Characterization of Cetacean Morbillivirus in Humpback Whales, Brazil.

Cetacean morbillivirus is an etiologic agent associated with strandings of live and dead cetacean species occurring sporadically or as epizootics worldwide. We report 2 cases of cetacean morbillivirus in humpback whales (Megaptera novaeangliae) in Brazil and describe the anatomopathological, immunohistochemical, and molecular characterization findings in the specimens.

Investigation of Chlamydia sp., Morbillivirus sp., Parvovirus sp., Leishmania sp. and Alphacoronavirus sp. in captive giant anteaters (Myrmecophaga tridactyla) / [Investigação de Chlamydia sp., Morbillivirus sp., Parvovirus sp., Leishmania sp. e Alphacoronavirus sp. em tamanduás-bandeira (Myrmecophaga tridactyla) em cativeiro]

This research aimed to investigate the occurrence of Chlamydia sp., Morbillivirus sp., Parvovirus sp., Leishmania sp. and Alphacoronavirus sp. in captive giant anteaters. Blood and fecal samples were taken from 16 animals in institutions from the states of Minas Gerais, Bahia and Distrito Federal, which had been in captivity for at least a year. A commercial rapid chromatographic immunoassay test was used for detecting coronavirus and parvovirus antigens, in addition to antibodies against leishmaniasis, all results being negative. In the case of the test for antibodies against distemper, four (4/16; 25%) anteaters had an average titration, two (2/16; 12.5%) a low titration and ten (10/16; 62.5%) were non-reactive. Using the DOT-ELISA (dot blotting) method for detection of immunoglobulin G, only one specimen obtained a 1 : 40 titration. For the polymerase chain reaction tests for Leishmania and Chlamydia, all samples were negative.

Esta pesquisa teve como objetivo investigar a ocorrência de Chlamydia sp., Morbillivirus sp., Parvovirus sp., Leishmania sp. e Alphacoronavirus sp. em tamanduás-bandeira cativos. Foram colhidas amostras de sangue e fezes de 16 animais em instituições dos estados de Minas Gerais, Bahia e Distrito Federal, que estavam em cativeiro há pelo menos um ano. Um teste comercial rápido de imunoensaio cromatográfico foi usado para detectar antígenos de coronavírus e parvovírus, além de anticorpos contra a leishmaniose, sendo todos os resultados negativos. No caso do teste para anticorpos contra a doença, quatro (4/16; 25%) tamanduás apresentaram titulação média, dois (2/16; 12,5%) uma titulação baixa e dez (10/16; 62,5%) não foram reativos. A partir do método DOT-ELISA (dot blotting) para detecção de imunoglobulina G, apenas um espécime obteve uma titulação de 1: 40. Para os testes de reação em cadeia da polimerase para Leishmania e Chlamydia, todas as amostras foram negativas.

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.

Molecular characterization of Feline paramyxovirus and Feline morbillivirus in cats from Brazil.

This study is aimed at detecting Feline paramyxovirus (FPaV) and Feline morbillivirus (FeMV) in 35 urine samples from domestic cats, collected in 2019, with or without clinical signs of uropathies using a reverse transcription-polymerase chain reaction (RT-PCR) followed by semi-nested polymerase chain reaction (SN-PCR) assays to amplify a partial paramyxovirus L gene. Eight (22.9%) out of the 35 urine samples were positive for paramyxoviruses. Sequencing and phylogenetic analyses revealed that three samples were positive for FPaV, four samples were positive for FeMV, and it was not possible to determine which virus was present in one RT-SN-PCR positive urine sample. FPaV strains showed 100% nucleotide (nt) identity with each other and 97% nt identity with a Japanese 163 FPaV strain. The FeMV strains showed 85.9% nt identity with each other; three strains were similar to previously described Brazilian FeMV strains, and one strain clustered in a different branch of the phylogenetic tree together with the first described Chinese FeMV strain. This study provides the first description of FPaV strains in cats from Brazil and provides new information about the molecular characteristics of FPaV and FeMV strains circulating in domestic cats in Brazil.

