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Paramyxoviruses, including members of the genus Morbillivirus, express accessory proteins with ancillary functions during viral replication. One of these, the C protein, is expressed from an alternative open reading frame (ORF) located in the P gene. The measles virus (MeV) C protein has been implicated in modulation of interferon signaling, but has more recently been shown to play a vital role in regulation of viral transcription and replication, preventing the excessive production of double-stranded RNA. Failure to do so, as seen with C-deficient MeV, leads to early activation of innate immune responses resulting in restriction of viral replication and attenuation in the host. One puzzling aspect of morbillivirus C protein biology has been the finding that a C-deficient canine distemper virus (CDV) generated by a similar mutagenesis strategy displayed no attenuation in ferrets, an animal model commonly used to evaluate CDV pathogenesis. To resolve how virus lacking this protein could maintain virulence, we revisited the CDV C protein and found that truncated C proteins are expressed from the CDV gene using alternative downstream start codons, even when the first start codon has been disrupted. We introduced an additional point mutation abrogating expression of these truncated C proteins. A new CDV with this mutation was attenuated in vitro and led to increased activation of protein kinase R. It was also strongly attenuated in ferrets, inducing only mild disease in infected animals, thus replicating the phenotype of C-deficient MeV. Our results demonstrate the crucial role of morbillivirus C proteins in pathogenesis. [ABSTRACT FROM AUTHOR]

Upon infection, morbilliviruses such as measles virus, rinderpest virus, and canine distemper virus (CDV) initially target immune cells via the signaling lymphocyte activation molecule (SLAM) before spreading to respiratory epithelia through the adherens junction protein nectin-4. However, the roles of these receptors in transmission from infected to naive hosts have not yet been formally tested. To experimentally addressing this question, we established a model of CDV contact transmission between ferrets. We show here that transmission of wild-type CDV sometimes precedes the onset of clinical disease. In contrast, transmission was not observed in most animals infected with SLAM- or nectin-4-blind CDVs, even though all animals infected with the nectin-4-blind virus developed sustained viremia. There was an unexpected case of transmission of a nectin-4-blind virus, possibly due to biting. Another unprecedented event was transient viremia in an infection with a SLAM-blind virus. We identified three compensatory mutations within or near the SLAM-binding surface of the attachment protein. A recombinant CDV expressing the mutated attachment protein regained the ability to infect ferret lymphocytes in vitro, but its replication was not as efficient as that of wild-type CDV. Ferrets infected with this virus developed transient viremia and fever, but there was no transmission to naive contacts. Our study supports the importance of epithelial cell infection and of sequential CDV H protein interactions first with SLAM and then nectin-4 receptors for transmission to naive hosts. It also highlights the in vivo selection pressure on the H protein interactions with SLAM. IMPORTANCE Morbilliviruses such as measles virus, rinderpest virus, and canine distemper virus (CDV) are highly contagious. Despite extensive knowledge of how morbilliviruses interact with their receptors, little is known about how those interactions influence viral transmission to naive hosts. In a ferret model of CDV contact transmission, we showed that sequential use of the signaling lymphocytic activation molecule (SLAM) and nectin-4 receptors is essential for transmission. In one animal infected with a SLAM-blind CDV, we documented mild viremia due to the acquisition of three compensatory mutations within or near the SLAM-binding surface. The interaction, however, was not sufficient to cause disease or sustain transmission to naive contacts. This work confirms the sequential roles of SLAM and nectin-4 in morbillivirus transmission and highlights the selective pressure directed toward productive interactions with SLAM. [ABSTRACT FROM AUTHOR]

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. IMPORTANCE When 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. [ABSTRACT FROM AUTHOR]

The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the liveattenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains. [ABSTRACT FROM AUTHOR]

To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression. [ABSTRACT FROM AUTHOR]

Paramyxovirus glycoproteins are posttranslationally modified by the addition of N-linked glycans, which are often necessary for correct folding, processing, and cell surface expression. To establish the contribution of N glycosylation to morbillivirus attachment (H) protein function and overall virulence, we first determined the use of the potential N-glycosylation sites in the canine distemper virus (CDV) H proteins. Biochemical characterization revealed that the three sites conserved in all strains were N glycosylated, whereas only two of the up to five additional sites present in wild-type strains are used. A wild-type virus with an H protein reproducing the vaccine strain N-glycosylation pattern remained lethal in ferrets but with a prolonged course of disease. In contrast, introduction of the vaccine H protein in the wild-type context resulted in complete attenuation. To further characterize the role of N glycosylation in CDV pathogenesis, the N-glycosylation sites of wild-type H proteins were successively deleted, including a nonstandard site, to ultimately generate a nonglycosylated H protein. Despite reduced expression levels, this protein remained fully functional. Recombinant viruses expressing N-glycan-deficient H proteins no longer caused disease, even though their immunosuppressive capacities were retained, indicating that reduced N glycosylation contributes to attenuation without affecting immunosuppression. [ABSTRACT FROM AUTHOR]