First detection of Feline morbillivirus infection in white-eared opossums (Didelphis albiventris, Lund, 1840), a non-feline host.

Feline Morbillivirus (FeMV) was first detected in 2012 in domestic cats from Hong Kong and was found to be associated with tubulointerstitial nephritis and chronic kidney disease. In subsequent studies in other countries, FeMV was detected in asymptomatic cats. However, it is not clear whether FeMV plays a role as a pathogen in the kidney diseases of cats, and other epidemiological data are still unknown. To date, studies have reported the presence of FeMV exclusively in domestic cats. This study is the first molecular detection of the FeMV RNA associated with pathological and immunohistochemical findings in a synanthropic marsupial, the white-eared opossum (Didelphis albiventris), inhabiting peri-urban areas of north-central Parana, Southern Brazil. Molecular techniques identified the viral RNA in the lungs and kidneys. Histopathologic evaluation of these tissues revealed interstitial pneumonia in the lungs with lymphocytic nephritis and tubular necrosis in the kidneys. Immunohistochemistry assays detected positive intralesional immunoreactivity to N protein of FeMV within the lungs and kidneys. A FeMV opossum strain was isolated in Crandell Rees feline kidney lineage cells, resulting in syncytia formation and cell death. Therefore, these results support the ability of FeMV to infect other mammal species and reinforce the possibility of the opossum to be a disseminator of this virus among domestic and wild animals.

Systematic beach monitoring as a health assessment tool: Cetacean morbillivirus under non-epizootic circumstances in stranded dolphins.

Cetacean morbillivirus (CeMV) was identified as the etiologic agent of several epizootic episodes worldwide. Most of these studies are based on unusual mortality events or identification of new viral strains. We investigated the occurrence of CeMV under non-epizootic circumstances at a world heritage in Southern Brazil by a combination of pathologic, immunohistochemical and molecular assays. From 325 stranded cetaceans, 40 were included. Guiana dolphin (Sotalia guianensis) was the most frequent species. Interstitial pneumonia and non-suppurative encephalitis were the main pathologic findings associated with CeMV infection. Intracytoplasmic immunolabelling anti-CeMV was observed mainly in lungs and lymph nodes. All samples were negative in reverse transcription polymerase chain reaction assay. Diagnosis of CeMV is challenging in areas where epizootic episodes have not been recorded and due to post-mortem changes. We observed a CeMV prevalence of 27.5%. The results described here increase the knowledge about CeMV under non-epizootic conditions in Brazil and worldwide.

Novel cetacean morbillivirus in a rare Fraser's dolphin (Lagenodelphis hosei) stranding from Maui, Hawai'i.

Cetacean morbillivirus (CeMV) is a global threat to cetaceans. We report a novel morbillivirus from a Fraser's dolphin (Lagenodelphis hosei) that stranded in Maui, Hawaii in 2018 that is dissimilar to the beaked whale morbillivirus previously identified from Hawaii and to other CeMV strains. Histopathological findings included intranuclear inclusions in bile duct epithelium, lymphoid depletion, rare syncytial cells and non-suppurative meningitis. Cerebellum and lung tissue homogenates were inoculated onto Vero.DogSLAMtag cells for virus isolation and cytopathic effects were observed, resulting in the formation of multinucleated giant cells (i.e., syncytia). Transmission electron microscopy of infected cell cultures also revealed syncytial cells with intracytoplasmic and intranuclear inclusions of viral nucleocapsids, consistent with the ultrastructure of a morbillivirus. Samples of the cerebellum, lung, liver, spleen and lymph nodes were positive for morbillivirus using a reverse transcription-polymerase chain reaction. The resulting 559 bp L gene sequence had the highest nucleotide identity (77.3%) to porpoise morbillivirus from Northern Ireland and the Netherlands. The resulting 248 bp P gene had the highest nucleotide identity to porpoise morbillivirus in Northern Ireland and the Netherlands and to a stranded Guiana dolphin (Sotalia guianensis) in Brazil (66.9%). As Fraser's dolphins are a pelagic species that infrequently strand, a novel strain of CeMV may be circulating in the central Pacific that could have additional population impacts through transmission to other small island-associated cetacean species.

Cetacean morbillivirus in Humpback whales' exhaled breath.

