Herpesvirus surveillance in stranded striped dolphins (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus) from Italy with emphasis on neuropathological characterization.

Herpesvirus (HV) is widely distributed among cetacean populations, with the highest prevalence reported in the Mediterranean Sea. In this study, a comprehensive analysis was conducted, including epidemiological, phylogenetic, and pathological aspects, with particular emphasis on neuropathology, to better understand the impact of HV in these animals. Our results show a higher presence of HV in males compared to females, with males exhibiting a greater number of positive tissues. Additionally, adults were more frequently affected by HV infection than juveniles, with no infections detected in calves or neonates. The affected species were striped (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus). The highest positivity rates were observed in the genital system, cerebrum, and skin tissues. Phylogenetic analysis indicated a higher occurrence of Gammaherpesvirus (GHV) sequences but increased genetic diversity within Alphaherpesvirus (AHV). Key neuropathological features included astro-microgliosis (n = 4) and meningitis with minimal to mild perivascular cuffing (n = 2). The presence of concurrent infections with other pathogens, particularly cetacean morbillivirus (CeMV), underscores the complex nature of infectious diseases in cetaceans. However, the presence of lesions at the Central Nervous System (CNS) with molecular positivity for GHV, excluding the involvement of other potential neurotropic agents, would confirm the potential of this HV subfamily to induce neurological damage. Pathological examination identified lesions in other organs that could potentially be associated with HV, characterized by lymphoid depletion and tissue inflammation. These findings enhance our understanding of HV in odontocetes and highlight the need for ongoing research into the factors driving these infections and their broader implications.

Detection of Antibodies against Feline Morbillivirus by Recombinant Matrix Enzyme-Linked Immunosorbent Assay.

Feline morbillivirus (FeMV) has been associated with feline health, although its exact role in pathogenesis is still debated. In this study, an indirect enzyme-linked immunosorbent assay (i-ELISA) targeting a recombinant matrix protein of FeMV (rFeMV-M) was developed and assessed in comparison to a Western blotting (WB) assay. The i-ELISA was evaluated using blood samples from 136 cats that were additionally tested with real-time reverse-transcription PCR (RT-qPCR). The i-ELISA exhibited a sensitivity of 90.1%, specificity of 75.6%, positive predictive value of 88.2%, and negative predictive value of 79.1%. The agreement between i-ELISA and WB analyses was substantial (a κ coefficient of 0.664 with a 95% confidence interval of 0.529 to 0.799). Within the study group, 68.4% (93/136) of the cats were serologically positive in the i-ELISA and 66.9% (91/136) in the WB assay, with 11.8% (11/93) of false positivity with the i-ELISA. However, only 8.1% (11/136) of the cats tested positive for FeMV using RT-qPCR (p < 0.001). The developed i-ELISA proved effective in identifying FeMV-infected cats and indicated the prevalence of FeMV exposure. Combining FeMV antibody detection through i-ELISA with FeMV RT-qPCR could offer a comprehensive method to determine and monitor FeMV infection status. Nevertheless, this assay still requires refinement due to a significant number of false positive results, which can lead to the misdiagnosis of cats without antibodies as having antibodies. This study also provided the first evidence of seroprevalence against FeMV among cat populations in Thailand, contributing valuable insights into the geographic distribution and prevalence of this virus.

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.

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.

Detection of feline morbillivirus in cats with symptoms of acute febrile infection.

