In Vitro Modeling of Feline Morbillivirus Infections Using Primary Feline Kidney Cells.

Domestic cats are the natural host of feline morbilliviruses (FeMV). Although other species can also be infected (such as dogs and opossums), no laboratory animal infection model is established so far. In vitro models for studying the molecular pathogenesis are therefore needed. For this purpose, propagation and titration of FeMV are key techniques. Unlike other morbilliviruses, such as canine distemper virus (CDV) or measles virus (MV), FeMV is a slow growing virus in cell culture and is difficult to titrate using classical plaque techniques. Here we describe methods for the efficient isolation of FeMV from natural sources (e.g., urine), the propagation of viral stocks, and their titration. In addition, we establish the generation of a three-dimensional infection model mimicking the feline tubular epithelium.

Systematic Determination of Herpesvirus in Free-Ranging Cetaceans Stranded in the Western Mediterranean: Tissue Tropism and Associated Lesions.

The monitoring of herpesvirus infection provides useful information when assessing marine mammals' health. This paper shows the prevalence of herpesvirus infection (80.85%) in 47 cetaceans stranded on the coast of the Valencian Community, Spain. Of the 966 tissues evaluated, 121 tested positive when employing nested-PCR (12.53%). The largest proportion of herpesvirus-positive tissue samples was in the reproductive system, nervous system, and tegument. Herpesvirus was more prevalent in females, juveniles, and calves. More than half the DNA PCR positive tissues contained herpesvirus RNA, indicating the presence of actively replicating virus. This RNA was most frequently found in neonates. Fourteen unique sequences were identified. Most amplified sequences belonged to the Gammaherpesvirinae subfamily, but a greater variation was found in Alphaherpesvirinae sequences. This is the first report of systematic herpesvirus DNA and RNA determination in free-ranging cetaceans. Nine (19.14%) were infected with cetacean morbillivirus and all of them (100%) were coinfected with herpesvirus. Lesions similar to those caused by herpesvirus in other species were observed, mainly in the skin, upper digestive tract, genitalia, and central nervous system. Other lesions were also attributable to concomitant etiologies or were nonspecific. It is necessary to investigate the possible role of herpesvirus infection in those cases.

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.

Zinc finger antiviral protein (ZAP) inhibits small ruminant morbillivirus replication in vitro.

Small ruminant morbillivirus (SRMV) is a highly contagious and economically important viral disease of small domestic and wild ruminants. Difficulty with its stable proliferation in ovis aries-derived cells has led to a relative lag in the study of its natural immunity and pathogenesis. Here we report the antiviral properties of ZAP against SRMV, a single-stranded negative-stranded RNA virus of the genus Morbillivirus. ZAP expression was significantly induced in sheep endometrial epithelial cells following SRMV infection. ZAP inhibited SRMV replication in cells after infection, while its overexpression in Vero-SLAM cells significantly increased their resistance to SRMV replication. The ZAP protein co-localized with SRMV RNA in the cytoplasm and ZAP-responsive elements were mapped to the 5' untranslated region of SRMV nucleocapsid, phosphoprotein, matrix, and fusion. In summary, ZAP confers resistance to SRMV infection by directly targeting viral RNA and inhibiting viral replication. Our findings further extend the ranges of viral targets of ZAP and help elucidate the mechanism of SRMV replication.

High seroprevalence of feline morbilliviruses in free-roaming domestic cats in Chile.

Feline morbillivirus infections have gained increased attention due to repeated reports of their association with urinary tract disease in cats. In the present study, 112 serum samples from free-roaming domestic cats in Chile were tested for antibodies against feline morbillivirus genotypes 1 and 2 (FeMV-1 and FeMV-2) using an indirect immunofluorescence assay. In total, 63% of the animals showed antibodies against one or both FeMV genotypes. Antibodies directed exclusively against FeMV-2 were significantly more prevalent in male cats. The correlation of sex and FeMV-2 infection might give insight into potential routes of transmission. We provide, for the first time, serological data on FeMV in Chile.