The humpback whale (HW; Megaptera novaeangliae) population that seasonally resides along the Brazilian coast concentrates in the Abrolhos Bank (Bahia and Espírito Santo states) for breeding during austral winter and spring. Cetacean morbillivirus (CeMV, Paramyxoviridae family) is currently one of the most significant biological threats to cetaceans worldwide with high infection and mortality rates. CeMV is pleiotropic yet it has special tropism for the respiratory, lymphoid and nervous system and is primarily transmitted by the aerogenous route. A new lineage of CeMV, the Guiana dolphin morbillivirus (GDMV), is known to affect cetaceans off Brazil. GDMV was first detected in a Guiana dolphin (Sotalia guianensis) stranded in the Abrolhos Bank region, in 2010. In addition to pathologic examinations on stranded HW, pathogen survey of free-ranging HW may provide valuable insight into the epidemiology of diseases. We hypothesized that HW in the Brazilian breeding ground could be exposed to CeMV. Thus, in the present study, we investigated the presence of CeMV in exhaled breath condensates (EBC) of HW in the Abrolhos Bank. Overall, 73 samples of EBC from 48 groups of HW were collected during the breeding seasons of 2011 (n = 16) and 2012 (n = 57). One sample failed to have the reference gene amplified and was excluded from the study. CeMV was detected by a RT-qPCR method in 2 EBC samples, representing 2 whale groups. Phylogenetic analysis of partial morbillivirus phosphoprotein gene showed 100% homology to GDMV. Our results show that HW in Brazil are infected by CeMV with a relative prevalence of 4.3% (2/47) and demonstrate the suitability of using EBC and RT-qPCR as a non-invasive tool for CeMV survey in free-ranging whales. This pioneer study provides scientific basis for non-invasive CeMV monitoring of HW, suggests HW may play a role in the dynamics of CeMV and raises concern for potential conservation implications for this species.

High genetic diversity of paramyxoviruses infecting domestic cats in Western Brazil.

Feline morbillivirus was discovered in 2012 in cats from Hong Kong, and it was initially found to be associated with chronic kidney disease. Although subsequent molecular surveys showed a common occurrence in cat populations from distinct countries, there were controversial results regarding the relationship between viral shedding through urine and reduced kidney function. In this study, 276 domestic cats of diverse origins from Western Brazil had their urine evaluated for the presence of paramyxoviral RNA by reverse transcription seminested PCR and direct sequencing. Additionally, a selected Brazilian feline morbillivirus strain was isolated in Crandell Rees feline kidney cells, and a nearly complete genome sequence was obtained. To assess the kidney function of all cats, serum biochemistry screening and standard urinalysis were performed. Our results revealed a relatively high paramyxovirus-positive rate (34.7%) in the evaluated cats although there was not a statistical association between the shedding of viral RNA through urine and kidney disease. Direct sequencing of partial fragments of the L gene demonstrated high genetic diversity among strains detected in cats in this study, since both feline morbillivirus RNA and feline paramyxovirus RNA were frequently shed in urine. Phylogenetic reconstruction based on partial amino acid sequences of the L gene showed that Brazilian feline paramyxovirus strains were genetically diverse since they grouped into two distinct subclusters; one subcluster contained three strains identified in Germany, while the second contained Japanese strain 163, which was recently classified in the Jeilongvirus genus of the Paramyxoviridae family. In contrast, the Brazilian feline morbillivirus strain FeMV/BR_Boni, herein characterized by nearly complete genome sequencing, was classified in the Morbillivirus genus with other strains previously identified as genotype 1. In conclusion, urinary excretion of diverse paramyxoviral RNA is frequent in cats of different origins from Western Brazil, but viral infection is not related to altered kidney function.

Clinical signs in free-ranging Guiana dolphins Sotalia guianensis during a morbillivirus epidemic: case study in Sepetiba Bay, Brazil.

From November 2017 to March 2018, a cetacean morbillivirus (CeMV) outbreak caused an unprecedented mass mortality among Guiana dolphins Sotalia guianensis in Ilha Grande Bay and Sepetiba Bay, Rio de Janeiro, Brazil. Small boat surveys were conducted to document the behavior and clinical signs presented by diseased dolphins. We observed 5 abnormally behaving, disoriented Guiana dolphins on separate days, of which 1 died stranded and 2 sank. Signs of ataxia included difficulties with swimming and maintaining a course, balance and buoyancy. At least 40 other individuals were emaciated, and 10 photo-identified dolphins had miscellaneous skin lesions, some ulcerated. Labored breathing suggestive of airway obstruction was heard in several groups. These neurological, respiratory and cutaneous signs may comprise part of the clinical constellation of CeMV infection in dolphins. The combined threat of anthropogenic pressures and CeMV lethal disease is of concern for the survival of the Guiana dolphin population in Sepetiba Bay.