Feline morbillivirus (FeMV) was identified for the first time in cats in 2012 in Hong Kong. Although its association with chronic kidney disease in cats has attracted the attention of researchers, its clinical significance as an acute infection has not been reported. Previously, we reported FeMV detection using next-generation sequence-based comprehensive genomic analysis of plasma samples from cats with suspected acute febrile infections. Here, we conducted an epidemiological survey to detect FeMV by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using blood samples from cats in Japan. FeMV was detected in 32/102 blood samples (31.4%) from cats with suspected acute viral infections. Most of the FeMV-positive cats had clinical findings consistent with acute viral infections, including fever, leukopenia, thrombocytopenia and jaundice. No FeMV was detected in healthy cats or clinically ill cats that visited veterinary hospitals. Phylogenetic analysis classified FeMV L genes into various FeMV subtypes. We also necropsied a FeMV-positive cat that died of a suspected acute infection. On necropsy, FeMV was detected in systemic organs, including the kidneys, lymph nodes and spleen by qRT-PCR and immunohistochemical staining. These results suggest that FeMV infections may cause acute symptomatic febrile infections in cats. A limitation of this study was that the involvement of other pathogens that cause febrile illnesses could not be ruled out and this prevented a definitive conclusion that FeMV causes febrile disease in infected cats. Further studies that include experimental infections are warranted to determine the pathogenicity of FeMV in cats.

Comparison between Sampling Techniques for Virological Molecular Analyses: Dolphin Morbillivirus and Herpesvirus Detection from FTA® Card and Frozen Tissue.

Stranded animals offer valuable information on marine mammal physiology and pathology; however, the decomposition state of the carcasses and lack of a rigorous cold chain for sample preservation can sometimes discourage diagnostic analyses based on nucleic acid detection. The present paper aims at evaluating the reliability of FTA® card tissue imprints as an alternative matrix to frozen tissues for virological analyses based on biomolecular methods. Given the contribution of Cetacean morbillivirus (CeMV) to strandings and the increase of herpesvirus detection in cetaceans, these two pathogens were selected as representative of RNA and DNA viruses. Dolphin morbillivirus (DMV) and herpesvirus presence was investigated in parallel on tissue imprints on FTA® cards and frozen tissues collected during necropsy of dolphins stranded in Italy. Samples were analysed by nested RT-PCR for DMV and nested-PCR for herpesvirus. Only one animal was positive for herpesvirus, hampering further considerations on this virus. DMV was detected in all animals, both in FTA® card imprints and tissue samples, with differences possibly related to the decomposition condition category of the carcasses. Tissue sampling on FTA® cards seems a promising alternative to frozen tissues for biomolecular analyses, especially when ensuring adequate storage and shipment conditions for frozen tissues is difficult.

Feline Morbillivirus: Clinical Relevance of a Widespread Endemic Viral Infection of Cats.

Feline morbillivirus (FeMV) was first isolated in 2012 from stray cats in Hong Kong. It has been found in association with tubulointerstitial nephritis (TIN), the most common cause of feline chronic kidney disease (CKD). However, viral host spectrum and virus tropism go beyond the domestic cat and kidney tissues. The viral genetic diversity of FeMV is extensive, but it is not known if this is clinically relevant. Urine and kidney tissues have been widely tested in attempts to confirm associations between FeMV infection and renal disease, but samples from both healthy and sick cats can test positive and some cross-sectional studies have not found associations between FeMV infection and CKD. There is also evidence for acute kidney injury following infection with FeMV. The results of prevalence studies differ greatly depending on the population tested and methodologies used for detection, but worldwide distribution of FeMV has been shown. Experimental studies have confirmed previous field observations that higher viral loads are present in the urine compared to other tissues, and renal TIN lesions associated with FeMV antigen have been demonstrated, alongside virus lymphotropism and viraemia-associated lymphopenia. Longitudinal field studies have revealed persistent viral shedding in urine, although infection can be cleared spontaneously.

Feline morbillivirus infection associated with fatal encephalitis in a Bengal cat.

Feline morbillivirus (FeMV) is a recently discovered morbillivirus of the family Paramyxoviridae, which include several highly contagious viruses with zoonotic potential. In this case report we describe the detection of FeMV in archived brain tissue of a 2-month-old Bengal cat with nonsuppurative encephalitis from the year 2011 in Switzerland by high-throughput sequencing (HTS). Our metagenomics approach was able to obtain a full-length sequence covering the entire FeMV genome. Phylogenetic analysis showed that our FeMV strain clustered within FeMV genotype 1. We were able to detect FeMV RNA by in situ hybridization (ISH) in brain sections with inflammatory lesions and demonstrated its potential neurotropism and association with encephalitis. Our results provide further insight into this recently discovered morbillivirus and encourage further investigations into the pathogenesis and epidemiology of associated diseases in cats and potentially other species.

