Molecular Analysis of Copy-Back Defective Interfering RNAs of Morbilliviruses.

Copy-back defective interfering RNAs are major contaminants of viral stock preparations of morbilliviruses and other negative strand RNA viruses. They are hybrid molecules of positive sense antigenome and negative sense genome. They possess perfectly complementary ends allowing the formation of extremely stable double-stranded RNA panhandle structures. The presence of the 3'-terminal promoter allows replication of these molecules by the viral polymerase. They thereby negatively interfere with replication of standard genomes. In addition, the double-stranded RNA stem structures are highly immunostimulatory and activate antiviral cell-intrinsic innate immune responses. Thus, copy-back defective interfering RNAs severely affect the virulence and pathogenesis of morbillivirus stocks. We describe two biochemical methods to analyze copy-back defective interfering RNAs in virus-infected samples, or purified viral RNA. First, we present our Northern blotting protocol that allows accurate size determination of defective interfering RNA molecules and estimation of the relative contamination level of virus preparations. Second, we describe a PCR approach to amplify defective interfering RNAs specifically, which allows detailed sequence analysis.

Generation of Defective Interfering Particles of Morbilliviruses Using Reverse Genetics.

RNA viruses generate defective genomes naturally during virus replication. Defective genomes that interfere with the infection dynamics either through resource competition or by interferon stimulation are known as defective interfering (DI) genomes. DI genomes can be successfully packaged into virus-like-particles referred to as defective interfering particles (DIPs). Such DIPs can sustainably coexist with the full-length virus particles and have been shown to negatively impact virus replication in vitro and in vivo. Here, we describe a method to generate a clonal DI genome population by reverse genetics. This method is applicable to other RNA viruses and will enable assessment of DIPs for their antiviral properties.

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.

Rational attenuation of canine distemper virus (CDV) to develop a morbillivirus animal model that mimics measles in humans.

Morbilliviruses are members of the family Paramyxoviridae and are known for their ability to cause systemic disease in a variety of mammalian hosts. The prototypic morbillivirus, measles virus (MeV), infects humans and still causes morbidity and mortality in unvaccinated children and young adults. Experimental infection studies in non-human primates have contributed to the understanding of measles pathogenesis. However, ethical restrictions call for the development of new animal models. Canine distemper virus (CDV) infects a wide range of animals, including ferrets, and its pathogenesis shares many features with measles. However, wild-type CDV infection is almost always lethal, while MeV infection is usually self-limiting. Here, we made five recombinant CDVs, predicted to be attenuated, and compared their pathogenesis to the non-attenuated recombinant CDV in a ferret model. Three viruses were insufficiently attenuated based on clinical signs, fatality, and systemic infection, while one virus was too attenuated. The last candidate virus caused a self-limiting infection associated with transient viremia and viral dissemination to all lymphoid tissues, was shed transiently from the upper respiratory tract, and did not result in acute neurological signs. Additionally, an in-depth phenotyping of the infected white blood cells showed lower infection percentages in all lymphocyte subsets when compared to the non-attenuated CDV. In conclusion, infection models using this candidate virus mimic measles and can be used to study pathogenesis-related questions and to test interventions for morbilliviruses in a natural host species.IMPORTANCEMorbilliviruses are transmitted via the respiratory route but cause systemic disease. The viruses use two cellular receptors to infect myeloid, lymphoid, and epithelial cells. Measles virus (MeV) remains an important cause of morbidity and mortality in humans, requiring animal models to study pathogenesis or intervention strategies. Experimental MeV infections in non-human primates are restricted by ethical and practical constraints, and animal morbillivirus infections in natural host species have been considered as alternatives. Inoculation of ferrets with wild-type canine distemper virus (CDV) has been used for this purpose, but in most cases, the virus overwhelms the immune system and causes highly lethal disease. Introduction of an additional transcription unit and an additional attenuating point mutation in the polymerase yielded a candidate virus that caused self-limiting disease with transient viremia and virus shedding. This rationally attenuated CDV strain can be used for experimental morbillivirus infections in ferrets that reflect measles in humans.

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.

Global genetic diversity of measles virus (Paramyxoviridae: Morbillivirus: Morbillivirus hominis): historical aspects and current state.

Monitoring the circulation of the measles virus and studying its genetic diversity is an important component of the measles elimination program. A methodological approach to molecular genetic studies and their interpretation in the measles surveillance was developed in the early 2000s. During its development, clear areas of circulation of each genotype of the virus were identified, therefore, the determination of viruses' genotypes was proposed to monitor circulation and identify transmission pathways. However, in the future, due to a significant decrease in the number of active genotypes, an approach based on sub-genotyping was proposed: determining not only the genotype of the virus, but also its genetic lineage/genetic variant. The Global Measles and Rubella Laboratory Network (GMRLN) systematically monitors the circulation of the measles virus at the sub-genotypic level, depositing the results in a specialized database MeaNS2. It is this database that is the most complete and reliable source of information about the genetic characteristic of measles viruses. This review presents both historical information and the latest data on the global genetic diversity of the measles virus.

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.

Gut bacterial communities in Atlantic bottlenose dolphins (Tursiops truncatus) throughout a disease-driven (Morbillivirus) unusual mortality event.

