Canine distemper virus (CDV) can cause fatal infections in giant pandas. Vaccination is crucial to prevent CDV infection in giant pandas. In this study, two bacterium-like particle vaccines F3-GEM and H4-GEM displaying the trimeric F protein or tetrameric H protein of CDV were constructed based on the Gram-positive enhanced-matrix protein anchor (GEM-PA) surface display system. Electron microscopy and Western blot results revealed that the F or H protein was successfully anchored on the surface of GEM particles. Furthermore, one more bacterium-like particle vaccine F3 and H4-GEM was also designed, a mixture consisting of F3-GEM and H4-GEM at a ratio of 1:1. To evaluate the effect of the three vaccines, mice were immunized with F3-GEM, H4-GEM or F3 and H4-GEM. It was found that the level of IgG-specific antibodies and neutralizing antibodies in the F3 and H4-GEM group was higher than the other two groups. Additionally, F3 and H4-GEM also increased the secretion of Th1-related and Th2-related cytokines. Moreover, F3 and H4-GEM induce IgG and neutralizing antibodies\' response in dogs. Conclusions: In summary, F3 and H4-GEM can provoke better immune responses to CDV in mice and dogs. The bacterium-like particle vaccine F3 and H4-GEM might be a potential vaccine candidate for giant pandas against CDV infection.

Canine distemper (CD) caused by canine distemper virus (CDV) is considered a highly contagious and acutely febrile disease in various animals around the world. Endoplasmic reticulum-associated protein degradation (ERAD) is an important biological effect induced by endoplasmic reticulum (ER) stress (ERS) for the degradation of unfolded/misfolded proteins in the ER of cells. CDV H glycoprotein is translocated into the ER for post-translational modifications. The effects of CDV H and ER on each other are unclear. In this study, we found that CDV H protein induced ERS through the PERK-mediated signaling pathway. The inhibition of ERS by 4-Phenylbutyric acid (4-PBA) increased the H protein amounts of an attenuated CDV, which was reduced by dithiothreitol (DTT)-induced ERS. Further, the H protein levels were increased when ERAD was inhibited by using Eeyarestatin I or interfering E3 ligase Hrd1 in ERAD, suggesting that the attenuated CDV H protein is degraded via ERAD. ERAD involved ubiquitin-dependent proteasome degradation (UPD) and/or autophagic-lysosome degradation (ALD). The attenuated CDV H protein was ubiquitinated and significantly increased after treatment with UPD inhibitor MG132 but not ALD inhibitor chloroquine (CQ), suggesting that ERAD degrading the attenuated CDV H protein selectively depends on UPD. Moreover, the inhibition of the degradation of CDV H protein with 4-PBA or MG132 treatment increased viral replication, whereas treatment with DTT promoting degradation of H protein was found to reduce viral replication. These findings suggest that the degradation of CDV H protein via ERAD negatively affects viral replication and provide a new idea for developing CDV prevention and control strategies.

Canine distemper (CD) is a highly contagious and an acutely febrile disease caused by canine distemper virus (CDV), which greatly threatens the dog and fur industry in many countries. Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality control system for the degradation of misfolded proteins in the ER. In this study, a proteomic approach was performed, and results found the E3 ubiquitin ligase 3-hydroxy-3-methylglutaryl reductase degradation protein 1 (Hrd1), which is involved in ERAD, as one of the CDV H-interacting proteins. The interaction of Hrd1 with CDV H protein was further identified by Co-IP assay and confocal microscopy. Hrd1 degraded the CDV H protein via the proteasome pathway dependent on its E3 ubiquitin ligase activity. Hrd1 catalyzed the K63-linked polyubiquitination of CDV H protein at lysine residue 115 (K115). Hrd1 also exhibited a significant inhibitory effect on CDV replication. Together, the data demonstrate that the E3 ligase Hrd1 mediates the ubiquitination of CDV H protein for degradation via the proteasome pathway and inhibits CDV replication. Thus, targeting Hrd1 may represent a novel prevention and control strategy for CDV infection.