POSTMORTEM COMPUTED TOMOGRAPHY AND MAGNETIC RESONANCE IMAGING FINDINGS IN A CASE OF COINFECTION OF DOLPHIN MORBILLIVIRUS AND ASPERGILLUS FUMIGATUS IN A JUVENILE BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS).

A freshly dead juvenile bottlenose dolphin (Tursiops truncatus), recovered from the waters near Sand Key, Clearwater, FL, was imaged postmortem using computed tomography and magnetic resonance imaging prior to conventional necropsy. The pattern of imaging findings in the brain was compatible with severe multifocal meningoencephalitis with intralesional necrosis and/or hemorrhage, and the pattern of imaging findings in the lungs was compatible with severe multifocal bronchopneumonia. The subsequent investigation included necropsy, histology, culture, and molecular diagnostics and demonstrated disseminated coinfection of dolphin morbillivirus and Aspergillus fumigatus. This is the first report documenting the cross-sectional imaging findings of this important cetacean comorbidity and demonstrates advances in modern, cooperative investigations of marine mammal mortality events.

Feline Morbillivirus, a New Paramyxovirus Possibly Associated with Feline Kidney Disease.

Feline morbillivirus (FeMV) was first isolated in stray cats in Hong Kong in 2012. Since its discovery, the virus has been reported in domestic cats worldwide, including in Hong Kong, Japan, Italy, US, Brazil, Turkey, UK, Germany, and Malaysia. FeMV is classified in the Morbillivirus genus within the Paramyxoviridae family. FeMV research has focused primarily on determining the host range, symptoms, and characteristics of persistent infections in vitro. Importantly, there is a potential association between FeMV infection and feline kidney diseases, such as tubulointerstitial nephritis (TIN) and chronic kidney diseases (CKD), which are known to significantly affect feline health and survival. However, the tropism and viral entry mechanism(s) of FeMV remain unknown. In this review, we summarize the FeMV studies up to date, including the discoveries of various FeMV strains, basic virology, pathogenicity, and disease signs.

First report of feline morbillivirus in mainland China.

Feline morbillivirus (FeMV) is an emerging member of the family Paramyxoviridae that is suspected to be involved in chronic kidney disease (CKD). FeMV was first discovered in Hong Kong in 2012 and has subsequently been detected in many countries. However, the prevalence of FeMV in mainland China is still unclear. To clarify the present status and examine the genetic diversity of FeMV in mainland China, in this study, we collected cat urine samples in veterinary hospitals in Guangdong Province in 2017 and 2018. Using reverse transcription (RT)-PCR, we found that the urine of six out of 64 cats tested positive for FeMV RNA. Sequencing and genetic analysis of the FeMV L gene showed that FeMV in mainland China is genetically diverse, and phylogenetic analysis showed that the viruses segregated into two clusters. Two isolates, GD5 and GD6, grouped in a branch that was separate from the one containing other previously reported FeMV isolates. These results will contribute to a better understanding of the evolution of FeMV in China.

Animal morbilliviruses and their cross-species transmission potential.

Like measles virus (MV), whose primary hosts are humans, non-human animal morbilliviruses use SLAM (signaling lymphocytic activation molecule) and PVRL4 (nectin-4) expressed on immune and epithelial cells, respectively, as receptors. PVRL4's amino acid sequence is highly conserved across species, while that of SLAM varies significantly. However, non-host animal SLAMs often function as receptors for different morbilliviruses. Uniquely, human SLAM is somewhat specific for MV, but canine distemper virus, which shows the widest host range among morbilliviruses, readily gains the ability to use human SLAM. The host range for morbilliviruses is also modulated by their ability to counteract the host's innate immunity, but the risk of cross-species transmission of non-human animal morbilliviruses to humans could occur if MV is successfully eradicated.

