Structure and supramolecular organization of the canine distemper virus attachment glycoprotein.

Canine distemper virus (CDV) is an enveloped RNA morbillivirus that triggers respiratory, enteric, and high incidence of severe neurological disorders. CDV induces devastating outbreaks in wild and endangered animals as well as in domestic dogs in countries associated with suboptimal vaccination programs. The receptor-binding tetrameric attachment (H)-protein is part of the morbilliviral cell entry machinery. Here, we present the cryo-electron microscopy (cryo-EM) structure and supramolecular organization of the tetrameric CDV H-protein ectodomain. The structure reveals that the morbilliviral H-protein is composed of three main domains: stalk, neck, and heads. The most unexpected feature was the inherent asymmetric architecture of the CDV H-tetramer being shaped by the neck, which folds into an almost 90° bent conformation with respect to the stalk. Consequently, two non-contacting receptor-binding H-head dimers, which are also tilted toward each other, are located on one side of an intertwined four helical bundle stalk domain. Positioning of the four protomer polypeptide chains within the neck domain is guided by a glycine residue (G158), which forms a hinge point exclusively in two protomer polypeptide chains. Molecular dynamics simulations validated the stability of the asymmetric structure under near physiological conditions and molecular docking showed that two receptor-binding sites are fully accessible. Thus, this spatial organization of the CDV H-tetramer would allow for concomitant protein interactions with the stalk and head domains without steric clashes. In summary, the structure of the CDV H-protein ectodomain provides new insights into the morbilliviral cell entry system and offers a blueprint for next-generation structure-based antiviral drug discovery.

Genetic diversity accelerates canine distemper virus adaptation to ferrets.

RNA viruses adapt rapidly to new host environments by generating highly diverse genome sets, so-called "quasispecies." Minor genetic variants promote their rapid adaptation, allowing for the emergence of drug-resistance or immune-escape mutants. Understanding these adaptation processes is highly relevant to assessing the risk of cross-species transmission and the safety and efficacy of vaccines and antivirals. We hypothesized that genetic memory within a viral genome population facilitates rapid adaptation. To test this, we investigated the adaptation of the Morbillivirus canine distemper virus to ferrets and compared an attenuated, Vero cell-adapted virus isolate with its recombinant derivative over consecutive ferret passages. Although both viruses adapted to the new host, the reduced initial genetic diversity of the recombinant virus resulted in delayed disease onset. The non-recombinant virus gradually increased the frequencies of beneficial mutations already present at very low frequencies in the input virus. In contrast, the recombinant virus first evolved de novo mutations to compensate for the initial fitness impairments. Importantly, while both viruses evolved different sets of mutations, most mutations found in the adapted non-recombinant virus were identical to those found in a previous ferret adaptation experiment with the same isolate, indicating that mutations present at low frequency in the original virus stock serve as genetic memory. An arginine residue at position 519 in the carboxy terminus of the nucleoprotein shared by all adapted viruses was found to contribute to pathogenesis in ferrets. Our work illustrates the importance of genetic diversity for adaptation to new environments and identifies regions with functional relevance.IMPORTANCEWhen viruses encounter a new host, they can rapidly adapt to this host and cause disease. How these adaptation processes occur remains understudied. Morbilliviruses have high clinical and veterinary relevance and are attractive model systems to study these adaptation processes. The canine distemper virus is of particular interest, as it exhibits a broader host range than other morbilliviruses and frequently crosses species barriers. Here, we compared the adaptation of an attenuated virus and its recombinant derivative to that of ferrets. Pre-existing mutations present at low frequency allowed faster adaptation of the non-recombinant virus compared to the recombinant virus. We identified a common point mutation in the nucleoprotein that affected the pathogenesis of both viruses. Our study shows that genetic memory facilitates environmental adaptation and that erasing this genetic memory by genetic engineering results in delayed and different adaptation to new environments, providing an important safety aspect for the generation of live-attenuated vaccines.

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.

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.

Representative measles virus infection requires appropriate airway epithelia culture conditions.

IMPORTANCE: For many years, measles virus (MeV) was assumed to first enter the host via the apical surface of airway epithelial cells and subsequently spread systemically. We and others reported that MeV has an overwhelming preference for entry at the basolateral surface of airway epithelial cells, which led to a fundamental new understanding of how MeV enters a human host. This unexpected observation using well-differentiated primary cultures of airway epithelia from human donors contradicted previous studies using immortalized cultured cells. Here, we show that appropriate differentiation and cell morphology of primary human airway epithelial cells are critical to recapitulate MeV infection patterns and pathogenesis of the in vivo airways. By simply culturing primary cells in media containing serum or passaging primary cultures, erroneous results quickly emerge. These results have broad implications for data interpretation related to respiratory virus infection, spread, and release from human airway epithelial cells.

