2013; Verna et al., 2017), real time RT-PCR (qRT-PCR) to differentiate between DMV and PMV strains by using primers designed for the hypervariable C-terminal region of the N gene for each strain for which DMV N gene set of primers could detect down to 102 viral RNA copies (Cycle threshold (CT) value = 17.99) while the PMV set of primers could detect down to 103 viral RNA copies (CT value = 25.72) (Grant et al., 2009). Novel techniques include new generation sequencing analysis (Peletto et al., 2018) and qRT-PCR followed by high resolution melting assay (HRM) for genotyping viral RNA obtained from FFPE samples which are not suitable for the regular RT-PCR CeMV detection protocols due to the chemical characteristics (Yang et al., 2016). These molecular techniques are sensitive and provide trustworthy results that can be used to diagnose the morbillivirus infection and to develop phylogenetic relationships and evolution analysis; however, it is important to combine detection techniques to provide further information regarding the stage of the infection and the pathology.

4.5. Difficulties

Difficulties in the detection and diagnosis techniques could be associated to the information gap in South America. Several factors can affect the detection and diagnosis of a morbilliviral infection, such as low viral titer that could reduce the possibilities of detecting the morbilliviral RNA in qRT-PCR. For that reason, RNA extraction must be performed using a pool of tissue homogenates (i.e. usually lung, brain, pulmonary lymph node and mesenteric lymph node) to increase the viral load and the probability of detecting the virus, followed by individual organ testing (de Medeiros Bento et al., 2016; Di Guardo et al., 2011). Moreover, other detection techniques such as immunohistochemistry and the pathological analyses require optimal sample conditions. Body condition is assigned according to stablish

17 protocols and a scale from 1 to 5 as follows: 1) alive, 2) freshly dead, 3) moderate decomposition (i.e. organs intact), 4) advanced decomposition (i.e. organs not recognizable) and 5) mummified or skeletal remains. These techniques can be performed with samples obtained from organisms whose body condition is between 1 and 3 (Casalone et al., 2014; Geraci & Lounsbury, 1993). However, the lack of organizations and effort at responding to strandings in south America plays a major role in the lack of information regarding morbilliviral infections, as happens in Colombia and in the southern region of Chile.

Furthermore, as it was mentioned before, asymptomatic or sub-acute infections are difficult to determine and diagnose as there is no clear evidence of the morbilliviral infection, leading to a great risk of acting as a reservoir or vector, disseminating the virus specially if the species is gregarious and migratory (Beffagna, Centelleghe, Franzo, Di Guardo, & Mazzariol, 2017; Jo et al., 2018). This condition is difficult to study because the organisms are not likely to strand or be found death due to the morbilliviral disease, and therefore, it should be studied in wild free-ranging organisms. Besides, strandings and response program should include Supplementary On-Site Information such as pre-stranding animal behavior when possible, because external clinical signs such as neurological changes, apathy and disorientation can be evident (Geraci & Lounsbury, 1993; Jauniaux et al., 2000; van Elk et al., 2014).

The issue of stranding can be addressed as a difficulty to study and diagnose morbillivirus infections as well. Several hypothesis have been proposed to explain why animals strand, instead of falling to the deep sea and provide the ecosystem with massive pulses of organic enrichment, as it mostly occurs (Roman et al., 2014). These events could be caused by anthropogenic stressors like pollution, fishing, vessel collision or interactions (Meynecke & Meager, 2016) or by several natural factors such as at-sea mortality, diseases, unusual weather events, changes in magnetic fields, oceanographic and abiotic factors such as sea temperature anomalies, prevailing winds, currents and “death acoustic zones”, and shore topography. Social strandings are of great importance as species that are gregarious have a strong social component and they can mass strand (Authier et al., 2014; Brabyn & Frew,

18 1994; Brabyn & McLean, 1992; Chan, Tsui, & Kot, 2017; Ferrari, 2016; Meynecke & Meager, 2016; Pierce, Santos, Smeenk, Saveliev, & Zuur, 2007; Sundaram, Poje, Veit, & Nganguia, 2006). If associated to CeMV infection, some of this possible causes of stranding could be related to the severe encephalitis that organisms could present due to the viral infection, leading them to difficulties to orientate, perceive echolocation signals, guiding the herd, among others. All these factors can cause or explain the strandings or organisms in beaches; however, morbillivirus infections cannot be correlated to strandings until now. For this, stranding protocols should include assessing RNA presence in a pool of tissue homogenates and when positive, individual organ testing, to determine not only the clinical signings associated to the cause the death but also if CeMV is the etiologic agents.

