Canine distemper virus N protein induces autophagy to facilitate viral replication.

BACKGROUND: Canine distemper virus (CDV) is one of the most contagious and lethal viruses known to the Canidae, with a very broad and expanding host range. Autophagy serves as a fundamental stabilizing response against pathogens, but some viruses have been able to evade or exploit it for their replication. However, the effect of autophagy mechanisms on CDV infection is still unclear. RESULTS: In the present study, autophagy was induced in CDV-infected Vero cells as demonstrated by elevated LC3-II levels and aggregation of green fluorescent protein (GFP)-LC3 spots. Furthermore, CDV promoted the complete autophagic process, which could be determined by the degradation of p62, co-localization of LC3 with lysosomes, GFP degradation, and accumulation of LC3-II and p62 due to the lysosomal protease inhibitor E64d. In addition, the use of Rapamycin to promote autophagy promoted CDV replication, and the inhibition of autophagy by Wortmannin, Chloroquine and siRNA-ATG5 inhibited CDV replication, revealing that CDV-induced autophagy facilitated virus replication. We also found that UV-inactivated CDV still induced autophagy, and that nucleocapsid (N) protein was able to induce complete autophagy in an mTOR-dependent manner. CONCLUSIONS: This study for the first time revealed that CDV N protein induced complete autophagy to facilitate viral replication.

A Single Oral Immunization with Replication-Competent Adenovirus-Vectored Vaccine Induces a Neutralizing Antibody Response in Mice against Canine Distemper Virus.

Canine Distemper Virus (CDV) is a fatal and highly contagious pathogen of multiple carnivores. While injectable vaccines are very effective in protecting domestic animals, their use in the wild is unrealistic. Alternative vaccines are therefore needed. Adenovirus (AdV) vectors are popular vaccine vectors due to their capacity to elicit potent humoral and cellular immune responses against the antigens they carry. In parallel, vaccines based on live human AdV-4 and -7 have been used in U.S. army for several decades as replicative oral vaccines against respiratory infection with the same viruses. Based on these observations, the use of oral administration of replication competent AdV-vectored vaccines has emerged as a promising tool especially for wildlife vaccination. Developing this type of vaccine is not easy, however, given the high host specificity of AdVs and their very low replication in non-target species. To overcome this problem, the feasibility of this approach was tested using mouse adenovirus 1 (MAV-1) in mice as vaccine vectors. First, different vaccine vectors expressing the entire or part H or F proteins of CDV were constructed. These different strains were then used as oral vaccines in BALB/c mice and the immune response to CDV was evaluated. Only the strain expressing the full length CDV H protein generated a detectable and neutralizing immune response to CDV. Secondly, using this strain, we were able to show that although this type of vaccine is sensitive to pre-existing immunity to the vector, a second oral administration of the same vaccine is able to boost the immune response against CDV. Overall, this study demonstrates the feasibility of using replicating AdVs as oral vaccine vectors to immunize against CDV in wildlife carnivores.

Multiple Receptors Involved in Invasion and Neuropathogenicity of Canine Distemper Virus: A Review.

The canine distemper virus (CDV) is a morbillivirus that infects a broad range of terrestrial carnivores, predominantly canines, and is associated with high mortality. Similar to another morbillivirus, measles virus, which infects humans and nonhuman primates, CDV transmission from an infected host to a naïve host depends on two cellular receptors, namely, the signaling lymphocyte activation molecule (SLAM or CD150) and the adherens junction protein nectin-4 (also known as PVRL4). CDV can also invade the central nervous system by anterograde spread through olfactory nerves or in infected lymphocytes through the circulation, thus causing chronic progressive or relapsing demyelination of the brain. However, the absence of the two receptors in the white matter, primary cultured astrocytes, and neurons in the brain was recently demonstrated. Furthermore, a SLAM/nectin-4-blind recombinant CDV exhibits full cell-to-cell transmission in primary astrocytes. This strongly suggests the existence of a third CDV receptor expressed in neural cells, possibly glial cells. In this review, we summarize the recent progress in the study of CDV receptors, highlighting the unidentified glial receptor and its contribution to pathogenicity in the host nervous system. The reviewed studies focus on CDV neuropathogenesis, and neural receptors may provide promising directions for the treatment of neurological diseases caused by CDV. We also present an overview of other neurotropic viruses to promote further research and identification of CDV neural receptors.