Molecular detection and characterisation of feline morbillivirus in domestic cats in Malaysia.

Feline morbillivirus (FeMV), a novel virus from the family of Paramyxoviridae, was first identified in stray cat populations. The objectives of the current study were to (i) determine the molecular prevalence of FeMV in Malaysia; (ii) identify risk factors associated with FeMV infection; and (iii) characterise any FeMV isolates by phylogenetic analyses. Molecular analysis utilising nested RT-PCR assay targeting the L gene of FeMV performed on either urine, blood and/or kidney samples collected from 208 cats in this study revealed 82 (39.4%) positive cats. FeMV-positive samples were obtained from 63/124 (50.8%) urine and 20/25 (80.0%) kidneys while all blood samples were negative for FeMV. In addition, from the 35 cats that had more than one type of samples collected (blood and urine; blood and kidney; blood, urine and kidney), only one cat had FeMV RNA in the urine and kidney samples. Risk factors such as gender, presence of kidney-associated symptoms and cat source were also investigated. Male cats had a higher risk (p = 0.031) of FeMV infection than females. In addition, no significant association (p = 0.083) was observed between the presence of kidney-associated symptoms with FeMV status. From the 82 positive samples, FeMV RNA was detected from 48/82 (58.5%) pet cats and 34/126 (27.0%) shelter cats (p < 0.0001). Partial L and N gene sequencing of the RT-PCR-positive samples showed 85-99% identity to the published FeMV sequences and it was significantly different from all other morbilliviruses. A phylogenetic analysis of the identified Malaysian FeMVs was performed with isolates from Japan, Thailand and China. Molecular characterisation revealed high relatedness of the Malaysian isolates with other Asian FeMVs, indicating that the virus had been circulating only within the region. Therefore, this study confirmed the existence of FeMV among domestic cats in Malaysia. The findings suggest further characterisation of the local isolates, including the whole genome sequencing and that studies at determining the direct consequences of FeMV infection in domestic cats are needed.

Isolation and genome sequences of two Feline Morbillivirus genotype 1 strains from Italy.

Feline morbillivirus (FeMV) is a novel viral paramyxovirus detected in cats. FeMV is suspected to be associated to tubulointerstitial nephritis, but its pathogenic role is far to be clearly understood.  In this short communication, we report the whole genome coding sequences of the first two FeMV strains isolated in Italy.

Marine Morbilliviruses: Diversity and Interaction with Signaling Lymphocyte Activation Molecules.

Epidemiological reports of phocine distemper virus (PDV) and cetacean morbillivirus (CeMV) have accumulated since their discovery nearly 30 years ago. In this review, we focus on the interaction between these marine morbilliviruses and their major cellular receptor, the signaling lymphocyte activation molecule (SLAM). The three-dimensional crystal structure and homology models of SLAMs have demonstrated that 35 residues are important for binding to the morbillivirus hemagglutinin (H) protein and contribute to viral tropism. These 35 residues are essentially conserved among pinnipeds and highly conserved among the Caniformia, suggesting that PDV can infect these animals, but are less conserved among cetaceans. Because CeMV can infect various cetacean species, including toothed and baleen whales, the CeMV-H protein is postulated to have broader specificity to accommodate more divergent SLAM interfaces and may enable the virus to infect seals. In silico analysis of viral H protein and SLAM indicates that each residue of the H protein interacts with multiple residues of SLAM and vice versa. The integration of epidemiological, virological, structural, and computational studies should provide deeper insight into host specificity and switching of marine morbilliviruses.

Comparative sequence analysis of morbillivirus receptors and its implication in host range expansion.