Many organisms could be dying at off-shore open sea and there is any evidence of this, on the contrary, several places are considered stranding hotspots in which necropsies reveal different causes of death, but they have a higher probability of detecting cetacean morbillivirus infections than single and rare stranding events (Figure 1.). This coupled with other factors could explain the lack of information and morbillivirus cases or stranding events in South America. Most of the times, carcasses beached when they are already dead, so the morbillivirus infection cannot be attributed to that geographic area as unique, isolated, non-reproductible events are caused by the action of sea currents that transport the death bodies and sometimes can take them out of their normal distribution range (Barreto, Moraes, Sperb, & Bughi, 2006). Other plausible explanation may be the coastal topography associated mostly to alive but sick individuals, because gently slope and sandy beaches are prone to cause strandings (Brabyn & McLean, 1992), besides some migratory routes are now dry land and several beaches and bays are “acoustic dead zones’’ due to the topography, bathymetry and geometry of the area, interfering echolocation and increasing the probability of stranding (Sundaram et al., 2006). Finally, cetaceans move and migrate by using magnetic receptors that sense the geomagnetic field that varies latitudinally and locally and temporal magnetic disturbances or alterations in the organism health can lead to disorientation as they can no longer monitor their position, leading to stranding (Brabyn & Frew, 1994; Ferrari, 2016).

19 The combination of the above-mentioned factors can result in single or mass stranding events in South America. Organisms could be healthy carriers of the disease, there is no concern about the infection as the mortality rate is low compared to Europe and North America and probably, there is no research on this topic.

5. HOST AFFECTED SPECIES

Cetacean Morbillivirus affects a great number of cetaceans of both Odontoceti (toothed whales) and Mysticeti (baleen whales) groups, having several possible hosts for its replication. This has led to diversification and specialization of the virus, however, PMV, DMV, PWMV, BWMV and the new strains found, maintain a multi-host transmission cycle in which DMV is the most prevalent and aggressive strain (Jo et al., 2018) affecting mainly species from the Delphinidae family, that have been reported as the most vulnerable to the CeMV infection, possibly due to their affinity for SLAM or their gregarious habits. Below are the species for which CeMV has been reported and the distribution of the species in order to analyze which populations in South America are vulnerable to the disease, together with a graphic representation of the stranding events (Figure 1.) and the information condensed in Table 1.

5.1. Mysticetes

Family Balaenopteridae

• Fin Whale (Balaenoptera physalus): this baleen whale has presented DMV infections leading to strandings in Iceland, Belgium, France and Italy (Blixenkrone-Moller et al., 1994; Casalone et al., 2014; Jauniaux et al., 2000; Mazzariol et al., 2012; Mazzariol et al., 2016; Profeta et al., 2015). This rorqual has a cosmopolitan distribution, mostly found at Southern hemisphere temperate waters; however, Fin whales are found in the North Atlantic, North Pacific, Gulf of California and commonly in the Mediterranean living in groups of three to seven individuals (Carwardine, 2002) so they are not common in South America.

20 • Minke Whale (Balaenoptera acutorostrata): one stranding case was reported in Tuscany, Italy with an unknow strain of CeMV (Di Guardo et al., 1995). These whales can be found alone worldwide but not year-round as it has a migratory behavior, and the three different populations are found in the North Pacific, North Atlantic and southern hemisphere. Mediterranean is not included in the distribution range, but it has been reported sporadic sightings in short incursions (Carwardine, 2002) this means, it is technically possible to find them in South America.

