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.

Historical isolates of dolphin morbillivirus: origin and new data.

Dolphin morbillivirus (DMV) was isolated in striped dolphins Stenella coeruleoalba from the Mediterranean Sea stranded along the coast of Spain during a lethal epidemic that killed thousands of individuals in 1990-1992. Though some of these isolates (MUC, 16A and the reference strain) have been extensively characterised, details on their origin were not reported in the literature, and records for these isolates are often difficult to trace and are, sometimes, erroneous. Here, we provide unpublished biological and histopathological data for these isolates, summarize the literature on their characterization and make suggestions for future studies.

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.

Establishment and characterization of a novel kidney cell line derived from the common bottlenose dolphin.

Common bottlenose dolphin (Tursiops truncatus) is a well-known cetacean species that inhabits temperate and tropical seas worldwide. Limited supply and poor quality of samples hinder the investigation of the effects of various pathogens and environmental pollutants on this cetacean species. Cultured cells are useful for experimental studies; however, no cell lines derived from cetaceans are generally available. Therefore, in this study, we established a novel kidney cell line, TK-ST, derived from T. truncatus. Primary cells exhibited the morphological characteristics of epithelial and fibroblast cells, but their immortalization and passaging resulted in a predominantly epithelial cell morphology. TK-ST was immortalized using the large T SV40 antigen and human telomerase reverse transcriptase and exhibited long-term stable cell growth. TK-ST cells are generally cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37°C and 5% CO2 but can also be cultured in 5-20% fetal bovine serum and several other classical media commonly used for common animal cell culture. TK-ST cells were found to be susceptible to several viruses, including the dolphin morbillivirus (most important virus in cetaceans), and exhibited cytopathic effects, facilitating the replication of the dolphin morbillivirus. Furthermore, mRNA expression levels of cytokine genes were increased in TK-ST cells after stimulation with lipopolysaccharides and poly(I:C). Therefore, the novel TK-ST cell line derived in this study can potentially be used for further in vitro studies on cetaceans.

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.

Canine Distemper Virus Alters Defense Responses in an Ex Vivo Model of Pulmonary Infection.

Canine distemper virus (CDV), belonging to the genus Morbillivirus, is a highly contagious pathogen. It is infectious in a wide range of host species, including domestic and wildlife carnivores, and causes severe systemic disease with involvement of the respiratory tract. In the present study, canine precision-cut lung slices (PCLSs) were infected with CDV (strain R252) to investigate temporospatial viral loads, cell tropism, ciliary activity, and local immune responses during early infection ex vivo. Progressive viral replication was observed during the infection period in histiocytic and, to a lesser extent, epithelial cells. CDV-infected cells were predominantly located within the bronchial subepithelial tissue. Ciliary activity was reduced in CDV-infected PCLSs, while viability remained unchanged when compared to controls. MHC-II expression was increased in the bronchial epithelium on day three postinfection. Elevated levels of anti-inflammatory cytokines (interleukin-10 and transforming growth factor-ß) were observed in CDV-infected PCLSs on day one postinfection. In conclusion, the present study demonstrates that PCLSs are permissive for CDV. The model reveals an impaired ciliary function and an anti-inflammatory cytokine response, potentially fostering viral replication in the lung during the early phase of canine distemper.

Genetic Characteristics of Measles Viruses Isolated in Taiwan between 2015 and 2020.

A genetic analysis of circulating measles virus (MeV) provides strong evidence of an interruption in endemic measles and supports the elimination status of this disease. This study investigated 219 MeVs isolated between 2015 and 2020. Based on the 450 nucleotide sequences of the nucleoprotein gene (N-450), three genotypes of the H1, D8 and B3 with 8, 18 and 6 different N-450 sequences, respectively, were identified. The H1 genotype virus has not circulated in Taiwan since 2017, and the D8 and B3 genotype MeVs became dominant between 2018 and 2019. Different D8 genotype variants were imported from neighboring countries, and the majority of MeV variants were detected only for a short period. However, MVs/Gir Somnath.IND/42.16[D8], a named strain designated by the World Health Organization (WHO), was detected over 2 years. To explore whether the endemic transmission of measles has been underestimated, another sequence window of the hypervariable, noncoding regions between the matrix (M) and fusion (F) genes (MF-NCR) was introduced to clarify the transmission chain. From the chronological sequence analysis of MeVs with N-450 and MF-NCR sequence windows, no endemic MeV variants lasted over 4 weeks, providing strong evidence to support the contention that Taiwan has reached the status for measles elimination.

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.

History of measles.

Measles is a highly contagious viral disease transmitted by aerosols through human-to-human contact. It is often considered as a benign disease, although mortality remains high in developing countries (>5%). Frequent complications (diarrhea, otitis, pneumonia, encephalitis) can be observed. The disease mainly affects young children from 5 to 6 months of age with a mortality peak in the first three years of life. The Persian physician Rhazes gave the first clinical description of the disease in the 10th century, clearly differentiating it from smallpox. Measles spread worldwide from the Renaissance. Its epidemiology was remarkably studied in 1846 by a Danish physician, Peter Panum, during an epidemic in the Faroe Islands. The viral nature of this disease was demonstrated in 1911 and the virus was identified in 1954 by Thomas Peebles and John Enders. It is a morbillivirus (family Paramyxoviridae), also responsible for rinderpest (ovine, bovine), distemper (canine, feline), and epidemic diseases of dolphins, porpoises and seals. The current measles virus appeared recently from the rinderpest virus around the 6th century BCE. It has adapted perfectly to the human species, becoming strictly human, without animal reservoirs. A live attenuated vaccine was developed in 1958 by Enders' team after numerous passages in cell cultures. The vaccine was licensed in the United States in 1963 and is used on a large scale by the WHO throughout the world. This highly effective and well-tolerated vaccine has greatly reduced the number of measles cases and saved millions of lives. Measles remains a major public health concern, causing over 100,000 deaths per year worldwide. Today, the most affected continents remain Africa, South America and Asia.

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.

Novel and classical morbilliviruses: Current knowledge of three divergent morbillivirus groups.

Currently, seven species of morbillivirus have been classified. Six of these species (Measles morbillivirus, Rinderpest morbillivirus, Small ruminant morbillivirus, Canine morbillivirus, Phocine morbillivirus, and Cetacean morbillivirus) are highly infectious and cause serious systemic diseases in humans, livestock, domestic dogs, and wild animals. These species commonly use the host proteins signaling lymphocytic activation molecule (SLAM) and nectin-4 as receptors, and this usage contributes to their virulence. The seventh species (Feline morbillivirus: FeMV) is phylogenetically divergent from the six SLAM-using species. FeMV differs from the SLAM-using morbillivirus group in pathogenicity and infectivity, and is speculated to use non-SLAM receptors. Recently, novel species of morbilliviruses have been discovered in bats, rodents, and domestic pigs. Because the ability to use SLAM and nectin-4 is closely related to the infectivity and pathogenicity of morbilliviruses, investigation of the potential usage of these receptors is useful for estimating infectivity and pathogenicity. The SLAM-binding sites in the receptor-binding protein show high similarity among the SLAM-using morbilliviruses. This feature may help to estimate whether novel morbillivirus species can use SLAM as a receptor. A novel morbillivirus species isolated from wild mice diverged from the classified morbilliviruses in the phylogenetic tree, forming a third group separate from the SLAM-using morbillivirus group and FeMV. This suggests that the novel rodent morbillivirus may exhibit a different risk from the SLAM-using morbillivirus group, and analyses of its viral pathogenicity and infectivity toward humans are warranted.