First description of a lesion in the upper digestive mucosa associated with a novel gammaherpesvirus in a striped dolphin (Stenella coeruleoalba) stranded in the Western Mediterranean Sea.

BACKGROUND: A wide variety of lesions have been associated with herpesvirus in cetaceans. However, descriptions of herpesvirus infections in the digestive system of cetaceans are scarce. CASE REPORT: A young female striped dolphin stranded in the Valencian Community (Spain) on the 6th August 2021. The animal showed external macroscopic lesions suggestive of an aggressive interaction with bottlenose dolphins (rake marks in the epidermis). Internally, the main findings included congestion of the central nervous system and multiple, well-defined, whitish, irregularly shaped, proliferative lesions on the oropharyngeal and laryngopharyngeal mucosa. Histopathology revealed lymphoplasmacytic and histiocytic meningoencephalitis, consistent with neuro brucellosis. The oropharyngeal and laryngopharyngeal plaques were comprised histologically of focally extensive epithelial hyperplasia. As part of the health surveillance program tissue samples were tested for cetacean morbillivirus using a real-time reverse transcription-PCR, for Brucella spp. using a real-time PCR, and for herpesvirus using a conventional nested PCR. All samples were negative for cetacean morbillivirus; molecular positivity for Brucella spp. was obtained in pharyngeal tonsils and cerebrospinal fluid; herpesvirus was detected in a proliferative lesion in the upper digestive mucosa. Phylogenetic analysis showed that the herpesvirus sequence was included in the Gammaherpesvirinae subfamily. This novel sequence showed the greatest identity with other Herpesvirus sequences detected in skin, pharyngeal and genital lesions in five different species. CONCLUSIONS: To the best of the authors' knowledge, this is the first report of a proliferative lesion in the upper digestive mucosa associated with gammaherpesvirus posititvity in a striped dolphin (Stenella coeruleoalba).

Short-Finned Pilot Whale Strandings Associated with Pilot Whale Morbillivirus, Brazil.

Cetacean morbillivirus (CeMV) causes illness and death in cetaceans worldwide; the CeMV strains circulating in the Southern Hemisphere are poorly known. We detected a pilot whale CeMV strain in 3 short-finned pilot whales (Globicephala macrorhynchus) stranded in Brazil during July-October 2020. Our results confirm this virus circulates in this species.

A South American Mouse Morbillivirus Provides Insight into a Clade of Rodent-Borne Morbilliviruses.

Morbilliviruses are negative-sense single-stranded monosegmented RNA viruses in the family Paramyxoviridae (order Mononegavirales). Morbilliviruses infect diverse mammals including humans, dogs, cats, small ruminants, seals, and cetaceans, which serve as natural hosts. Here, I report the identification and characterization of novel viruses detected in public RNAseq datasets of South American long-haired and olive field mice. The divergent viruses dubbed Ratón oliváceo morbillivirus (RoMV) detected in renal samples from mice collected from Chile and Argentina are characterized by an unusually large genome including long intergenic regions and the presence of an accessory protein between the F and H genes redounding in a genome architecture consisting in 3'-N-P/V/C-M-F-hp-H-L-5'. Structural and functional annotation, genetic distance, and evolutionary insights suggest that RoMV is a member of a novel species within genus Morbillivirus tentatively named as South American mouse morbillivirus. Phylogenetic analysis suggests that this mouse morbillivirus is closely related to and clusters into a monophyletic group of novel rodent-borne morbilliviruses. This subclade of divergent viruses expands the host range, redefines the genomic organization and provides insights on the evolutionary history of genus Morbillivirus.

FeMV is a cathepsin-dependent unique morbillivirus infecting the kidneys of domestic cats.