SLAM (CD150) and nectin-4 are the major morbillivirus receptors responsible for virus pathogenesis and host range expansion. Recently, morbillivirus infections have been reported in unnatural hosts, including endangered species, posing a threat to their conservation. To understand the host range expansion of morbilliviruses, we generated the full-length sequences of morbillivirus receptors (goat, sheep, and dog SLAM, and goat nectin-4) and tried to correlate their role in determining host tropism. A high level of amino acid identity was observed between the sequences of related species, and phylogenetic reconstruction showed that the receptor sequences of carnivores, marine mammals, and small ruminants grouped separately. Analysis of the ligand binding region (V region; amino acid residues 52-136) of SLAM revealed high amino acid identity between small ruminants and bovine SLAMs. Comparison of canine SLAM with ruminants and non-canids SLAM revealed appreciable changes, including charge alterations. Significant differences between feline SLAM and canine SLAM have been reported. The binding motifs of nectin-4 genes (FPAG motif and amino acid residues 60, 62, and 63) were found to be conserved in sheep, goat, and dog. The differences reported in the binding region may be responsible for the level of susceptibility or resistance of a species to a particular morbillivirus.

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.

Association of feline morbillivirus infection with defined pathological changes in cat kidney tissues.

Feline morbillivirus (FeMV) is an emerging member of morbillivirus discovered in 2012. Although association of FeMV infection with kidney diseases in cats has been suggested, the pathogenicity of the virus has not been clear to date. To study the association between FeMV infection and pathological changes in kidney tissues of infected cats, we performed immunohistochemistry and immunofluorescent assays to detect FeMV antigens and analyzed the effect of FeMV infection on the pathological changes in the kidney tissues. In 38 kidney tissue samples from cats, some tissue injury scores were significantly higher when the FeMV antigens were detected, especially those for the tubular tissues in which the FeMV antigens were mostly localized. Pathological changes associated with the FeMV antigens included the ones typically found in chronic kidney diseases, such as interstitial cell infiltration, glomerulosclerosis, tubular atrophy and fibrosis. We also detected some feline IgG localizations in glomerular tissues, though co-localization or significant association with the FeMV antigens were not found. Our study confirms the association of FeMV infection with some kidney tissue injuries and provides additional information about roles of FeMV infection in chronic kidney diseases.

Morbillivirus infection in Risso's dolphin Grampus griseus: a phylogenetic and pathological study of cases from the Canary Islands.

The earliest evidence of cetacean morbillivirus (CeMV) infection dates from 1982, when the dolphin morbillivirus strain (DMV) was identified in bottlenose dolphins Tursiops truncatus stranded in the mid-Atlantic region. Since then, CeMV has been detected globally in at least 26 species of mysticetes and odontocetes, causing widespread mortality and a wide range of pathological effects. In the Canary Islands, DMV and pilot whale morbillivirus have been detected in cetacean species, including short-finned pilot whales Globicephala macrorhynchus and bottlenose dolphins. Risso's dolphins Grampus griseus have been reported year-round in waters of the Canary Islands and are considered a resident species. No information is currently available on CeMV prevalence in this species in this ocean region. We searched for evidence of CeMV infection in 12 Risso's dolphins stranded in the Canary Islands from 2003 to 2015 by means of histopathology, PCR and immunohistochemistry. PCR revealed 2 CeMV-positive animals (16.6%). Phylogenetic analysis showed that the strains from the 2 positive specimens were phylogenetically quite distant, proving that more than 1 strain infects the Risso's dolphin population in this region. We also determined that the strain detected in one of the specimens mainly circulated in the northeastern Atlantic Ocean from 2007 to 2013.

A real-time RT-PCR assay for molecular identification and quantitation of feline morbillivirus RNA from biological specimens.

The aim of this study was to develop a real-time RT-PCR to detect and quantitate feline morbillivirus (FeMV) RNA in biological samples. Primers and probe were targeted on a conserved region of FeMV P/V/C gene. To validate the assay with field samples, a total number of specimens of cats have been recruited including 264 urine and blood samples and compared with a generic RT-PCR targeting the L protein encoding gene of morbilliviruses. In addition, 385 tissue samples from 35 carcasses of cats have been also employed. RNA titres were low in all tested samples. Results also indicated the absence of cross-reaction with related morbilliviruses and existing pathogens of cats. In tissues with low levels of FeMV RNA, the presence of viral antigen was also evidenced by immunohistochemistry targeting the N viral protein. This newly described assay allows for a rapid, accurate and reliable quantitative detection of FeMV RNA that can be applied for diagnostics and research studies.