Nanoparticles of conformation-stabilized canine distemper virus hemagglutinin are highly immunogenic and induce robust immunity.

BACKGROUND: Canine distemper virus (CDV) infection of ferrets, dogs, and giant pandas causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system. In this study, we tested a new candidate CDV vaccine-CDV nanoparticles-based on hemagglutinin protein. METHODS: The nanoparticles were generated from conformation-stabilized CDV hemagglutinin tetramers. Immune responses against CDV were evaluated in mice. Immunization was initiated 6 weeks after birth and boosted two times with 4-week intervals. The blood and mucosal samples were collected 2 weeks after each immunization. RESULTS: Vaccination with CDV nanoparticles elicited high levels of IgG antibody titers in mice (approximately sevenfold to eightfold higher than that obtained with soluble CDV H protein) and mucosal immune responses and developed increased CDV-specific neutralizing antibody. The mice that received nanoparticles showed significantly higher IFN-γ- and IL-4-secreting cell population in the spleen and lymph node compared with mice immunized with soluble H protein. The co-stimulatory molecular expression of CD80 and CD86 on the surface of DCs was also upregulated. CONCLUSION: The results demonstrate that self-assembly into nanoparticles can increase the immunogenicity of vaccine antigens, and nanoparticles assembled from conformation-stabilized CDV H protein can serve as a new CDV vaccine.

Sustained Replication of Synthetic Canine Distemper Virus Defective Genomes In Vitro and In Vivo.

Defective interfering (DI) genomes restrict viral replication and induce type I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity, and determine their long-term effects. To address this, we generated a recombinant canine distemper virus (rCDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques, and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate that these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues, which are a major site of CDV replication. Our established pipeline for detection, generation, and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome. IMPORTANCE Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes might be harnessed therapeutically. Negative-strand RNA virus research has provided a rich pool of natural DI genomes over many years, and they are probably the best understood in vitro. Here, we demonstrate the identification, synthesis, production, and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection.

Comparable Infection Level and Tropism of Measles Virus and Canine Distemper Virus in Organotypic Brain Slice Cultures Obtained from Natural Host Species.

Measles virus (MV) and canine distemper virus (CDV) are closely related members of the family Paramyxoviridae, genus Morbillivirus. MV infection of humans and non-human primates (NHPs) results in a self-limiting disease, which rarely involves central nervous system (CNS) complications. In contrast, infection of carnivores with CDV usually results in severe disease, in which CNS complications are common and the case-fatality rate is high. To compare the neurovirulence and neurotropism of MV and CDV, we established a short-term organotypic brain slice culture system of the olfactory bulb, hippocampus, or cortex obtained from NHPs, dogs, and ferrets. Slices were inoculated ex vivo with wild-type-based recombinant CDV or MV expressing a fluorescent reporter protein. The infection level of both morbilliviruses was determined at different times post-infection. We observed equivalent infection levels and identified microglia as main target cells in CDV-inoculated carnivore and MV-inoculated NHP brain tissue slices. Neurons were also susceptible to MV infection in NHP brain slice cultures. Our findings suggest that MV and CDV have comparable neurotropism and intrinsic capacity to infect CNS-resident cells of their natural host species.

Central nervous system lesions caused by canine distemper virus in 4 vaccinated dogs.

We examined the cerebellum and cerebrum of 4 vaccinated dogs, 3-60-mo-old, that displayed clinical signs of canine distemper virus (CDV) infection, and died 7-40 d after developing neurologic signs. The main histologic lesions were demyelination, gliosis, meningitis, perivascular lymphocytic cuffing, and inclusion bodies. These lesions were similar in all 4 cases regardless of the time since vaccination, except that meningoencephalitis and gliosis were subacute in 3 dogs and chronic in 1 dog. However, these differences did not appear to be related to their vaccination status. Immunohistologically, a CDV-positive immunoreaction was seen mainly in astrocytes, neurons and their axons, lymphocytes around and in the blood vessels of the pia mater and choroid plexus, ependymal cells of each ventricle, and the cells of the choroid plexus. The histologic and immunohistologic changes were similar in the cerebellum and cerebrum. The genetic characterization of the virus strains in 2 of these naturally occurring canine distemper cases confirmed that they were South American wild-type strains (Kiki and Uy251) belonging to the EU1/SA1 lineage. These strains are not included in the commercial CDV vaccines available in Uruguay.