Feline morbillivirus (FeMV) is a recently discovered pathogen of domestic cats and has been classified as a morbillivirus in the Paramyxovirus family. We determined the complete sequence of FeMVUS5 directly from an FeMV-positive urine sample without virus isolation or cell passage. Sequence analysis of the viral genome revealed potential divergence from characteristics of archetypal morbilliviruses. First, the virus lacks the canonical polybasic furin cleavage signal in the fusion (F) glycoprotein. Second, conserved amino acids in the hemagglutinin (H) glycoprotein used by all other morbilliviruses for binding and/or fusion activation with the cellular receptor CD150 (signaling lymphocyte activation molecule [SLAM]/F1) are absent. We show that, despite this sequence divergence, FeMV H glycoprotein uses feline CD150 as a receptor and cannot use human CD150. We demonstrate that the protease responsible for cleaving the FeMV F glycoprotein is a cathepsin, making FeMV a unique morbillivirus and more similar to the closely related zoonotic Nipah and Hendra viruses. We developed a reverse genetics system for FeMVUS5 and generated recombinant viruses expressing Venus fluorescent protein from an additional transcription unit located either between the phospho-protein (P) and matrix (M) genes or the H and large (L) genes of the genome. We used these recombinant FeMVs to establish a natural infection and demonstrate that FeMV causes an acute morbillivirus-like disease in the cat. Virus was shed in the urine and detectable in the kidneys at later time points. This opens the door for long-term studies to address the postulated role of this morbillivirus in the development of chronic kidney disease.

In Vitro Growth, Receptor Usage and Pathogenesis of Feline Morbillivirus in the Natural Host.

Feline morbillivirus (FeMV) is a recently discovered virus belonging to the genus Morbillivirus of the virus family Paramyxoviridae. Often, the virus has been detected in urine of cats with a history of urinary disease and has a worldwide distribution. Currently, it is unclear which receptor the virus uses to enter the target cells. Furthermore, many aspects of FeMV biology in vivo, including tissue tropism, pathogenesis, and virus excretion in the natural host remain unclear. In this study we analyzed the replication of FeMV in various cell lines. Secondly, we tested if the presence of feline SLAMF1 (Signaling Lymphocytic Activation Molecule family 1/CD150, principal entry receptor for other members of the Morbillivirus genus) improved FeMV replication efficiency in vitro. Finally, to elucidate in vivo biology in cats, as a natural host for FeMV, we experimentally infected a group of cats and monitored clinical symptoms, viremia, and excretion of the virus during the course of 56 days. Our study showed that FeMV shares some features with other morbilliviruses like the use of the SLAMF1 receptor. For the first time, experimental infection of SPF cats showed that FeMV does not induce an acute clinical disease like other morbilliviruses but can induce lesions in the kidneys, including tubulointerstitial nephritis. Further investigations are needed to confirm the site and dynamics of replication of FeMV in the urinary tract and the longer-term impact of FeMV-induced lesions on the renal function. Whether FeMV infection can result in chronic kidney disease will require the monitoring of cats over a longer period.

Molecular signatures in cetacean morbillivirus and host species proteomes: Unveiling the evolutionary dynamics of an enigmatic pathogen?

Cetacean morbillivirus (CeMV) infects marine mammals often causing a fatal respiratory and neurological disease. Recently, CeMV has expanded its geographic and host species range, with cases being reported worldwide among dolphins, whales, seals, and other aquatic mammalian species, and therefore has emerged as the most threatening nonanthropogenic factor affecting marine mammal's health and conservation. Extensive research efforts have aimed to understand CeMV epidemiology and ecology, however, the molecular mechanisms underlying its transmission and pathogenesis are still poorly understood. In particular, the field suffers from a knowledge gap on the structural and functional properties of CeMV proteins and their host interactors. Nevertheless, the body of scientific literature produced in recent years has inaugurated new investigational trends, driving future directions in CeMV molecular research. In this mini-review, the most recent literature has been summarized in the context of such research trends, and categorized into four priority research topics, such as (1) the interaction between CeMV glycoprotein and its host cell receptors across several species; (2) the CeMV molecular determinants responsible for different disease phenotype; (3) the host molecular determinants responsible for differential susceptibility to CeMV infection; (4) the CeMV molecular determinants responsible for difference virulence among circulating CeMV strains. Arguably, these are the most urgent topics that need to be investigated and that most promisingly will help to shed light on the details of CeMV evolutionary dynamics in the immediate future.

