perspectives of morbillivirus infections in cetaceans: a

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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

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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.

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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).

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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,

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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;

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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

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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

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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).

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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).

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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

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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

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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

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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,

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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.

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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).

16

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),

17

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

18

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,

19

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).

20

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.

21

• 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).

22

• 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

23

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).

24

• 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,

25

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

26

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

27

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.

28

!

!

!

!

!

!

!

!

!!

!

!

!

!

!

!

!

!

!

!

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!!

!!

!!

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8 5 3 2 1

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

29

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

Species Ocean Years Countries Epidemiological Status

Virus Strain Literature Cited

MYSTICETI Family Balaenopteridae

Balaenoptera physalus

Eastern Atlantic &

North Sea

1983 Iceland Unknown CeMV (Blixenkrone-Moller et al., 1994)

Eastern Atlantic &

North Sea

1997-

1998

Belgium, France Periodic

mortalities

Unknown (Jauniaux et al., 2000)

Mediterranean Sea 2011 Tuscany, Italia Periodic

mortalities

DMV (Casalone et al., 2014; Mazzariol et al., 2012, 2016;

Profeta et al., 2015)

Balaenoptera acutorostrata

Mediterranean Sea 1993 Tuscany, Italia Unknown Unknown (Di Guardo et al., 1995)

ODONTOCETI Family Phocoenidae

Phocoena phocoena

Eastern Atlantic &

North Sea

1988-

1990

N. Ireland, UK,

Netherlands

Periodic

mortalities

PMV (Kennedy et al., 1991; McCullough et al., 1991; Van

Bressem et al., 2001; Visser et al., 1993)

Northwestern Atlantic 1993-

1994

East coast, Canada Unknown CeMV (Duignan et al., 1995)

Family Physeteridae

Physeter macrocephalus

Eastern Pacific 2011 Hawaii, USA Unknown BWMV (West et al., 2015)

Family Kogiidae

Kogia breviceps


1991

Southeast coast USA Unknown CeMV (Duignan et al., 1995)

Western Pacific 2009 SW Taiwan Periodic

mortalities

DMV (Yang et al., 2006)

Family Ziphiidae

Indopacetus pacificus

Eastern Pacific 2010 Hawaii, USA Unknown BWMV (West et al., 2013)

Southern Ocean 2013 New Caledonia Unknown DMV (Garrigue et al., 2016)

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

30

Family Delphinidae

Delphinus delphis

Eastern Atlantic &

North Sea

1988-

1990

UK, Netherlands,

Germany

Unknown CeMV (Van Bressem et al., 2001; Visser et al., 1993)

Eastern Atlantic &

North Sea

2007 Canary Islands Periodic

mortalities

DMV (Sierra et al., 2014; Van Bressem et al., 2001)

Mediterranean Sea 1990 Italy Unknown CeMV (Van Bressem et al., 1993)


1994

Northeast coast USA Possibly endemic CeMV (Duignan et al., 1995)

Eastern Pacific 1995-

1997

California, USA Unknown CeMV (Reidarson et al., 1998; Taubenberger et al., 2000b)

Southern Ocean 2013 South Australia Unknown CeMV (Kemper et al., 2013)

Indian Ocean 1999 East London, South

Africa

Unknown CeMV (Van Bressem et al., 2001)

Delphinus capensis Eastern Pacific 1993-

1995

Central Peru Endemic CeMV (Van Bressem et al., 1998)

Feresa attenuata Northwestern Atlantic 1983-

1991

Southeast coast USA Unknown CeMV (Duignan et al., 1995)

Globicephala macrorhynchus

Eastern Atlantic &

North Sea

1996,

2015

Canary Islands Periodic

mortalities

PWMV (Sierra et al., 2016)


1994

Florida, USA Endemic CeMV (Duignan et al., 1995)

Globicephala melas

Mediterranean Sea 2006-

2007,

2013

Spain, France, Italy Epidemic DMV (Casalone et al., 2014; Fernández et al., 2008; Profeta

et al., 2015)

Eastern Atlantic &

North Sea


mortalities

CeMV (Sierra et al., 2016)


1993

Northeast coast USA Endemic CeMV (Barrett et al., 1993; Duignan et al., 1995)

Northwestern Atlantic late

nineties

New Jersey, USA Periodic

mortalities

PWMV (Taubenberger et al., 2000b)