Antiviral Screen against Canine Distemper Virus-Induced Membrane Fusion Activity.

Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide.

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

Myocarditis caused by naturally acquired canine distemper virus infection in 4 dogs.

Canine distemper virus (CDV) has long been recognized as a cause of myocarditis; however, cases of myocarditis caused by naturally acquired CDV infection have been reported only rarely in dogs. We describe here our retrospective study of naturally acquired systemic CDV infection in 4 dogs, 4-7 wk old, that had myocarditis, with myocardial necrosis and fibrosis. One of the 4 dogs had intracytoplasmic eosinophilic inclusion bodies in cardiomyocytes. Other lesions included bronchointerstitial pneumonia (4 of 4), necrotizing hepatitis (2 of 4), splenic lymphoid necrosis (2 of 4), encephalitis (1 of 3; brain was not submitted in 1 case), and necrotizing gastroenteritis (1 of 4). The presence of CDV in the heart was confirmed by immunohistochemistry in all 4 dogs.

Isolation and molecular characterizations of canine distemper virus from a naturally infected Korean dog using Vero cells expressing dog signaling lymphocyte activation molecule.

BACKGROUND: Canine distemper virus (CDV) infection results in high morbidity and mortality in dogs. There has been no report about isolation of Korean CDV since 1980 in Korea. OBJECTIVES: To investigate the biological properties and the genetic characterization of Korean CDV. METHODS: Vero cells expressing dog signaling lymphocyte activation molecule (dSLAM) gene named as Vero/dSLAM were used to isolate CDV using 17 samples. Diagnostic methods such as cytopathic effects, immunofluorescence assay, peroxidase linked assay, electron microscopy, rapid immunodiagnostic assay, and reverse transcription polymerase chain reaction were used to confirm the Korean CDV isolate as a CDV. The genetic analysis was performed through cloning and sequencing of hemagglutinin gene of CDV isolate. RESULTS: A virus propagated in Vero/dSLAM cell was confirmed as CDV (CD1901 strain) based on the above methods. The CD1901 strain showed the highest viral titer (105.5 50% tissue culture infectious dose [TCID50]/mL) in the Vero/dSLAM cells at 4 days post inoculation, but did not form a fork on chorioallantoic membrane of 7-day-old egg. Ribavirin, a nucleotide analogue anti-viral agent, inhibits moderately the Korean CDV propagation in the Vero/dSLAM cells. The nucleotide and amino acid sequences of the H gene of CD1901 strain were compared with those of other CDV strains. The CD1901 strain belonged to Asia 1 group and had the highest similarity (99.9%) with the BA134 strain, which was isolated in China in 2008. CONCLUSIONS: We constructed successfully Vero/dSLAM and isolated one Korean CDV isolate (CD1901 strain) from a naturally infected dog. The CD1901 strain belonged to Asia 1 genotype.

Clustered Lysine Residues of the Canine Distemper Virus Matrix Protein Regulate Membrane Association and Budding Activity.