Development and characterization of a novel nanobody with SRMV neutralizing activity.

Peste des petits ruminants (PPR) is an acute, contact infectious disease caused by the small ruminant morbillivirus (SRMV), and its morbidity in goats and sheep can be up to 100% with significant mortality. Nanobody generated from camelid animals such as alpaca has attracted wide attention because of its unique advantages compared with conventional antibodies. The main objective of this study was to produce specific nanobodies against SRMV and identify its characteristics. To obtain the coding gene of SRMV-specific nanobodies, we first constructed an immune phage-displayed library from the VHH repertoire of alpaca that was immunized with SRMV-F and -H proteins. By using phage display technology, the target antigen-specific VHHs can be obtained after four consecutive rounds of biopanning. Results showed that the size of this VHH library was 2.26 × 1010 CFU/mL and the SRMV-F and -H specific phage particles were greatly enriched after four rounds of biopanning. The positive phage clones were selected and sequenced, and total of five independent different sequences of SRMV-specific nanobodies were identified. Subsequently, the DNA fragments of the five nanobodies were cloned into E. coli BL21(DE3), respectively, and three of them were successfully expressed and purified. Specificity and affinity towards inactivated SRMV of these purified nanobodies were then evaluated using the ELISA method. Results demonstrated that NbSRMV-1-1, NbSRMV-2-10, and NbSRMV-1-21 showed no cross-reactivity with other antigens, such as inactivated BTV, inactivated FMDV, His-tag labeled protein, and BSA. The ELISA titer of these three nanobodies against inactivated SRMV was up to 1:1000. However, only NbSRMV-1-21 displayed SRMV neutralizing activity at a maximum dilution of 1:4. The results indicate that the nanobodies against SRMV generated in this study could be useful in future applications. This study provided a novel antibody tool and laid a foundation for the treatment and detection of SRMV.

California sea lion (Zalophus californianus) lymph-node explant reveals involvement and possible transcriptional regulation of SLAM and nectin-4 during phocine distemper virus infection.

Phocine distemper virus (PDV) is a significant cause of mortality for phocid seals; however, the susceptibility of otariids to this virus is poorly understood. The authors used a lymph-node explant culture system from California sea lions (Zalophus californianus, CSL) to investigate: (1) the role of signaling lymphocyte activation molecule (SLAM) and nectin-4 in PDV infection and their cellular expression patterns, (2) if PDV induces transcriptional regulation of cell-entry receptors, and (3) the involvement of apoptosis in PDV infection. PDV replicated in the lymph-node explants with peak replication 3 days post-infection (dpi), but the replication was not sustained 4 to 5 dpi. The PDV+ cells co-localized SLAM and nectin-4. These cells expressed IBA1, indicating a histiocytic lineage. Comparison of receptor expression between infected and mock-infected lymph nodes suggested transcriptional downregulation of both receptors during the initial stage of infection and upregulation during the late stage of infection, but the values lack of statistical significance. Cleaved caspase-3+ cells were slightly increased in the infected lymph nodes compared with the mock-infected lymph node from 1 to 4 dpi, but without statistical significance, and a few apoptotic cells co-expressed PDV. The results suggest that lymph-node explants might be an important model to study PDV pathogenesis. CSLs have the potential to be infected with PDV, as they express both cell-entry receptors in histiocytes. The lack of statistical significance in the PDV replication, transcriptional regulation of viral receptors, and changes in apoptosis suggest that although CSL might be infected by PDV, they might be less susceptible than phocid species.

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.

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.

Sequestration and Destruction of Rinderpest Virus-Containing Material 10 Years after Eradication.

In 2021, the world marked 10 years free from rinderpest. The United Nations Food and Agriculture Organization and World Organisation for Animal Health have since made great strides in consolidating, sequencing, and destroying stocks of rinderpest virus-containing material, currently kept by only 14 known institutions. This progress must continue.

Concentrating all helper protein functions on a single entity allows rescue of recombinant measles virus by transfection of just two plasmids.