5.2. Odontocetes

Family Delphinidae

• Common Dolphin (Delphinus delphis): this species has a huge distribution range, including tropical, subtropical and warm temperate waters all around the globe in huge groups of 10 to 500 individuals; they travel long distances and can associate with individuals from other species (Carwardine, 2002), increasing the probability of spreading the disease due to their gregarious behavior. It is present in South America but never associated to a morbilliviral epidemic until now. This leads to several stranding events due to DMV strain in different areas such as eastern Atlantic and North Sea (Sierra et al., 2014; Van Bressem et al., 2001; I. K. G. Visser et al., 1993), Mediterranean Sea (Van Bressem et al., 1993), Northwestern Atlantic (Duignan et al., 1995), Eastern Pacific (Reidarson et al., 1998; Taubenberger et al., 2000b), Southern Ocean (Kemper et al., 2013) and Indian Ocean (Van Bressem et al., 2001). • Long-beaked Common Dolphin (Delphinus capensis): this species has similar distribution and size groups as the Common Dolphin, however, bigger groups up to 2,000 individuals in the Eastern tropical Pacific has been reported (Carwardine, 2002). This dolphin species corresponds to one of the few reports of CeMV in South America, which happened in Peru (Van Bressem et al., 1998); however, the taxonomy of D. capensis in South America has recently been debated (Farías- Curtidor et al., 2017).

21 • Pygmy Killer Whale (Feresa attenuata): The species is distributed in tropical and subtropical offshore deep waters in groups of 15 to 25 individuals, mostly in areas such Japan, Hawaii, Indian Ocean and the Caribbean (Carwardine, 2002). However, Morbillivirus infection has been only reported in the Southeast coast of USA (Duignan et al., 1995). • Short-finned Pilot Whale (Globicephala macrorhynchus): PWMV was reported in the Canary Islands in which periodic mortalities occur (Sierra et al., 2016) and in Florida, USA where the infection is considered endemic (Duignan et al., 1995). This species moves in groups of 10 to 30 or even 50 individuals in tropical, warm temperate and subtropical waters. They perform nomadic movements with no fixed migrations, but they are common year-round in Hawaii and Canary Islands. This species could be found in the Northeast Pacific ins South American waters (Carwardine, 2002). • Long-finned Pilot Whale (Globicephala melas): several stranding events have been reported for this species associated to DMV and PWMV infections in different places worldwide like Spain, France, Italy (Casalone et al., 2014; Fernández et al., 2008; Profeta et al., 2015), Canary Islands in which periodic mortalities are reported (Sierra et al., 2016), Northeast USA coast where is considered endemic (Barrett et al., 1993; Duignan et al., 1995) New Jersey, USA periodic mortalities are common (Taubenberger et al., 2000b) and in Northland, New Zealand (Van Bressem et al., 2001). This pilot whale species is important in the dissemination of the morbilliviral infection as they are found in big groups of 10 to 50 or even 100 individuals in subpolar and cold temperate waters in the south hemisphere and in the North Atlantic; in deep waters. This species is one for which more mass strandings are reported (Carwardine, 2002). • Risso’s Dolphin (Grampus griseus): this species if found in big groups of 3 to 50 and sometimes even 150 individuals in occasional gatherings, and it has a wide distribution in deep tropical and subtropical waters around the world (Carwardine, 2002). Even though, there is only one stranding associated to CeMV infection in this

22 species and was found in the Mediterranean coast of Spain (Kemper et al., 2013; Van Bressem et al., 2001). • Fraser’s Dolphin (Lagenodelphis hosei): the distribution of this species is poorly known although they travel in big groups of 100 to 500 or bigger when found offshore, then inhabit tropical and warm temperate waters and appears to be more common in the equator region (Carwardine, 2002). However, strandings of CeMV infected organisms have been reported in the Gulf of Mexico (Duignan et al., 1995), Northeast Australia (Stone et al., 2012), Puerto Madryn in Argentina and Rio de Janeiro in Brazil (Van Bressem et al., 2001). • White-beaked Dolphin (Lagenorhynchus albirostris): periodic mortalities of DMV infected organisms have been reported in Germany and Netherlands (Elk et al., 2014; Visser et al., 1993). The distribution of this species is in cool temperate and subarctic waters in the North Atlantic, and the group size is of 2 to 30 individuals (Carwardine, 2002). South America is not part of their distribution range. • Atlantic White-sided Dolphin (Lagenorhynchus acutus): it is distributed in cool temperate and subarctic waters in the North Atlantic and it performs inshore- offshore movements in groups of 5 to 50 individuals and huge groups of 1,000 have been reported occasionally (Carwardine, 2002) and it has been reported CeMV infection in the Northeast coast of USA (Duignan et al., 1995). • Dusky Dolphin (Lagenorhynchus obscurus): the distribution of this dolphin is in the costal temperate waters in South America, South Africa and New Zealand and usually they are found in small groups of 2 to 15 individuals and least common in big groups that can reach numbers of 1,000 (Carwardine, 2002). CeMV has been reported in individuals found in Peru (Van Bressem et al., 1998). • Pacific White-sided Dolphin (Lagenorhynchus obliquidens): individuals from this species have been infected with CeMV in Japan (Uchida et al., 1999), which is part of the distribution area of the species. Individuals from this species can be found in deep waters in the North Pacific in groups of 10 to 100 individuals (Carwardine, 2002).