Renal epitheliotropism of feline morbillivirus in two cats.

The association of feline morbillivirus (FeMV) with kidney disease in cats is controversial. Two cats with a history of severe hematuria had eosinophilic inclusion-like bodies in the renal tubular epithelial cells, without any inflammatory cellular reaction. Ultrastructurally, aggregations of electron-dense viral-like particles were found where the inclusion-like bodies were located. Immunohistochemistry (IHC) using antibodies against FeMV matrix protein labeled these inclusion-like bodies, and also labeled the cytoplasm of tracheal and bronchiolar epithelial cells, and lymphocytes and macrophages in spleen and mesenteric lymph node. Using double IHC, FeMV antigen was detected in astroglia and oligodendroglia but not in microglia. Phylogenetic characterization of the fusion and hemagglutinin gene sequences revealed FeMV-1A genotypes in both cats. These findings indicated an active viral infection with FeMV. We propose that FeMV is a renal epitheliotropic virus and also localizes in various other tissues.

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.

Feline morbillivirus-1 in dogs with respiratory diseases.

Feline morbillivirus-1 (FeMV-1) is a viral pathogen associated with kidney disease in domestic cats and wild felids. We initially identified the FeMV-1 from the lung of a necropsied dog with severe pulmonary disease by the reverse transcription polymerase chain reaction (RT-PCR). Thereafter, we investigated FeMV-1 in nasal and oral swab samples from 73 healthy and 113 dogs with respiratory illnesses. We found polymerase chain reaction (PCR)-positive FeMV-1 from only 14/113 (12.39%) dogs with respiratory disease (p = .001). Of these 14 dogs, six were co-infected with other canine respiratory viruses (6/14; 42.86%). Two independent immunohistochemistry procedures, using antibodies against matrix and phosphoprotein of FeMV-1, confirmed the presence of FeMV-1 in lung tissues of two necropsied dogs (out of a total of 22 dogs, 9.09%) that died from respiratory disease. This finding corresponded to transmission electron microscopy findings that paramyxoviral particles exist in lung epithelia. FeMV-1 antigen localization was also evident in the kidney, lymphoid and brain tissues of two deceased dogs. FeMV-1 was successfully isolated from a necropsied dog and from two living dogs, all with respiratory illnesses, which supports FeMV infection in dogs. The detection of FeMV-1 in dog tissues expands the known tropism of this virus to a non-felid host. Our findings indicate that FeMV-1, alone or in co-infection with other viral pathogens, might contribute to respiratory illness and death in dogs.

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.

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.

Feline Morbillivirus in Southern Italy: Epidemiology, Clinico-Pathological Features and Phylogenetic Analysis in Cats.

Feline morbillivirus (FeMV) was isolated for the first time in 2012 with an association with chronic kidney disease (CKD) suggested. This study aimed at investigating in cats from southern Italy FeMV prevalence and risk factors for exposure to FeMV, including the relationship with CKD; sequencing amplicons and analyzing phylogeny of PCR positive samples. Blood serum, K3EDTA blood and urine samples from 223 cats were investigated. Ten carcasses were also evaluated. FeMV RNA was detected in 2.4% (5/211) blood and 16.1% (36/223) urine samples. One carcass tested positive by qPCRFeMV from kidney, urinary bladder, and submandibular lymph nodes. Antibodies against FeMV were detected in 14.5% (28/193) cats. We followed up 27 cats (13 FeMV positive cats) and documented in some cases urine shedding after up to 360 days. Older and foundling cats and cats living in rescue catteries, were more frequently infected with FeMV. A significant correlation between FeMV and higher serum creatinine values or low urine specific gravity was found. FeMV positivity was significantly associated with retroviral infection, and the presence of some clinical signs apart from CKD clinicopathological markers. Our study highlights the possibility of a link between FeMV exposure and CKD and a general impairment of feline health.