The generation of recombinant measles virus (MeV) from manipulated genomes on plasmid DNA is quite a complex and inefficient process. As a member of the order Mononegavirales its single-stranded ssRNA genome in negative sense orientation is not infectious, but requires co-availability of the viral RNA-dependent RNA polymerase L, the polymerase co-factor phosphoprotein P, and the nucleocapsid protein N in defined relative amounts to establish infectious centres in transfected cell cultures that release replication-competent recombinant MeV particles. For this so-called rescue, different rescue systems were developed that rely on at least four different components. In this work, we establish a functional MeV rescue system just being composed of two components: the plasmid encoding the (modified) viral genome, and a one-helper-plasmid bundling all helper functions. In contrast to a rescue-system for Newcastle Disease Virus, another paramyxovirus, co-expression of all helper proteins by the same promoter failed. Instead, adaptation of the strength of the respective promoters to drive each helper gene´s expression to the relative expression found in MeV-infected cells or other rescue systems, which indeed adjusted respective mRNA and protein expression, yielded success, albeit not yet to the same efficacy as the four-component system. Thereby, our study paves the way for the development of easier and, after further optimization, more efficient rescue systems to generate recombinant MeV for e.g. the application as a vaccine platform or oncolytic virus, for example.

Development and Validation of a Pan-Genotypic Real-Time Quantitative Reverse Transcription-PCR Assay To Detect Canine Distemper Virus and Phocine Distemper Virus in Domestic Animals and Wildlife.

Canine distemper virus (CDV) is an animal morbillivirus belonging to the family Paramyxoviridae and has caused major epizootics with high mortality levels in susceptible wildlife species. In recent years, the documented genetic diversity of CDV has expanded, with new genotypes identified in India, the Caspian Sea, and North America. However, no quantitative real-time PCR (RT-qPCR) that has been validated for the detection of all genotypes of CDV is currently available. We have therefore established and characterized a pan-genotypic probe-based RT-qPCR assay based on the detection of a conserved region of the phosphoprotein (P) gene of CDV. This assay has been validated using virus strains representative of six genotypes of CDV in different sample types, including frozen tissue, formalin-fixed paraffin-embedded tissue sections, and virus isolates. The primers and probe target sequences were sufficiently conserved to also enable detection of the phocine distemper virus strains responsible for epizootics in harbor seals in the North Sea in 1988 and 2002. Comparison with two recently published RT-qPCR assays for CDV showed that under equivalent conditions the primers and probe set reported in this study were more sensitive in detecting nucleic acids from an Asia-4 genotype, which displays sequence variation in primer and probe binding sites. In summary, this validated new pan-genotypic RT-qPCR assay will facilitate screening of suspected distemper cases caused by novel genotypes for which full genome sequences are unavailable and have utility in detecting multiple CDV strains in geographical regions where multiple genotypes cocirculate in wildlife.

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.

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.

Development and characterization of mouse monoclonal antibodies to canine morbillivirus.

Canine morbillivirus is a highly contagious multi-host pathogen with high morbidity and mortality. Timely diagnosis is of utmost importance to effectively control such a dreadful disease. Monoclonal antibodies (mAbs) serve as a high throughput diagnostics and applied tools for research and development (R&D). In the present study, a total of six mouse monoclonal antibodies were developed. All the mAbs generated belonged to IgG class. Of the six mAbs, two of them, namely CD-2F8 and CD-3D8 were directed against the nucleocapsid protein of CDV as determined in western blotting. The reactivity of all the mAbs was checked in indirect-ELISA and cell-ELISA using different morbilliviruses. The mAbs could broadly be categorized as; CDV specific (CD-3D8 and CD-2F8), cross-reactive to PPR virus (CD-AB3 and CD-4D6) and cross-reactive to both PPR virus and measles virus (CD-5D10 and CD-6E5). The characterized mAbs were used for antigenic profiling of CDV, PPR virus and measles virus. Based on the reactivity pattern; a close antigenic relationship was found among CDV and PPR virus as compared to measles virus. A pair of CDV specific mAbs namely CD-2F8 and CD-3D8 were identified which did not cross-react with measles and PPR viruses and thus could be used for diagnostic applications.

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.

Distribution of canine distemper virus and nectin-4 in raccoon (Procyon lotor) skin.

Raccoons (Procyon lotor) are abundant in urban/wildland interfaces and are key sources of canine distemper virus (CDV) outbreaks in domestic, zoo, and free-ranging wildlife species. CDV is pantropic, which provides multiple potential routes of transmission (urine, respiratory secretions, feces), but the specific role of skin as a target of infection, as a diagnostic sample, or as a potential source of environmental persistence and transmission is unknown. We have characterized the distribution of CDV and its known receptor, nectin-4, in skin samples of 36 raccoons. Even with skin samples that were grossly and histologically normal, immunohistochemistry of skin was useful in the diagnosis of CDV infection, which was found in both epithelium and endothelium. Nectin-4 was codistributed with cellular targets of viral infection. Skin secretions, shed keratinocytes, and hair of CDV infected raccoons are all potential environmental fomites.