Canine distemper virus (CDV) infects many sensitive species worldwide and its host range is expanding. The hemagglutinin (H) protein, the major neutralizing target, binds to cellular receptors and subsequently triggers fusion for initial viral infection. So it\'s necessary to clarify the precise neutralizing epitopes of H protein and extend the knowledge of mechanisms of virus neutralization. In this study, a neutralizing monoclonal antibody (mAb) 2D12 against CDV H protein, which had different reactivity with different CDV strains, was generated and characterized. A series of truncated H proteins were screened to define the minimal linear epitope 238DIEREFD244 recognized by 2D12. Further investigation revealed that the epitope was highly conserved in America-1 vaccine lineage of CDV strains, but different substitutions in the epitope appeared in CDV strains of the other lineages and two substitutions (D238Y and R241G) caused the change of antigenicity. Thus, the epitope represents a novel lineage-specific neutralizing target on H protein of CDV for differentiation of America-1 vaccine lineage and the other lineages of CDV strains. The epitope was identified to localize at the surface of H protein in two different positions in a three-dimensional (3D) structure, but not at the position of the receptor-binding site (RBS), so the mAb 2D12 that recognized the epitope did not inhibit binding of H protein to the receptor. But mAb 2D12 interfered with the H-F interaction for inhibiting membrane fusion, suggesting that the mAb plays key roles for formation of H-F protein oligomeric structure. Our data will contribute to the understanding of the structure, function, and antigenicity of CDV H protein and mechanisms of virus neutralization.

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.

The glycine cleavage system (GCS) is a fundamental component of life, widely existing in microbes, plants, animals, and humans. A better understanding of the functionality and working mechanisms, and the engineering of the GCS have both scientific and practical impacts, which may lead to new knowledge and findings in life sciences, improved biomass production and human/animal health, efficient biosynthesis of chemicals, effective carbon fixation and global climate change mitigation. In this chapter, the GCS is first discussed in the context of the reductive glycine pathway (rGlyP), a recently proposed and appealing assimilation pathway of CO2 and formate, and its implementation and optimization in microorganisms for formatotrophic growth. Then, the present knowledge about the components, reactions, and working mechanisms of the GCS and related enzymes is reviewed. Particular emphasis is also placed on the conformational and structural features of the GCS proteins, especially the different forms of lipoylated H protein and its lipoylation by lipoate protein ligase (LplA). Subsequently, existing analytic methods for the components and reactions of the GCS and recent advances in quantitatively understanding and purposefully engineering the GCS are presented. Finally, perspectives of current state of the art in the GCS research are given and future research needs are highlighted.

Glycine cleavage system (GCS) plays a central role in one-carbon (C1) metabolism and receives increasing interest as a core part of the recently proposed reductive glycine pathway (rGlyP) for assimilation of CO2 and formate. Despite decades of research, GCS has not yet been well understood and kinetic data are barely available. This is to a large degree because of the complexity of GCS, which is composed of four proteins (H, T, P, and L) and catalyzes reactions involving different substrates and cofactors. In vitro kinetics of reconstructed microbial multi-enzyme glycine cleavage/synthase system is desired to better implement rGlyP in microorganisms like Escherichia coli for the use of C1 resources. Here, we examined in vitro several factors that may affect the rate of glycine synthesis via the reverse GCS reaction. We found that the ratio of GCS component proteins has a direct influence on the rate of glycine synthesis, namely higher ratios of P protein and especially H protein to T and L proteins are favorable, and the carboxylation reaction catalyzed by P protein is a key step determining the glycine synthesis rate, whereas increasing the ratio of L protein to other GCS proteins does not have significant effect and the ratio of T protein to other GCS proteins should be kept low. The effect of substrate concentrations on glycine synthesis is quite complex, showing interdependence with the ratios of GCS component proteins. Furthermore, adding the reducing agent dithiothreitol to the reaction mixture not only results in great tolerance to high concentration of formaldehyde, but also increases the rate of glycine synthesis, probably due to its functions in activating P protein and taking up the role of L protein in the non-enzymatic reduction of Hox to Hred. Moreover, the presence of some monovalent and divalent metal ions can have either positive or negative effect on the rate of glycine synthesis, depending on their type and their concentration.