The canine distemper virus (CDV) matrix (M) protein is multifunctional; it orchestrates viral assembly and budding, drives the formation of virus-like particles (VLPs), regulates viral RNA synthesis, and may support additional functions. CDV M may assemble into dimers, where each protomer is constituted by N-terminal and C-terminal domains (NTD and CTD, respectively). Here, to investigate whether electrostatic interactions between CDV M and the plasma membrane (PM) may contribute to budding activity, selected surface-exposed positively charged lysine residues, which are located within a large basic patch of CTD, were replaced by amino acids with selected properties. We found that some M mutants harboring amino acids with neutral and positive charge (methionine and arginine, respectively) maintained full functionality, including proper interaction and localization with the PM as well as intact VLP and progeny virus production as demonstrated by employing a cell exit-complementation system. Conversely, while the overall structural integrity remained mostly unaltered, most of the nonconservative M variants (carrying a glutamic acid; negatively charged) exhibited a cytosolic phenotype secondary to the lack of interaction with the PM. Consequently, such M variants were entirely defective in VLP production and viral particle formation. Furthermore, the proteasome inhibitor bortezomib significantly reduced wild-type M-mediated VLP production. Nevertheless, in the absence of the compound, all engineered M lysine variants exhibited unaffected ubiquitination profiles, consistent with other residues likely involved in this functionally essential posttranslational modification. Altogether, our data identified multiple surface-exposed lysine residues located within a basic patch of CDV M-CTD, critically contributing to PM association and ensuing membrane budding activity.IMPORTANCE Although vaccines against some morbilliviruses exist, infections still occur, which can result in dramatic brain disease or fatal outcome. Postexposure prophylaxis with antivirals would support global vaccination campaigns. Unfortunately, there is no efficient antiviral drug currently approved. The matrix (M) protein of morbilliviruses coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, molecular mechanisms supporting these functions remain poorly understood, which preclude the rationale design of inhibitors. Here, to investigate potential interactions between canine distemper virus (CDV) M and the plasma membrane (PM), we combined structure-guided mutagenesis of selected surface-exposed lysine residues with biochemical, cellular, and virological assays. We identified several lysines clustering in a basic patch microdomain of the CDV M C-terminal domain, which contributed to PM association and budding activity. Our findings provide novel mechanistic information of how morbilliviruses assemble and egress from infected cells, thereby delivering bases for future antiviral drug development.

Viral Pathogenesis, Recombinant Vaccines, and Oncolytic Virotherapy: Applications of the Canine Distemper Virus Reverse Genetics System.

Canine distemper virus (CDV) is a highly contagious pathogen transmissible to a broad range of terrestrial and aquatic carnivores. Despite the availability of attenuated vaccines against CDV, the virus remains responsible for outbreaks of canine distemper (CD) with significant morbidity and mortality in domesticated and wild carnivores worldwide. CDV uses the signaling lymphocytic activation molecule (SLAM, or CD150) and nectin-4 (PVRL4) as entry receptors, well-known tumor-associated markers for several lymphadenomas and adenocarcinomas, which are also responsible for the lysis of tumor cells and apparent tumor regression. Thus, CDV vaccine strains have emerged as a promising platform of oncolytic viruses for use in animal cancer therapy. Recent advances have revealed that use of the CDV reverse genetic system (RGS) has helped increase the understanding of viral pathogenesis and explore the development of recombinant CDV vaccines. In addition, genetic engineering of CDV based on RGS approaches also has the potential of enhancing oncolytic activity and selectively targeting tumors. Here, we reviewed the host tropism and pathogenesis of CDV, and current development of recombinant CDV-based vaccines as well as their use as oncolytic viruses against cancers.

Phocine distemper virus uses phocine and other animal SLAMs as a receptor but not human SLAM.

Morbilliviruses use the signaling lymphocyte activation molecule (SLAM) as a receptor to infect their hosts. Seals are almost the only animal species that show apparent infection with phocine distemper virus (PDV). Seal SLAM functioned as a PDV receptor. However, dolphin- and dog-SLAM molecules, but not human SLAM, were also fully functional PDV receptors. These data suggest that the host range of PDV is not simply determined by its SLAM usage. However, human nonsusceptibility to PDV infection may be at least partly attributable to the inability of PDV to use human SLAM as a receptor.

RNA sequencing analyses of gene expressions in a canine macrophages cell line DH82 infected with canine distemper virus.

Virulent morbillivirus infections, including Meals Virus (MeV) and Canine Distemper Virus (CDV), caused severe immune suppression and leukopenia, while attenuated vaccine strains developed protective host immune responses. However, the detailed molecular foundations of host antiviral responses were poorly characterized. In order to better understand the interactions between attenuated vaccine and host antiviral responses, the global gene expression changes in CDV-11-infected DH82 cells, a macrophage-derived cell line from canine, were investigated by transcriptomic analysis, and portions of results were confirmed with quantitative RT-PCR. The results exhibited that 372 genes significantly up-regulated (p < .01) and 119 genes were significantly down-regulated (p < .01) in CDV-infected macrophages DH82 at 48 h p.i.. The enriched functions of the significantly up-regulated (p < .01) genes were closely associated with interferon stimulated genes (ISGs), chemokine genes and pro-inflammatory factor genes. Gene ontology and pathway analysis of differentially expressed genes (DEGs) revealed that the most significantly involved pathways in CDV-infected DH82 cells were NF-κB and TNF signaling pathway, cytokine-cytokine receptor interaction, and pathogen associated molecular patterns (PAMPs), such as Toll-like, RIG-I-like and NOD-like receptor signalings. Thus, the findings indicated that pattern recognition receptors (PRRs) possibly mediated host innate and protective antiviral immune responses in CDV-11 infected DH82 cells.