Gut microbiomes are important determinants of animal health. In sentinel marine mammals where animal and ocean health are connected, microbiome impacts can scale to ecosystem-level importance. Mass mortality events affect cetacean populations worldwide, yet little is known about the contributory role of their gut bacterial communities to disease susceptibility and progression. Here, we characterized bacterial communities from fecal samples of common bottlenose dolphins, Tursiops truncatus, across an unusual mortality event (UME) caused by dolphin Morbillivirus (DMV). 16S rRNA gene sequence analysis revealed similar diversity and structure of bacterial communities in individuals stranding before, during, and after the 2013-2015 Mid-Atlantic Bottlenose Dolphin UME and these trends held in a subset of dolphins tested by PCR for DMV infection. Fine-scale shifts related to the UME were not common (10 of 968 bacterial taxa) though potential biomarkers for health monitoring were identified within the complex bacterial communities. Accordingly, acute DMV infection was not associated with a distinct gut bacterial community signature in T. truncatus. However, temporal stratification of DMV-positive dolphins did reveal changes in bacterial community composition between early and late outbreak periods, suggesting that gut community disruptions may be amplified by the indirect effects of accumulating health burdens associated with chronic morbidity.

Metagenomics-enabled reverse-genetics assembly and characterization of myotis bat morbillivirus.

Morbilliviruses are among the most contagious viral pathogens of mammals. Although previous metagenomic surveys have identified morbillivirus sequences in bats, full-length morbilliviruses from bats are limited. Here we characterize the myotis bat morbillivirus (MBaMV) from a bat surveillance programme in Brazil, whose full genome was recently published. We demonstrate that the fusion and receptor binding protein of MBaMV utilize bat CD150 and not human CD150, as an entry receptor in a mammalian cell line. Using reverse genetics, we produced a clone of MBaMV that infected Vero cells expressing bat CD150. Electron microscopy of MBaMV-infected cells revealed budding of pleomorphic virions, a characteristic morbillivirus feature. MBaMV replication reached 103-105 plaque-forming units ml-1 in human epithelial cell lines and was dependent on nectin-4. Infection of human macrophages also occurred, albeit 2-10-fold less efficiently than measles virus. Importantly, MBaMV is restricted by cross-neutralizing human sera elicited by measles, mumps and rubella vaccination and is inhibited by orally bioavailable polymerase inhibitors in vitro. MBaMV-encoded P/V genes did not antagonize human interferon induction. Finally, we show that MBaMV does not cause disease in Jamaican fruit bats. We conclude that, while zoonotic spillover into humans may theoretically be plausible, MBaMV replication would probably be controlled by the human immune system.

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.

Classification of new morbillivirus and jeilongvirus sequences from bats sampled in Brazil and Malaysia.

As part of a broad One Health surveillance effort to detect novel viruses in wildlife and people, we report several paramyxovirus sequences sampled primarily from bats during 2013 and 2014 in Brazil and Malaysia, including seven from which we recovered full-length genomes. Of these, six represent the first full-length paramyxovirid genomes sequenced from the Americas, including two that are the first full-length bat morbillivirus genome sequences published to date. Our findings add to the vast number of viral sequences in public repositories, which have been increasing considerably in recent years due to the rising accessibility of metagenomics. Taxonomic classification of these sequences in the absence of phenotypic data has been a significant challenge, particularly in the subfamily Orthoparamyxovirinae, where the rate of discovery of novel sequences has been substantial. Using pairwise amino acid sequence classification (PAASC), we propose that five of these sequences belong to members of the genus Jeilongvirus and two belong to members of the genus Morbillivirus. We also highlight inconsistencies in the classification of Tupaia virus and Mòjiang virus using the same demarcation criteria and suggest reclassification of these viruses into new genera. Importantly, this study underscores the critical importance of sequence length in PAASC analysis as well as the importance of biological characteristics such as genome organization in the taxonomic classification of viral sequences.

Origin and spreading of canine morbillivirus in South America.

Canine distemper virus (CDV) is a Morbillivirus (Canine morbillivirus) that greatly impacts domestic and wildlife carnivores worldwide. The CDV RNA genome has high genetic variability, evidenced by several lineages that follow a global geographic pattern. The evolutionary trajectories and population dynamics of CDV lineages are still unclear and debatable, particularly in South America, where relatively few sequences are available. We performed phylogenetic and Bayesian analyses using an updated dataset of the highly variable hemagglutinin (H) gene, including seven South American countries. The time to the most recent common ancestor (tMRCA) of the current CDV lineages was dated to the early 1900s in North America. Maximum likelihood and Bayesian maximum clade credibility phylogenies showed similar topologies with two main branches (L1 and L2) corresponding to the NA1 lineage (L1) and the remaining lineages worldwide (L2). The four circulating lineages in South America (EU1/SA1, SA2, SA3, NA4/SA4) arose from independent migration events from North America and Europe. North American strains colonized most northern South American countries via Ecuador and then Colombia and Peru, originating the SA3 and NA4/SA4 lineages during their spread. The entry and expansion in the southern part of South America (Argentina, Brazil, Chile, and Uruguay) occurred through three independent migration events and gave rise to the EU1/SA1 and SA2 lineages. South American lineages have specific combinations of amino acids under positive selection that constitute signatures of taxonomic and evolutionary relevance. Our findings provide a comprehensive scenario for the origin and migration routes of Canine morbillivirus in South America and highlight the importance of phylodynamics in understanding the geographic patterns of modern genetic variability.

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.

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.

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.