A novel real-time PCR to detect Cetacean morbillivirus in Atlantic cetaceans.

Cetacean morbillivirus (CeMV, family Paramyxoviridae) is a re-emergent pathogen associated with severe epizootic outbreaks causing high mortality among cetaceans worldwide. Recently, CeMV caused an unusual mortality event of Guiana dolphins (Sotalia guianensis) in Brazil. Partial sequence of the viral phosphoprotein (P) gene showed that the Guiana dolphin morbillivirus (GDMV) might represent a new lineage of CeMV. This study aimed to develop a molecular technique to detect the most common CeMV strains known to circulate in the Atlantic Ocean: GDMV, Dolphin morbillivirus (DMV) and Pilot-whale morbillivirus (PWMV). A sensible real-time reverse transcription polymerase chain reaction (RT-qPCR) method based on intercalating dye, targeting the P gene was described. This assay successfully detected GDMV, PWMV and DMV from field samples. Its performance was compared to a RT-qPCR method that specifically detects GDMV. Both assays had high sensibility and excellent intra- and inter-assay reproducibility. A total of 109 field samples from 32 Guiana dolphins were screened for CeMV by conventional RT-PCR in parallel with the RT-qPCR assay. The detection rate increased from 32% to 60% by use of the novel RT-qPCR. The RT-qPCR assay described herein allows rapid and sensitive detection of Atlantic CeMV strains, and is potentially suitable for screening of CeMV globally.

Cetacean morbillivirus in Southern Right Whales, Brazil.

Cetacean morbillivirus (CeMV) has caused repeated epizootics and interepizootic fatalities in a variety of cetacean species worldwide. Recently, a novel CeMV strain (GD-CeMV) was linked to a mass die-off of Guiana dolphins (Sotalia guianensis) in Brazil. Southern right whales (SRWs; Eubalaena australis) migrate to the southern Brazilian coast during austral winter and spring (June through November) for breeding and calving. Because unexplained high calf mortality rates have recurrently been documented in SRWs, we hypothesized they could be infected with CeMV. We developed a novel real-time RT-PCR method based on SYBR® GREEN for detection of CeMV and identified the virus in three out of five stranded SRWs from Santa Catarina state, Brazil. The partial sequences of the morbillivirus phosphoprotein gene suggest that the virus is similar to the GD-CeMV strain. Our results indicate CeMV can infect SRWs and should be considered in the differential aetiologic diagnosis of infectious diseases in this species. It also raises concern for potential conservation implications for this species in its main coastal breeding area off Southern Brazil.

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.

Coinfection by Cetacean morbillivirus and Aspergillus fumigatus in a juvenile bottlenose dolphin (Tursiops truncatus) in the Gulf of Mexico.

A recently deceased juvenile male bottlenose dolphin (Tursiops truncatus) was found floating in the Gulf of Mexico, off Sand Key in Clearwater, Florida. At autopsy, we identified pneumonia and a focus of malacia in the right cerebrum. Cytologic evaluation of tissue imprints from the right cerebrum revealed fungal hyphae. Fungal cultures of the lung and brain yielded Aspergillus fumigatus, which was confirmed by amplification of a portion of the fungal nuclear ribosomal internal transcribed spacer 2 region sequence. Microscopic pulmonary lesions of bronchiolar epithelial cell syncytia with intracytoplasmic and intranuclear inclusions within bronchiolar epithelial cells were suggestive of Cetacean morbillivirus (CeMV) infection. The occurrence of CeMV infection was supported by positive immunohistochemical staining for morbillivirus antigen. CeMV detection was confirmed by amplification and sequencing a portion of the morbilliviral RNA-dependent RNA polymerase gene from lung tissue. This case provides CeMV sequence data available from the Gulf of Mexico and underscores the need for genomic sequencing across diverse host, temporospatial, and population (i.e., single animal vs. mass mortality events) scales to improve our understanding of these globally emerging pathogens.