First report of feline morbillivirus in South America.

Feline morbillivirus was first identified in healthy and diseased stray cats captured in Hong Kong. Recently, it was demonstrated that the virus circulates within cat populations in Japan, Italy, Germany, and the USA. Importantly, an association between feline morbillivirus infection and chronic kidney disease was suggested by histological analysis of kidney tissue of infected cats. The aim of this study was to verify the presence and examine the genetic diversity of feline morbilliviruses associated with infections of domestic cats in Brazil. Seventeen cats without clinical manifestations of urinary tract diseases from a multi-cat household and 35 random client-owned cats admitted to the Teaching Veterinary Hospital for a variety of reasons were evaluated for paramyxoviral infection and the presence of uropathy. A fragment of the paramyxoviral L gene was amplified from urine samples using a reverse transcription semi-nested PCR assay. For the first time, we detected a feline morbillivirus strain that was genetically related to viral strains previously characterized in Japan in urine samples from cats in South America, in Brazil. This together with the recent description of feline morbillivirus identification within cat populations in the USA, suggests a possible widespread distribution of this viral agent on the American continent. Our data demonstrated feline morbillivirus RNA shedding mostly in the urine of cats without clinical, laboratorial, or ultrasonographic signs of urinary tract diseases. In contrast to previously published findings that associated feline morbillivirus infection with chronic kidney disease, we did not observe a clear relationship between feline morbillivirus RNA shedding in urine and kidney disease in the cats evaluated.

A simultaneous diagnosis and genotyping method for global surveillance of cetacean morbillivirus.

Cetacean morbillivirus (CeMV) is considered one of the most important viral pathogens in cetaceans. CeMV outbreaks of lethal disease have repeatedly been observed in Europe, the Americas, and Australia, while large herds of gregarious species were found to be the likely reservoirs and sources of CeMV infection to susceptible species in the Atlantic and Pacific Oceans. Furthermore, three new strains were detected recently in Hawaii, Brazil and Australia. To clarify the real global distribution of CeMV and possible carriers, we showed a novel technique successfully diagnosing and distinguishing different virus strains (DMV, PWMV and novel CeMVs) using FFPE samples from 1996 to 2011. This efficient method that combines qRT-PCR and high resolution melting (HRM) could be applied to the future retrospective global studies for better understanding of different prevalence and outbreak conditions among ocean basins and the mechanism of variable host response to pathogens.

Fetal distress and in utero pneumonia in perinatal dolphins during the Northern Gulf of Mexico unusual mortality event.

An unusual mortality event (UME) involving primarily common bottlenose dolphins Tursiops truncatus of all size classes stranding along coastal Louisiana, Mississippi, and Alabama, USA, started in early 2010 and continued into 2014. During this northern Gulf of Mexico UME, a distinct cluster of perinatal dolphins (total body length <115 cm) stranded in Mississippi and Alabama during 2011. The proportion of annual dolphin strandings that were perinates between 2009 and 2013 were compared to baseline strandings (2000-2005). A case-reference study was conducted to compare demographics, histologic lesions, and Brucella sp. infection prevalence in 69 UME perinatal dolphins to findings from 26 reference perinates stranded in South Carolina and Florida outside of the UME area. Compared to reference perinates, UME perinates were more likely to have died in utero or very soon after birth (presence of atelectasis in 88 vs. 15%, p < 0.0001), have fetal distress (87 vs. 27%, p < 0.0001), and have pneumonia not associated with lungworm infection (65 vs. 19%, p = 0.0001). The percentage of perinates with Brucella sp. infections identified via lung PCR was higher among UME perinates stranding in Mississippi and Alabama compared to reference perinates (61 vs. 24%, p = 0.01), and multiple different Brucella omp genetic sequences were identified in UME perinates. These results support that from 2011 to 2013, during the northern Gulf of Mexico UME, bottlenose dolphins were particularly susceptible to late-term pregnancy failures and development of in utero infections including brucellosis.

An insight into the epidemiology of dolphin morbillivirus worldwide.