23 • Melon-headed Whale (Peponocephala electra): CeMV is considered endemic in the Northeast of Australia in which strandings of this species have been reported (Stone et al., 2012). It is found in big groups of 50 to 500 and rarely estimates of even 1,500 have been reported, in tropical and subtropical waters all around the globe, but are more common in Australia, Philippines and year-round in Hawaii (Carwardine, 2002). • False Killer Whale (Pseudorca crassidens): a stranding associated to morbilliviral infections was reported in the Southeast USA coast (Duignan et al., 1995). This species lives in tropical, subtropical and warm temperate waters worldwide but mostly offshore as they inhabit deep waters in groups of 10 to 50 individuals (Carwardine, 2002). • Guiana Dolphin (Sotalia guianensis): this species is found in South America inshore near estuaries along the North Atlantic coast all the way to the Caribbean up to Nicaragua and Honduras (Flores & da Silva, 2009). A CeMV infection case was reported recently in Espirito Santo, Brazil in 2010 (Groch et al., 2014). • Striped Dolphin (Stenella coeruleoalba): this cetacean is the most affected species by morbillivirus infections, reaching mortality numbers of more than 400 carcasses in one single stranding event (Aguilar & Raga, 1993). They conform big groups of 10 to 50 and even thousands of individuals and it has a wide distribution in tropical, subtropical and warm temperate waters all around the globe including South America (Carwardine, 2002). Mortality events associated to DMV infections have been reported in a variety of places including the Eastern Atlantic at the Canary Islands (Sierra et al., 2014), in the Mediterranean Sea in several countries like Spain, France, Italy and Greece (Aguilar & Raga, 1993; Casalone et al., 2014; Di Guardo et al., 1995; Di Guardo, Di Francesco et al., 2013; Profeta et al., 2015; Raga et al., 2008; Van Bressem et al., 1993) and in the Northeast cost of USA (Duignan et al., 1995). • Atlantic Spotted Dolphin (Stenella frontalis): a morbillivirus-associated stranding was reported in the Northeast coast of USA (Duignan et al., 1995), as it is distributed in warm temperate, subtropical and tropical waters in the North and South Atlantic,

24 forming groups usually of 5 to 15 that move closer to the shore during summer and pelagic populations have also been reported (Carwardine, 2002). • Bottlenose Dolphin (Tursiops truncatus): it is a widely distributed species, in cold temperate, tropical and subtropical seas around the world, with normal group size of 1 to 10 individuals for the inshore populations and 1 to 25 or even 500 may occur in offshore populations (Carwardine, 2002). For this reason, several morbillivirus infections have been reported in a wide variety of places including the Northeast Atlantic in the United Kingdom (Van Bressem et al., 2001), the Eastern Atlantic in the Canary Islands (Sierra et al., 2014), in different countries of the Mediterranean Sea like Israel, Spain, France, Italy (Casalone et al., 2014; Duignan et al., 1995; Profeta et al., 2015), at the Northwestern Atlantic in several locations in the USA (Bossart et al., 2010, 2011; Krafft et al., 1995; Rowles et al., 2011; Schulman et al., 1997; Shimizu et al., 2013; Taubenberger et al., 1996) and in the South and Western Pacific in Australia (Kemper et al., 2013; Stone et al., 2011, 2012). • Indo-Pacific Bottlenose Dolphin (Tursiops aduncus): this species has stranded in different occasions in Australia (Kemper et al., 2013; Stephens et al., 2014; Stone et al., 2012; Van Bressem et al., 2001), but there are no reports of Morbillivirus infection. Its distribution is in the warm temperate to tropical waters in the Indian and the West Pacific Ocean (Moller & Beheregaray, 2001).