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.

Cetacean morbillivirus: current knowledge and future directions.

We review the molecular and epidemiological characteristics of cetacean morbillivirus (CeMV) and the diagnosis and pathogenesis of associated disease, with six different strains detected in cetaceans worldwide. CeMV has caused epidemics with high mortality in odontocetes in Europe, the USA and Australia. It represents a distinct species within the Morbillivirus genus. Although most CeMV strains are phylogenetically closely related, recent data indicate that morbilliviruses recovered from Indo-Pacific bottlenose dolphins (Tursiops aduncus), from Western Australia, and a Guiana dolphin (Sotalia guianensis), from Brazil, are divergent. The signaling lymphocyte activation molecule (SLAM) cell receptor for CeMV has been characterized in cetaceans. It shares higher amino acid identity with the ruminant SLAM than with the receptors of carnivores or humans, reflecting the evolutionary history of these mammalian taxa. In Delphinidae, three amino acid substitutions may result in a higher affinity for the virus. Infection is diagnosed by histology, immunohistochemistry, virus isolation, RT-PCR, and serology. Classical CeMV-associated lesions include bronchointerstitial pneumonia, encephalitis, syncytia, and lymphoid depletion associated with immunosuppression. Cetaceans that survive the acute disease may develop fatal secondary infections and chronic encephalitis. Endemically infected, gregarious odontocetes probably serve as reservoirs and vectors. Transmission likely occurs through the inhalation of aerosolized virus but mother to fetus transmission was also reported.

Análise microscópica do miocárdio ventricular esquerdo em cães soropositivos para cinomose / Microscopic analysis of the left ventricular myocardium in positive serum dogs to distemper disease

Classificado no gênero Morbillivirus da família Paramixoviridae, o vírus da cinomose possui RNA de fita simples de polaridade negativa, é causador de doença multissistêmica, altamente contagiosa e grave dos cães e carnívoros selvagens, e com elevado índice de mortalidade em animais não vacinados ou com falhas vacinais. Com o objetivo de avaliar as alterações histopatológicas no coração, particularmente na região do miocárdio ventricular esquerdo, de cães naturalmente infectados com o vírus da cinomose, foram estudados 35 animais, de ambos os sexos e com idades variadas. Das 35 amostras enviadas ao Laboratório de Medicina Veterinária Preventiva do Hospital Veterinário de Uberaba, 100 por cento (35/35) mostrou-se soropositivas para a cinomose (técnica de imunoensaio em fase sólida) e tiveram no miocárdio ventricular esquerdo as seguintes alterações histopatológicas: miocardite, degeneração hialina, hiperemia e hemorragia, com 42,8 por cento (15/35), 31,4 por cento (11/35), 14,3 por cento (5/35) e 11,4 por cento (4/35), respectivamente. Tendo utilizado o teste Qui-Quadrado com nível de significância de 0,05, conclui-se que existe alta correlação (p=0,02) entre os animais infectados com o vírus da cinomose e as alterações histopatológicas observadas no miocárdio ventricular esquerdo.(AU)

Classified pertaining to the genus Morbillivirus of the Paramyxoviridae family, the canine distemper virus is a RNA single-stranded virus with negative polarity and causes a multisystemic disease, serious and highly contagious for dogs and wild carnivores, with a high mortality rate in non-vaccinated animals or with vaccine fails. With the objective to evaluate heart histopathological alterations, particularly in the left ventricular myocardium, in dogs naturally infected with canine distemper virus, 35 dogs, males and females of different ages, were studied. All the 35 samples sent to the Veterinary Hospital of Uberaba were serum-positive for distemper (immunoassay technique in solid phase) and had in the left ventricular myocardium the following histopathologic alterations: myocarditis, hyalin degeneration, hyperemia and hemorrhage, in 42.8 percent (15/35), 31.4 percent (11/35), 14.3 percent (5/35) and 11.4 percent (4/35), respectively. Having carried out the Qui-Quadrado test with a significancy level of 0.05, it can be concluded that there is a high correlation (p=0.02) between the infected animals with canine distemper virus and histopathological alterations found in the left ventricular myocardium.(AU)