Morbillivirus Pathogenesis and Virus-Host Interactions.

Despite the availability of safe and effective vaccines against measles and several animal morbilliviruses, they continue to cause regular outbreaks and epidemics in susceptible populations. Morbilliviruses are highly contagious and share a similar pathogenesis in their respective hosts. This review provides an overview of morbillivirus history and the general replication cycle and recapitulates Morbillivirus pathogenesis focusing on common and unique aspects seen in different hosts. It also summarizes the state of knowledge regarding virus-host interactions on the cellular level with an emphasis on viral interference with innate immune response activation, and highlights remaining knowledge gaps.

Measles vaccination: Threat from related veterinary viruses and need for continued vaccination post measles eradication.

Measles virus (MV) is the only human virus within the morbillivirus genus of the Paramyxoviridae. The veterinary members are canine distemper virus (CDV), peste des petits ruminants virus (PPRV), Rinderpest Virus (RPV) as well as the marine morbilliviruses phocine distemper virus (PDV), dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV). Morbilliviruses have a severe impact on humans and animal species. They confer diseases which have contributed to morbidity and mortality of the population on a global scale. There is substantial evidence from both natural and experimental infections that morbilliviruses can readily cross species barriers. Of most concern with regard to zoonosis is the more recently reported fatal infection of primates in Japan and China with strains of CDV which have adapted to this host. The close genetic relationship, shared cell entry receptors and similar pathogenesis between the morbilliviruses highlights the potential consequences of complete withdrawal of MV vaccination after eradication. Therefore, it would be prudent to continue the current MV vaccination. Ultimately development of novel, safe vaccines which have higher efficacy against the veterinary morbilliviruses is a priority. These would to protect the human population long term against the threat of zoonosis by these veterinary viruses.

Neuronal and astrocytic involvement in striped dolphins (Stenella coeruleoalba) with morbilliviral encephalitis.

Dolphin morbillivirus (DMV), a highly pathogenic agent, may cause peculiar, "brain-only" forms of infection (BOFDI), in which viral antigen and/or genome is found exclusively in the brain from striped dolphins (Stenella coeruleoalba). These BOFDIs show morphopathological similarities with subacute sclerosing panencephalitis and old dog encephalitis (ODE) in measles virus-infected patients and in canine distemper virus-infected dogs, respectively. The brain tissue from 3 BOFDI-affected striped dolphins was investigated by means of double labelling-indirect immunofluorescence (DL-IIF) and ultrastructurally, in order to characterize the DMV-targeted neuronal and non-neuronal cell populations, along with the associated submicroscopic findings. Viral colonization of calbindin-immunoreactive (IR) and nitric oxide synthase-IR neurons was detected in the cerebral parenchyma from the 3 DMV-infected dolphins under study, associated with nuclear (chromatin) and cytoplasmic (mitochondrial) ultrastructural changes. Furthermore, a limited viral targeting of brain astrocytes was found in these animals, all of which exhibited a prominent astrogliosis/astrocytosis. To the best of our knowledge, those herein reported should be the first submicroscopic pathology and neuropathogenetic data about BOFDI in striped dolphins. In this respect, the marked astrogliosis/astrocytosis and the low viral colonization of brain astrocytes in the 3 DMV-infected dolphins under investigation are of interest from the comparative pathology and viral neuropathogenesis standpoints, when compared with ODE-affected dogs, in whose brain a non-cytolytic, astrocyte-to-astrocyte infectious spread has been recently documented. Further studies aimed at characterizing the complex DMV-host interactions in BOFDI-affected striped dolphins are needed.