Family Kogiidae

• Pygmy sperm whale (Kogia breviceps): there are two stranding reports of stranded organisms infected with DMV in the Southeast coast of USA and Taiwan (Duignan et al., 1995; Yang, Pang, Jeng, Chou, & Chueh, 2006). These organisms are found normally in groups from three to six and prefer deep temperate, tropical and subtropical waters beyond the continental edge, mostly in the USA southeastern coast, South Africa, Australia, New Zealand and some areas in Asia (Carwardine, 2002). This means, this species is not common in South America, but some

25 strandings without necropsies have been reported in Colombia (Trujillo, Caicedo & Diazgranados, 2014).

Family Phocoenidae

• Harbor porpoises (Phocoena phocoena): is the only CeMV host of the family from which PMV was isolated in the first places, presenting periodic mortalities (Van Bressem et al., 2001; Visser et al., 1993). This species is found in the Northern Hemisphere in sub-polar and cold temperate waters in 2 to 5 individuals per group (Carwardine, 2002).

Family Physeteridae

• Sperm whale (Physeter macrocephalus): is the only species of the family and it has been reported BWMV infections in stranded animals in Hawaii (West et al., 2015). This organism is of great concern, as it has a gregarious behavior with a regular group size of 1 to 50 individuals and males tend to be solitary. Besides, it has a cosmopolitan distribution in which it inhabits deep waters worldwide, even in South America (Carwardine, 2002), being a risk as they can act as vectors, moving the infection from any area of the world.

Family Ziphiidae

• Longman's Beaked Whale (Indopacetus pacificus): BWMV and DMV have been detected in individuals stranded in Hawaii and New Caledonia, respectively (Garrigue et al., 2016; West et al., 2013). Little is known about the distribution about this species, but it is speculated that it has a great area range in the Indian and Pacific Ocean. It has never been observed somewhere near South America (Carwardine, 2002). • Cuvier’s Beaked Whale (Ziphius cavirostris): individuals from this species where found at Calabria, Italy in 2015 with DMV infection (Centelleghe et al., 2017). It has an enormous distribution, being cosmopolitan and inhabiting tropical, subtropical

26 and temperate waters and travels in groups sizes from one to ten individuals. It can be found in South America (Carwardine, 2002), but Italy is considered a stranding hotspot.

27

! !

!! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

! ! ! ! ! ! ! ! ! ! !

8 5 3 2 1

Figure 1. Stranding events associated to any CeMV strain infection worldwide.

28 Table 1. Stranding events associated to any CeMV strain infection worldwide. PMV = porpoise morbillivirus, CeMV = cetacean morbillivirus, DMV = dolphin morbillivirus, PWMV = pilot whale morbillivirus.

Epidemiological Virus Species Ocean Years Countries Literature Cited Status Strain MYSTICETI Family Balaenopteridae Eastern Atlantic & 1983 Iceland Unknown CeMV (Blixenkrone-Moller et al., 1994) North Sea Balaenoptera Eastern Atlantic & 1997- Belgium, France Periodic Unknown (Jauniaux et al., 2000) physalus North Sea 1998 mortalities Mediterranean Sea 2011 Tuscany, Italia Periodic DMV (Casalone et al., 2014; Mazzariol et al., 2012, 2016; mortalities Profeta et al., 2015) Balaenoptera Mediterranean Sea 1993 Tuscany, Italia Unknown Unknown (Di Guardo et al., 1995) acutorostrata ODONTOCETI Family Phocoenidae Eastern Atlantic & 1988- N. Ireland, UK, Periodic PMV (Kennedy et al., 1991; McCullough et al., 1991; Van North Sea 1990 Netherlands mortalities Bressem et al., 2001; Visser et al., 1993) Phocoena phocoena Northwestern Atlantic 1993- East coast, Canada Unknown CeMV (Duignan et al., 1995) 1994 Family Physeteridae Physeter Eastern Pacific 2011 Hawaii, USA Unknown BWMV (West et al., 2015) macrocephalus Family Kogiidae Northwestern Atlantic 1983- Southeast coast USA Unknown CeMV (Duignan et al., 1995) 1991 Kogia breviceps Western Pacific 2009 SW Taiwan Periodic DMV (Yang et al., 2006) mortalities Family Ziphiidae Indopacetus Eastern Pacific 2010 Hawaii, USA Unknown BWMV (West et al., 2013) pacificus Southern Ocean 2013 New Caledonia Unknown DMV (Garrigue et al., 2016)