Análise microscópica do miocárdio ventricular esquerdo em cães soropositivos para cinomose / Microscopic analysis of the left ventricular myocardium in positive serum dogs to distemper disease

Classificado no gênero Morbillivirus da família Paramixoviridae, o vírus da cinomose possui RNA de fita simples de polaridade negativa, é causador de doença multissistêmica, altamente contagiosa e grave dos cães e carnívoros selvagens, e com elevado índice de mortalidade em animais não vacinados ou com falhas vacinais. Com o objetivo de avaliar as alterações histopatológicas no coração, particularmente na região do miocárdio ventricular esquerdo, de cães naturalmente infectados com o vírus da cinomose, foram estudados 35 animais, de ambos os sexos e com idades variadas. Das 35 amostras enviadas ao Laboratório de Medicina Veterinária Preventiva do Hospital Veterinário de Uberaba, 100 por cento (35/35) mostrou-se soropositivas para a cinomose (técnica de imunoensaio em fase sólida) e tiveram no miocárdio ventricular esquerdo as seguintes alterações histopatológicas: miocardite, degeneração hialina, hiperemia e hemorragia, com 42,8 por cento (15/35), 31,4 por cento (11/35), 14,3 por cento (5/35) e 11,4 por cento (4/35), respectivamente. Tendo utilizado o teste Qui-Quadrado com nível de significância de 0,05, conclui-se que existe alta correlação (p=0,02) entre os animais infectados com o vírus da cinomose e as alterações histopatológicas observadas no miocárdio ventricular esquerdo.

Classified pertaining to the genus Morbillivirus of the Paramyxoviridae family, the canine distemper virus is a RNA single-stranded virus with negative polarity and causes a multisystemic disease, serious and highly contagious for dogs and wild carnivores, with a high mortality rate in non-vaccinated animals or with vaccine fails. With the objective to evaluate heart histopathological alterations, particularly in the left ventricular myocardium, in dogs naturally infected with canine distemper virus, 35 dogs, males and females of different ages, were studied. All the 35 samples sent to the Veterinary Hospital of Uberaba were serum-positive for distemper (immunoassay technique in solid phase) and had in the left ventricular myocardium the following histopathologic alterations: myocarditis, hyalin degeneration, hyperemia and hemorrhage, in 42.8 percent (15/35), 31.4 percent (11/35), 14.3 percent (5/35) and 11.4 percent (4/35), respectively. Having carried out the Qui-Quadrado test with a significancy level of 0.05, it can be concluded that there is a high correlation (p=0.02) between the infected animals with canine distemper virus and histopathological alterations found in the left ventricular myocardium.

Recent measles viral activity in Uruguai: serological and genetic approaches.

In the summer 1999, a measles outbreak occurred in Uruguai. During this outbreak 58 cases were recorded, 36 of which were laboratory confirmed as positive for measles virus (MV) IgM. The cases occurred in touristic places (Montevideo and Maldonado) predominantly among health facilities and tourist service personnel. Urine specimens collected between days 1 and 4 after the onset of the rash from seven cases were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR with primers specific for the carboxyl-terminal region of the nucleoprotein (N) gene. Three of these specimens/cases were positive for MV. Sequencing of 300 nucleotides (nt) of PCR products corresponding to a part of the carboxyl-terminal region of the MV N gene detected in these specimens MV of D6 genotype. The same nucleotide sequences and the same genotype were also previously observed for MV isolates from the 1997 epidemic in Brazil and the 1998 epidemic in Argentina, demonstrating that the D6 genotype was, and may be still circulating in South America.

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.