Serum samples from 288 cetaceans representing 25 species and originating from 11 different countries were collected between 1995 and 1999 and examined for the presence of dolphin morbillivirus (DMV)-specific antibodies by an indirect ELISA (iELISA) (N = 267) or a plaque reduction assay (N = 21). A total of 35 odontocetes were seropositive: three harbour porpoises (Phocoena phocoena) and a common dolphin (Delphinus delphis) from the Northeastern (NE) Atlantic, a bottlenose dolphin (Tursiops truncatus) from Kent (England), three striped dolphins (Stenella coeruleoalba), two Risso's dolphins (Grampus griseus) and a bottlenose dolphin from the Mediterranean Sea, one common dolphin from the Southwest (SW) Indian Ocean, three Fraser's dolphins (Lagenodelphis hosei) from the SW Atlantic, 18 long-finned pilot whales (Globicephala melas) and a bottlenose dolphin from the SW Pacific as well as a captive bottlenose dolphin (Tursiops aduncus) originally from Taiwan. The presence of morbillivirus antibodies in 17 of these animals was further examined in other iELISAs and virus neutralization tests. Our results indicate that DMV infects cetaceans worldwide. This is the first report of DMV-seropositive animals from the SW Indian, SW Atlantic and West Pacific Oceans. Prevalence of DMV-seropositives was 85.7% in 21 pilot whales from the SW Pacific and both sexually mature and immature individuals were infected. This indicates that DMV is endemic in these animals. The same situation may occur among Fraser's dolphins from the SW Atlantic. The prevalence of DMV-seropositives was 5.26% and 5.36% in 19 common dolphins and 56 harbour porpoise from the NE Atlantic, respectively, and 18.75% in 16 striped dolphins from the Mediterranean. Prevalence varied significantly with sexual maturity in harbour porpoises and striped dolphins; all DMV-seropositives being mature animals. The prevalence of seropositive harbour porpoise and striped dolphins appeared to have decreased since previous studies. These data suggest that DMV is not endemic within these populations, that they are losing their humoral immunity against the virus and that they may be vulnerable to new epidemics.

A review of virus infections of cataceans and the potential impact of morbilliviruses, poxviruses and papillomaviruses on host population dynamics.

Viruses belonging to 9 families have been detected in cetaceans. We critically review the clinical features, pathology and epidemiology of the diseases they cause. Cetacean morbillivirus (family Paramyxoviridae) induces a serious disease with a high mortality rate and persists in several populations. It may have long-term effects on the dynamics of cetacean populations either as enzootic infection or recurrent epizootics. The latter presumably have the more profound impact due to removal of sexually mature individuals. Members of the family Poxviridae infect several species of odontocetes, resulting in ring and tattoo skin lesions. Although poxviruses apparently do not induce a high mortality, circumstancial evidence suggests they may be lethal in young animals lacking protective immunity, and thus may negatively affect net recruitment. Papillomaviruses (family Papovaviridae) cause genital warts in at least 3 species of cetaceans. In 10% of male Burmeister's porpoises Phocoena spinipinnis from Peru, lesions were sufficiently severe to at least hamper, if not impede, copulation. Members of the families Herpesviridae, Orthomyxoviridae and Rhabdoviridae were demonstrated in cetaceans suffering serious illnesses, but with the exception of a 'porpoise herpesvirus' their causative role is still tentative. Herpes-like viruses and caliciviruses (Caliciviridae) give rise to cutaneous diseases in Monodontidae and Delphinidae. Antibodies to several serotypes of caliciviruses were found in odontocetes and mysticetes. An unrecognized Hepadnaviridae was detected by serology in a captive Pacific white-sided dolphin Lagenorhynchus obliquidens with chronic persistent hepatitis. Adenoviruses (Adenoviridae) were isolated from the intestinal tracts of mysticeti and a beluga Delphinapterus leucas but were not associated with any pathologies. We discuss the potential impact of Paramyxoviridae, Poxviridae and Papovaviridae on the dynamics of several odontocete populations.

Serological evidence of morbillivirus infection in small cetaceans from the Southeast Pacific.