Ziphius cavirostris Mediterranean Sea 2015 Calabria, Italy Unknown DMV (Centelleghe et al., 2017)

29 Family Delphinidae Eastern Atlantic & 1988- UK, Netherlands, Unknown CeMV (Van Bressem et al., 2001; Visser et al., 1993) North Sea 1990 Germany Eastern Atlantic & 2007 Canary Islands Periodic DMV (Sierra et al., 2014; Van Bressem et al., 2001) North Sea mortalities Mediterranean Sea 1990 Italy Unknown CeMV (Van Bressem et al., 1993) Northwestern Atlantic 18980- Northeast coast USA Possibly endemic CeMV (Duignan et al., 1995) Delphinus delphis 1994 Eastern Pacific 1995- California, USA Unknown CeMV (Reidarson et al., 1998; Taubenberger et al., 2000b) 1997 Southern Ocean 2013 South Australia Unknown CeMV (Kemper et al., 2013) Indian Ocean 1999 East London, South Unknown CeMV (Van Bressem et al., 2001) Africa Eastern Pacific 1993- Central Peru Endemic CeMV (Van Bressem et al., 1998) Delphinus capensis 1995 Northwestern Atlantic 1983- Southeast coast USA Unknown CeMV (Duignan et al., 1995) Feresa attenuata 1991 Eastern Atlantic & 1996, Canary Islands Periodic PWMV (Sierra et al., 2016) Globicephala North Sea 2015 mortalities macrorhynchus Northwestern Atlantic 1886- Florida, USA Endemic CeMV (Duignan et al., 1995) 1994 Mediterranean Sea 2006- Spain, France, Italy Epidemic DMV (Casalone et al., 2014; Fernández et al., 2008; Profeta 2007, et al., 2015) 2013 Eastern Atlantic & 2015 Canary Islands Periodic CeMV (Sierra et al., 2016) North Sea mortalities Globicephala melas Northwestern Atlantic 1982- Northeast coast USA Endemic CeMV (Barrett et al., 1993; Duignan et al., 1995) 1993 Northwestern Atlantic late New Jersey, USA Periodic PWMV (Taubenberger et al., 2000b) nineties mortalities Western Pacific 1997 Northland, New Endemic CeMV (Van Bressem et al., 2001) Zeland