Evolution and Interspecies Transmission of Canine Distemper Virus-An Outlook of the Diverse Evolutionary Landscapes of a Multi-Host Virus.

Canine distemper virus (CDV) is a worldwide distributed virus which belongs to the genus Morbillivirus within the Paramyxoviridae family. CDV spreads through the lymphatic, epithelial, and nervous systems of domestic dogs and wildlife, in at least six orders and over 20 families of mammals. Due to the high morbidity and mortality rates and broad host range, understanding the epidemiology of CDV is not only important for its control in domestic animals, but also for the development of reliable wildlife conservation strategies. The present review aims to give an outlook of the multiple evolutionary landscapes and factors involved in the transmission of CDV by including epidemiological data from multiple species in urban, wild and peri-urban settings, not only in domestic animal populations but at the wildlife interface. It is clear that different epidemiological scenarios can lead to the presence of CDV in wildlife even in the absence of infection in domestic populations, highlighting the role of CDV in different domestic or wild species without clinical signs of disease mainly acting as reservoirs (peridomestic and mesocarnivores) that are often found in peridomestic habits triggering CDV epidemics. Another scenario is driven by mutations, which generate genetic variation on which random drift and natural selection can act, shaping the genetic structure of CDV populations leading to some fitness compensations between hosts and driving the evolution of specialist and generalist traits in CDV populations. In this scenario, the highly variable protein hemagglutinin (H) determines the cellular and host tropism by binding to signaling lymphocytic activation molecule (SLAM) and nectin-4 receptors of the host; however, the multiple evolutionary events that may have facilitated CDV adaptation to different hosts must be evaluated by complete genome sequencing. This review is focused on the study of CDV interspecies transmission by examining molecular and epidemiological reports based on sequences of the hemagglutinin gene and the growing body of studies of the complete genome; emphasizing the importance of long-term multidisciplinary research that tracks CDV in the presence or absence of clinical signs in wild species, and helping to implement strategies to mitigate the infection. Integrated research incorporating the experience of wildlife managers, behavioral and conservation biologists, veterinarians, virologists, and immunologists (among other scientific areas) and the inclusion of several wild and domestic species is essential for understanding the intricate epidemiological dynamics of CDV in its multiple host infections.

Tropism and molecular pathogenesis of canine distemper virus.

BACKGROUND: Canine distemper virus (CDV), currently termed Canine morbillivirus, is an extremely contagious disease that affects dogs. It is identified as a multiple cell tropism pathogen, and its host range includes a vast array of species. As a member of Mononegavirales, CDV has a negative, single-stranded RNA genome, which encodes eight proteins. MAIN BODY: Regarding the molecular pathogenesis, the hemagglutinin protein (H) plays a crucial role both in the antigenic recognition and the viral interaction with SLAM and nectin-4, the host cells' receptors. These cellular receptors have been studied widely as CDV receptors in vitro in different cellular models. The SLAM receptor is located in lymphoid cells; therefore, the infection of these cells by CDV leads to immunosuppression, the severity of which can lead to variability in the clinical disease with the potential of secondary bacterial infection, up to and including the development of neurological signs in its later stage. CONCLUSION: Improving the understanding of the CDV molecules implicated in the determination of infection, especially the H protein, can help to enhance the biochemical comprehension of the difference between a wide range of CDV variants, their tropism, and different steps in viral infection. The regions of interaction between the viral proteins and the identified host cell receptors have been elucidated to facilitate this understanding. Hence, this review describes the significant molecular and cellular characteristics of CDV that contribute to viral pathogenesis.