Western Pacific 1997 Northland, New

Zeland

Endemic CeMV (Van Bressem et al., 2001)

31

Grampus griseus Mediterranean Sea 1997,19

99

Valencia, Spain Unknown CeMV (Casalone et al., 2014; Van Bressem et al., 2001)

Lagenodelphis hosei

Northwestern Atlantic 1994 Gulf of Mexico, USA Possibly endemic CeMV (Duignan et al., 1995)

Southwestern Atlantic 1999 Puerto Madryn,

Argentina

Unknown CeMV (Van Bressem et al., 2001)

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)

Lagenorhynchus albirostris

Eastern Atlantic &

North Sea

1988-

1990,

2007,

2011

Germany,

Netherlands

Periodic

mortalities

DMV (Elk et al., 2014; Visser et al., 1993)

Lagenorhynchus acutus


1993

Northeast coast USA Unknown CeMV (Duignan et al., 1995)

Lagenorhynchus obscurus


1995


Lagenorhynchus obliquidens

Western Pacific 1998 Miyazaki, Japan Unknown Unknown (Uchida et al., 1999)

Peponocephala electra

Western Pacific 2005-

2007

NE Australia Endemic CeMV (Stone et al., 2012)

Pseudorca crassidens


1988

Southeast coast USA Possibly endemic CeMV (Duignan et al., 1995)

Sotalia guianensis Southwestern Atlantic 2010 Espirito Santo, Brazil Unknown CeMV (Groch et al., 2014)

Stenella coeruleoalba

Eastern Atlantic &

North Sea

2002-

2011

Canary Islands Periodic

mortalities

DMV (Sierra et al., 2014)

Mediterranean Sea 1990–

1992

Spain, France, Italy,

Greece

Epidemic DMV (Aguilar & Raga, 1993; Di Guardo et al., 1995; Profeta

et al., 2015; Bressem et al., 1993)

Mediterranean Sea 2006-

2008,

Spain, France, Italy Epidemic DMV (Casalone et al., 2014; Di Guardo et al., 2013; Profeta

et al., 2015; Raga et al., 2008)


1993

Northeast cost USA Unknown CeMV (Duignan et al., 1995)

Stenella frontalis Northwestern Atlantic 1993 Northeast coast USA Unknown CeMV (Duignan et al., 1995)

32

Tursiops truncatus

Eastern Atlantic &

North Sea

1999 Kent, UK Unknown CeMV (Van Bressem et al., 2001)

Eastern Atlantic &

North Sea


mortalities

DMV (Sierra et al., 2014)

Mediterranean Sea 1994;

2007-

2008,

2011

Israel, Spain, France,

Italy

Periodic

mortalities

DMV (Casalone et al., 2014; Duignan et al., 1995; Profeta et

al., 2015)

Northwestern Atlantic 1982 Florida, USA Epidemic CeMV (Rowles et al., 2011)


1988

East coast USA Epidemic CeMV (Rowles et al., 2011; Schulman et al., 1997;

Taubenberger et al., 1996)


1994

Gulf of Mexico, USA Epidemic CeMV (Rowles et al., 2011; Taubenberger et al., 1996)


2007

Florida, USA Unknown CeMV (Bossart et al., 2010, 2011; Rowles et al., 2011)


2014

East coast USA Epidemic DMV (Rowles et al., 2011; Shimizu et al., 2013)


1994

east coast USA Endemic CeMV (Rowles et al., 2011)


1994

US Atlantic coast,

Gulf of Mexico

Unknown CeMV (Krafft et al., 1995; Rowles et al., 2011)


1995


Western Pacific 1997 Tasmania, Australia Unknown CeMV (Van Bressem et al., 2001)


2010

Queensland,

Australia

Periodic

mortalities

DMV (Stone et al., 2011, 2012)

Southern Ocean 2013 South Australia Unknown CeMV (Kemper et al., 2013)

Tursiops aduncus

Indian Ocean 2009 Western Australia Periodic

mortalities

CeMV (Stephens et al., 2014)


2010

NE Australia Unknown CeMV (Stone et al., 2012)

Southern Ocean 2012-

2013

South Australia Unknown CeMV (Kemper et al., 2013; Van Bressem et al., 2001)

33

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.

34

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.

35

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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

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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

perspectives of morbillivirus infections in cetaceans: a

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