Phenotypic and Transcriptional Changes of Pulmonary Immune Responses in Dogs Following Canine Distemper Virus Infection.

Canine distemper virus (CDV), a morbillivirus within the family Paramyxoviridae, is a highly contagious infectious agent causing a multisystemic, devastating disease in a broad range of host species, characterized by severe immunosuppression, encephalitis and pneumonia. The present study aimed at investigating pulmonary immune responses of CDV-infected dogs in situ using immunohistochemistry and whole transcriptome analyses by bulk RNA sequencing. Spatiotemporal analysis of phenotypic changes revealed pulmonary immune responses primarily driven by MHC-II+, Iba-1+ and CD204+ innate immune cells during acute and subacute infection phases, which paralleled pathologic lesion development and coincided with high viral loads in CDV-infected lungs. CD20+ B cell numbers initially declined, followed by lymphoid repopulation in the advanced disease phase. Transcriptome analysis demonstrated an increased expression of transcripts related to innate immunity, antiviral defense mechanisms, type I interferon responses and regulation of cell death in the lung of CDV-infected dogs. Molecular analyses also revealed disturbed cytokine responses with a pro-inflammatory M1 macrophage polarization and impaired mucociliary defense in CDV-infected lungs. The exploratory study provides detailed data on CDV-related pulmonary immune responses, expanding the list of immunologic parameters potentially leading to viral elimination and virus-induced pulmonary immunopathology in canine distemper.

Cryo-EM structure of the cetacean morbillivirus nucleoprotein-RNA complex.

Cetacean morbillivirus (CeMV) is an emerging and highly infectious paramyxovirus that causes outbreaks in cetaceans and occasionally in pinnipeds, representing a major threat to biodiversity and conservation of endangered marine mammal populations in both hemispheres. As for all non-segmented, negative-sense, single-stranded RNA (ssRNA) viruses, the morbilliviral genome is enwrapped by thousands of nucleoprotein (N) protomers. Each bound to six ribonucleotides, N protomers assemble to form a helical ribonucleoprotein (RNP) complex that serves as scaffold for nucleocapsid formation and as template for viral replication and transcription. While the molecular details on RNP complexes elucidated in human measles virus (MeV) served as paradigm model for these processes in all members of the Morbillivirus genus, no structural information has been obtained from other morbilliviruses, nor has any CeMV structure been solved so far. We report the structure of the CeMV RNP complex, reconstituted in vitro upon binding of recombinant CeMV N to poly-adenine ssRNA hexamers and solved to 4.0 Å resolution by cryo-electron microscopy. In spite of the amino acid sequence similarity and consequently similar folding of the N protomer, the CeMV RNP complex exhibits different helical parameters as compared to previously reported MeV orthologs. The CeMV structure reveals exclusive interactions leading to more extensive protomer-RNA and protomer-protomer interfaces. We identified twelve residues, among those varying between CeMV strains, as putatively important for the stabilization of the RNP complex, which highlights the need to study the potential of CeMV N mutations that modulate nucleocapsid assembly to also affect viral phenotype and host adaptation.

Receptor-mediated cell entry of paramyxoviruses: Mechanisms, and consequences for tropism and pathogenesis.

Research in the last decade has uncovered many new paramyxoviruses, airborne agents that cause epidemic diseases in animals including humans. Most paramyxoviruses enter epithelial cells of the airway using sialic acid as a receptor and cause only mild disease. However, others cross the epithelial barrier and cause more severe disease. For some of these viruses, the host receptors have been identified, and the mechanisms of cell entry have been elucidated. The tetrameric attachment proteins of paramyxoviruses have vastly different binding affinities for their cognate receptors, which they contact through different binding surfaces. Nevertheless, all input signals are converted to the same output: conformational changes that trigger refolding of trimeric fusion proteins and membrane fusion. Experiments with selectively receptor-blinded viruses inoculated into their natural hosts have provided insights into tropism, identifying the cells and tissues that support growth and revealing the mechanisms of pathogenesis. These analyses also shed light on diabolically elegant mechanisms used by morbilliviruses, including the measles virus, to promote massive amplification within the host, followed by efficient aerosolization and rapid spread through host populations. In another paradigm of receptor-facilitated severe disease, henipaviruses, including Nipah and Hendra viruses, use different members of one protein family to cause zoonoses. Specific properties of different paramyxoviruses, like neurotoxicity and immunosuppression, are now understood in the light of receptor specificity. We propose that research on the specific receptors for several newly identified members of the Paramyxoviridae family that may not bind sialic acid is needed to anticipate their zoonotic potential and to generate effective vaccines and antiviral compounds.