30 Mediterranean Sea 1997,19 Valencia, Spain Unknown CeMV (Casalone et al., 2014; Van Bressem et al., 2001) Grampus griseus 99 Northwestern Atlantic 1994 Gulf of Mexico, USA Possibly endemic CeMV (Duignan et al., 1995) Southwestern Atlantic 1999 Puerto Madryn, Unknown CeMV (Van Bressem et al., 2001) Lagenodelphis hosei Argentina Southwestern Atlantic 1999 Rio de Janeiro, Brazil Unknown CeMV (Van Bressem et al., 2001) Western Pacific 2006 NE Australia Unknown CeMV (Stone et al., 2012) Eastern Atlantic & 1988- Germany, Periodic DMV (Elk et al., 2014; Visser et al., 1993) Lagenorhynchus North Sea 1990, Netherlands mortalities albirostris 2007, 2011 Lagenorhynchus Northwestern Atlantic 1985- Northeast coast USA Unknown CeMV (Duignan et al., 1995) acutus 1993 Lagenorhynchus Eastern Pacific 1993- Central Peru Endemic CeMV (Van Bressem et al., 1998) obscurus 1995 Lagenorhynchus Western Pacific 1998 Miyazaki, Japan Unknown Unknown (Uchida et al., 1999) obliquidens Peponocephala Western Pacific 2005- NE Australia Endemic CeMV (Stone et al., 2012) electra 2007 Pseudorca Northwestern Atlantic 1982- Southeast coast USA Possibly endemic CeMV (Duignan et al., 1995) crassidens 1988 Sotalia guianensis Southwestern Atlantic 2010 Espirito Santo, Brazil Unknown CeMV (Groch et al., 2014) Eastern Atlantic & 2002- Canary Islands Periodic DMV (Sierra et al., 2014) North Sea 2011 mortalities Mediterranean Sea 1990– Spain, France, Italy, Epidemic DMV (Aguilar & Raga, 1993; Di Guardo et al., 1995; Profeta Stenella 1992 Greece et al., 2015; Bressem et al., 1993) coeruleoalba Mediterranean Sea 2006- Spain, France, Italy Epidemic DMV (Casalone et al., 2014; Di Guardo et al., 2013; Profeta 2008, et al., 2015; Raga et al., 2008) Northwestern Atlantic 1991- Northeast cost USA Unknown CeMV (Duignan et al., 1995) 1993 Stenella frontalis Northwestern Atlantic 1993 Northeast coast USA Unknown CeMV (Duignan et al., 1995)

31 Eastern Atlantic & 1999 Kent, UK Unknown CeMV (Van Bressem et al., 2001) North Sea Eastern Atlantic & 2005 Canary Islands Periodic DMV (Sierra et al., 2014) North Sea mortalities Mediterranean Sea 1994; Israel, Spain, France, Periodic DMV (Casalone et al., 2014; Duignan et al., 1995; Profeta et 2007- Italy mortalities al., 2015) 2008, 2011 Northwestern Atlantic 1982 Florida, USA Epidemic CeMV (Rowles et al., 2011) Northwestern Atlantic 1987- East coast USA Epidemic CeMV (Rowles et al., 2011; Schulman et al., 1997; 1988 Taubenberger et al., 1996) Northwestern Atlantic 1993- Gulf of Mexico, USA Epidemic CeMV (Rowles et al., 2011; Taubenberger et al., 1996) 1994 Tursiops truncatus Northwestern Atlantic 2003- Florida, USA Unknown CeMV (Bossart et al., 2010, 2011; Rowles et al., 2011) 2007 Northwestern Atlantic 2013- East coast USA Epidemic DMV (Rowles et al., 2011; Shimizu et al., 2013) 2014 Northwestern Atlantic 1992- east coast USA Endemic CeMV (Rowles et al., 2011) 1994 Northwestern Atlantic 1987- US Atlantic coast, Unknown CeMV (Krafft et al., 1995; Rowles et al., 2011) 1994 Gulf of Mexico Eastern Pacific 1993- Central Peru Endemic CeMV (Van Bressem et al., 1998) 1995 Western Pacific 1997 Tasmania, Australia Unknown CeMV (Van Bressem et al., 2001) Western Pacific 2009- Queensland, Periodic DMV (Stone et al., 2011, 2012) 2010 Australia mortalities Southern Ocean 2013 South Australia Unknown CeMV (Kemper et al., 2013) Indian Ocean 2009 Western Australia Periodic CeMV (Stephens et al., 2014) mortalities Western Pacific 2005- NE Australia Unknown CeMV (Stone et al., 2012) Tursiops aduncus 2010 Southern Ocean 2012- South Australia Unknown CeMV (Kemper et al., 2013; Van Bressem et al., 2001) 2013

32 6. DISCUSSION AND CONCLUSION

Significant findings and advances have been done regarding molecular biology, phylogeny and pathology of CeMV infections; however, further studies are needed to elucidate the host range, ecology and epidemiology of the disease. All of the affected species face anthropogenic threats like fishing, pollution, entanglements in fishing gear, ship collisions, habitat degradation (Meynecke & Meager, 2016), among others and most species are categorized in a conservation status of threatened. Furthermore, these species are vulnerable to a CeMV infection, a natural treat that could cause decreases in population numbers and affect the conservation status. For this reason, it is of great importance to study, characterize and diagnose morbilliviral infections, to understand the virus itself, the host and the interaction as a whole, the viral affinity, the environmental factors, the immune response, epidemiology, as well as the data, samples and effort when attending strandings (Echeverri-Zuluaga, Duque-García, & Ruiz-Saenz, 2015; Jo et al., 2018).