Fusion and hemagglutinin proteins of canine distemper virus promote osteoclast formation through NF-κB dependent and independent mechanisms.

Paget's disease (PD) features abnormal osteoclasts (OC) which sharply increase in number and size and then intensely induce bone resorption. The purpose of this study was to determine the direct effects of canine distemper virus (CDV) and its fusion protein and hemagglutinin protein (F + H) on receptor activator of nuclear factor kappa-B ligand (RANKL) induced OC formation in vitro. Immunofluorescence assay, OC morphological and functional detection, intracellular signaling pathway detection, Real-time PCR analysis and ELISA were applied in this study. Immunofluorescence assay provided the conclusive proof that CDV can infect and replicate in RAW264.7 mouse monocyte cell line, primary human peripheral blood mononuclear cells (PBMC) and their further fused OC. Both CDV and F + H significantly promoted OC formation and bone resorption ability induced by RANKL. Meanwhile, intracellular signaling transduction analysis revealed CDV and F + H specifically upregulated the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) induced by RANKL, respectively. Furthermore, without RANKL stimulation, both CDV and F + H slightly induced OC-like cells formation in RAW264.7 cell line even in the presence of NF-κB inhibitor. F + H upregulate OC differentiation and activity through modulation of NF-κB signaling pathway, and induce OC precursor cells merging dependent on the function of glycoproteins themselves. These results meant that F and H proteins play a pivotal role in CDV supporting OC formation. Moreover, this work further provide a new research direction that F and H proteins in CDV should be considered as a trigger during the pathogenesis of PD.

Primary resistance mechanism of the canine distemper virus fusion protein against a small-molecule membrane fusion inhibitor.

Morbilliviruses (e.g. measles virus [MeV] or canine distemper virus [CDV]) employ the attachment (H) and fusion (F) envelope glycoproteins for cell entry. H protein engagement to a cognate receptor eventually leads to F-triggering. Upon activation, F proteins transit from a prefusion to a postfusion conformation; a refolding process that is associated with membrane merging. Small-molecule morbilliviral fusion inhibitors such as the compound 3G (a chemical analog in the AS-48 class) were previously generated and mechanistic studies revealed a stabilizing effect on morbilliviral prefusion F trimers. Here, we aimed at designing 3G-resistant CDV F mutants by introducing single cysteine residues at hydrophobic core positions of the helical stalk region. Covalently-linked F dimers were generated, which highlighted substantial conformational flexibility within the stalk to achieve those irregular F conformations. Our findings demonstrate that "top-stalk" CDV F cysteine mutants (F-V571C and F-L575C) remained functional and gained resistance to 3G. Conversely, although not all "bottom-stalk" F cysteine variants preserved proper bioactivity, those that remained functional exhibited 3G-sensitivity. According to the recently determined prefusion MeV F trimer/AS-48 co-crystal structure, CDV residues F-V571 and F-L575 may directly interact with 3G. A combination of conformation-specific anti-F antibodies and low-resolution electron microscopy structural analyses confirmed that 3G lost its stabilizing effect on "top-stalk" F cysteine mutants thus suggesting a primary resistance mechanism. Overall, our data suggest that the fusion inhibitor 3G stabilizes prefusion CDV F trimers by docking at the top of the stalk domain.

Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering.

Morbillivirus (e.g., measles virus [MeV] and canine distemper virus [CDV]) host cell entry is coordinated by two interacting envelope glycoproteins, namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H proteins consists of stalk, connector, and head domains that assemble into functional noncovalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed linker) and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a nonconservative mutagenesis scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to the SLAM/CD150 receptor. Nondenaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H tetramerization per se was not the only mechanism preventing F activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine substitution of natural cysteines (H-C139A and H-C154A) and thus were unable to form covalently linked tetramers, were fusion activation defective. Our data suggest a dual regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.IMPORTANCE MeV and CDV remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). The current hypothesis postulates that tetrameric H ectodomains (composed of stalk, connector, and head domains) undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis scan analysis of both microdomains revealed that replacing amino acid 146 in the H-linker region with nonhydrophobic residues produced covalent H tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional pre-F-triggering H tetramers. Furthermore, such structures might be critical to convert receptor engagement into F activation.