Protein lipoylation is essential for the function of many key enzymes but barely studied kinetically. Here, the two-step reaction cascade of H protein lipoylation catalyzed by the multifunctional enzyme lipoate-protein ligase A (LplA) was quantitatively and differentially studied. We discovered new phenomena and unusual kinetics of the cascade: (a) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate lipoyl-AMP (Lip-AMP); (b) Lip-AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (c) both the lipoylation of H protein and its hydrolysis is enhanced by the apo and lipoylated forms of H protein and a mutant without the lipoylation site. A conceptual mechanistic model is proposed to explain these experimental observations in which conformational change of LplA upon interaction with H protein and competitive nucleophilic attacks play key roles.

Canine distemper virus (CDV) elicits a severe contagious disease in a broad range of hosts. CDV mortality rates are 50% in domestic dogs and 100% in ferrets. Its primary infection sites are respiratory and intestinal mucosa. This study aimed to develop an effective mucosal CDV vaccine using a non-antibiotic marked probiotic pPGΔCm-T7g10-EGFP-H/L. casei 393 strain expressing the CDV H protein. Its immunogenicity in BALB/c mice was evaluated using intranasal and oral vaccinations, whereas in dogs the intranasal route was used for vaccination. Our results indicate that this probiotic vaccine can stimulate a high level of secretory immunoglobulin A (sIgA)-based mucosal and IgG-based humoral immune responses in mice. SIgA levels in the nasal lavage and lungs were significantly higher in intranasally vaccinated mice than those in orally vaccinated mice. Both antigen-specific IgG and sIgA antibodies were effectively elicited in dogs through the intranasal route and demonstrated superior immunogenicity. The immune protection efficacy of the probiotic vaccine was evaluated by challenging the immunized dogs with virulent CDV 42 days after primary immunization. Dogs of the pPGΔCm-T7g10-EGFP-H/L. casei 393 group were completely protected against CDV. The proposed probiotic vaccine could be promising for protection against CDV infection in dogs.

Canine distemper virus (CDV) causes a highly contagious disease in a wide range of carnivores. The hemagglutinin (H) protein of viruses shows the highest variability and plays an important role in modulation of viral antigenicity, virulence, and receptor recognition. Since 2012, canine distemper (CD) outbreaks in fur-bearing animals (minks, foxes, raccoon dogs) caused by CDV variants with I542N and Y549H substitutions in the H protein have been frequently reported in China. To characterize the molecular evolutionary dynamics and epidemiological dynamics of CDV, 235 H gene sequences of CDV wild-type strains collected from 22 countries between 1975 and 2015, including 44 strains predominant in fur-bearing animals in China, were analyzed. The phylogenetic relationships and evolutionary rates of the CDV strains were determined by Bayesian phylogenetics. The CDV strains clustered into distinct geographic genotypes, irrespective of the species of isolation. All the variant strains formed a distinct monophyletic cluster and belonged to the F sub-genotype within the Asia-1 genotype-currently the predominant sub-genotype in fur-bearing animals in China. Evolutionary analysis suggested that the variant strains originated in 2006. Furthermore, the selection pressure analysis revealed that the Y549H substitution was under positive selection pressure for adaptation toward the fur-bearing animals. The residue at position 549 also showed structural interaction with the V domain of the mink signaling lymphocyte-activation molecule (SLAM) receptor based on the homology modeling of the H-SLAM complex. Our results suggested that the Y549H substitution contributed to the molecular adaptation of CDV variants in the fur-bearing animals during the viral evolutionary phase in China.