Therefore, it is necessary to conduct studies focused on evaluating the vulnerability of the species to the infection, not only the reported hosts but the sympatric or phylogenetically related species that are prone to infect with the virus but that have not been reported as hosts yet, as it was done for O. orca (Gaydos et al., 2004). This issue could be address by different methodologies, traditional ones by evaluating the possible pathogens and the effects that it can have in the health and the population by comparing with close related species or by assessing the environmental risks or by means of new techniques. The implementation of protein crystallography techniques will allow to model the H protein and the SLAM cetacean receptor in order study the interaction of these proteins, the specificity, viral fitness, and evolution as the function of synonymous and nonsynonymous substitutions could be elucidated. Besides, molecular docking and protein modeling could be useful to propose the possible vulnerability of species for which the infection has not been reported but that could be susceptible to the virus as the can be naïve populations (Beffagna et al., 2017); however, further molecular studies are needed to obtain SLAM sequences for different species.

33 As stranding areas or probabilities are difficult to predict in situ, other techniques involving geographic information systems (GIS) tools can be very helpful at studying cetacean strandings by means of currents, temperature, bathymetry, coastal topology, previous stranding and morbillivirus infections events, to predict possible epidemics and stranding areas, or to determine which oceanographic conditions do not allow animals to strand in the coast making the CeMV infection unnoticeable. Modelling and including special epidemiology is necessary to understand the risk and incidence of the disease, working as surveillance and monitoring of wild population and morbillivirus outbreaks (Authier et al., 2014; Barreto et al., 2006; Norman, 2008).

Lastly, an effort must be made in the implementation of strandings response network and database usage, in a regional, national or even globally scale. Databases are critical to preserve and manage information related to strandings, mortality events and infectious diseases or epidemics. Having access to stranding and necropsy data, etiologic agents, geographic ranges, provide valuable information and could be useful to understand mortalities worldwide. For this reason, existing databases should be centralized providing information about health status of several cetacean species and populations that act as ecosystem sentinel (Bossart, 2011; Chan, Tsui, & Kot, 2017b).

Morbillivirus infections should have a more important role when assessing species risk, health and population numbers. This information must be compiled with several other factors when stablishing conservation status and species management, as it supposes an important threat to the reported host and to several other species that can be healthy carriers or present sub-acute infections of CeMV.

34 8. REFERENCES

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45 ABBREVIATIONS TABLE CeMV Cetacean Morbillivirus MV Measles Virus CDV Canine Distemper Virus RPV Rinderpest Virus PPRV Peste-des-petits Ruminants Virus PDV Phocine Distemper Virus FmoPV Feline Morbillivirus PMV Porpoise Morbillivirus DMV Dolphin Morbillivirus PWMV Pilot Whale Morbillivirus BWMV Beaked Whale Morbillivirus N Nucleocapsid protein RNP Ribonucleoprotein complex P Phosphoprotein L Large protein M Matrix protein F Fusion protein H Hemagglutinin glycoprotein CPE Cytopathic Effect ORF Overlapping Reading Frame SLAM Signaling Lymphocytic Activated Molecule CNS Central Nervous System IgV Immunoglobulin Variable domain IgC2 Immunoglobulin Constant-2 domain PVRL4 Poliovirus-receptor-like 4 HV Herpes Virus PCBs Persistent Polychlorinated Biphenyls

46 PCR Polymerase Chain Reaction RT-PCR Reverse Transcription Polymerase Chain Reaction IHC Immunohistochemistry VN Virus Neutralization PR Plaque Reduction iELISAs Indirect Enzyme-Linked Immunosorbent Assays OD Optical Density FFPE Formalin-Fixed Paraffin-Embedded qRT-PCR Reverse Transcription - Quantitative Polymerase Chain Reaction HRM High Resolution Melting GIS Geographic Information Systems

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