The presence of morbillivirus-specific serum antibodies was examined by an indirect enzyme linked immunosorbent assay (iELISA) and virus neutralization tests in serum samples from 30 dusky dolphins (Lagenorhynchus obscurus), 8 long-snouted common dolphins (Delphinus capensis), 2 inshore and 6 offshore bottlenose dolphins (Tursiops truncatus) and 20 Burmeister's porpoises (Phocoena spinipinnis) taken in fisheries off central Peru in 1993-1995. The sera from six dusky dolphins, one common dolphin and three offshore bottlenose dolphins were positive on a coat of dolphin morbillivirus (DMV) antigen in the iELISA. Several of these sera were also positive when tested against peste des petits ruminants and rinderpest virus antigen. Porpoise morbillivirus and/or DMV neutralizing antibodies were detected in the sera of two bottlenose and three dusky dolphins that reacted positively with DMV antigen in iELISA and also in the sera of one common, one dusky and one bottlenose dolphin that were negative in the iELISA. These results strongly suggest that viruses closely related, or identical, to the cetacean morbillivirus present in the North Atlantic and the Mediterranean Sea infect several species of Delphinidae of the Southeastern Pacific. No convincing morbillivirus-specific antibody positive reactions were detected in the sera from either the Burmeister's porpoises or the inshore bottlenose dolphins.

Morbilliviral epizootic in bottlenose dolphins of the Gulf of Mexico.

Morbillivirus infection was diagnosed in 35/67 bottlenose dolphins (Tursiops truncatus) from the Gulf of Mexico that stranded from October 1993 through April 1994 in Alabama, Mississippi, and Texas (USA) during periods of increased dolphin strandings in each of the 3 states. Diagnosis was based on histologic lesions, immunohistochemical demonstration of mobilliviral antigen, and detection of morbilliviral RNA by a reverse transcriptase polymerase chain reaction (RT-PCR) test performed on formalin-fixed, paraffin-embedded tissue (5 dolphins), on histologic lesions and detection of morbilliviral RNA by RT-PCR performed on formalin-fixed, paraffin-embedded tissue (1 dolphin), and on detection of morbilliviral RNA by RT-PCR performed on unfixed lung samples collected from carcasses with advanced postmortem autolysis (29 dolphins). Histologic lesions included proliferative interstitial pneumonia with syncytial cells and eosinophilic intranuclear and intracytoplasmic inclusion bodies, lymphoid depletion and syncytial cells with eosinophilic intranuclear inclusion bodies in lymph nodes, eosinophilic intracytoplasmic inclusion bodies in transitional epithelium of urinary bladder, and a syncytial cell with eosinophilic intranuclear inclusion bodies in epidermis. Concomitant pulmonary aspergillosis was diagnosed histologically in 4 dolphins. This is the 5th reported morbilliviral epizootic of aquatic mammals and the 2nd involving bottlenose dolphins in the United States.

Postmortem diagnosis of morbillivirus infection in bottlenose dolphins (Tursiops truncatus) in the Atlantic and Gulf of Mexico epizootics by polymerase chain reaction-based assay.

Lung tissue from 39 bottlenose dolphins (Tursiops truncatus) found dead off the U.S. Atlantic and Gulf of Mexico coasts from 1987 to 1994 was examined for the presence of morbillivirus using a reverse transcriptase polymerase chain reaction (RT-PCR) technique. Of the Atlantic cases examined, six of six were positive using this assay; 18 of 25 Gulf of Mexico cases with amplifiable RNA also were found to be positive, and eight additional specimens had no amplifiable RNA. The RT-PCR allowed the diagnosis of morbillivirus infection to be made from either sections of paraffin-embedded formalin-fixed material or from unfixed tissue. Conformation of diagnosis was made by subsequent hybridization of the amplified products with a dolphin morbillivirus specific probe using the Southern blot technique. Application of this method to autolyzed post-mortem tissues allows diagnoses of morbillivirus infection to be made in specimens which cannot be evaluated by histologic and immunocytochemical techniques.

Morbilliviral disease in an Atlantic bottlenose dolphin (Tursiops truncatus) from the Gulf of Mexico.

A free-living adult female Atlantic bottlenose dolphin (Tursiops truncatus) found dead near Panama City, Florida (USA), had necrotizing and ulcerative tracheitis, suppurative and hemorrhagic pneumonia, and necrotizing myocarditis; fungal hyphae were present in these lesions. Additionally, lungs had multifocal proliferative interstitial pneumonia with occasional syncytial cells. Some syncytial cells and type II pneumocytes contained eosinophilic intranuclear or intracytoplasmic inclusion bodies, or both. Based on an immunoperoxidase technique, there was morbilliviral antigen within cytoplasm and nuclei of type II pneumocytes and syncytial cells: antigen also occurred in trachea, skin, liver, stomach, intestine, and uterus. Based on pathologic and immunocytochemical findings, the dolphin had morbillivirus-induced disease. This is the first report of morbilliviral disease in a marine mammal from the Gulf of Mexico.