Use of SLAM and PVRL4 and Identification of Pro-HB-EGF as Cell Entry Receptors for Wild Type Phocine Distemper Virus

Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.     

Canine distemper virus epithelial cell infection is required for clinical disease but not for immunosuppression

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.     

The V Protein of Canine Distemper Virus Is Required for Virus Replication in Human Epithelial Cells

Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.     

Canine Distemper Virus Associated with a Lethal Outbreak in Monkeys Can Readily Adapt To Use Human Receptors

A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.     

Nearby clusters of hemagglutinin residues sustain SLAM-dependent canine distemper virus entry in peripheral blood mononuclear cells

Signaling lymphocytic activation molecule (SLAM, CD150) is the universal morbillivirus receptor. Based on the identification of measles virus (MV) hemagglutinin (H) amino acids supporting human SLAM-dependent cell entry, we mutated canine distemper virus (CDV) H and identified residues necessary for efficient canine SLAM-dependent membrane fusion. These residues are located in two nearby clusters in a new CDV H structural model. To completely abolish SLAM-dependent fusion, combinations of mutations were necessary. We rescued a SLAM-blind recombinant CDV with six mutations that did not infect ferret peripheral blood mononuclear cells while retaining full infectivity in epithelial cells.     

Mesenchymal to epithelial transition driven by canine distemper virus infection of canine histiocytic sarcoma cells contributes to a reduced cell motility in vitro

Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV‐infected cells exhibited an increased expression of epithelial markers such as E‐cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.     

Macrophage Fc receptors control infectivity and neutralization of canine distemper virus-antibody complexes.

Dogs that are persistently infected or that become moribund after exposure to canine distemper virus (CDV) have antibody that neutralized CDV when tested in dog lung macrophage cultures but failed to neutralize CDV when tested in epithelial, fibroblastic, or lymphatic cells. The antibody attached to protein A and was found in the immunoglobulin G fraction. The antibody bound complement and lysed CDV-infected target cells. The neutralizing activity in macrophages could be abolished (i) by pepsin digestion and removal of Fc portions from the antibody, (ii) by blocking the Fc receptors of macrophages with heat-treated normal dog serum, and (iii) by binding of protein A to Fc portions of the antibody. It was concluded that attachment of the CDV-antibody complex to Fc receptors of macrophages was essential for virus neutralization. If this attachment was hindered, the CDV-antibody complex became infectious for macrophages. In contrast, serum from recovering dogs neutralized CDV when tested in epithelial, fibroblastic, or lymphatic cells as well as in macrophages.     

Canine distemper virus utilizes different receptors to infect chicken embryo fibroblasts and vero cells

Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts (CEF) is a common method to develop attenuated live vaccines with full security. Canine distemper virus (CDV) also does this, but the mechanisms and particular receptors remain unclear. Virus overlay protein blot assays were carried out on CEF membrane proteins, which were extracted respectively with a Mem-PER™ kit, a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method, and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells, indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.     

Antagonistic Pleiotropy and Fitness Trade-Offs Reveal Specialist and Generalist Traits in Strains of Canine Distemper Virus

Theoretically, homogeneous environments favor the evolution of specialists whereas heterogeneous environments favor generalists. Canine distemper is a multi-host carnivore disease caused by canine distemper virus (CDV). The described cell receptor of CDV is SLAM (CD150). Attachment of CDV hemagglutinin protein (CDV-H) to this receptor facilitates fusion and virus entry in cooperation with the fusion protein (CDV-F). We investigated whether CDV strains co-evolved in the large, homogeneous domestic dog population exhibited specialist traits, and strains adapted to the heterogeneous environment of smaller populations of different carnivores exhibited generalist traits. Comparison of amino acid sequences of the SLAM binding region revealed higher similarity between sequences from Canidae species than to sequences from other carnivore families. Using an in vitro assay, we quantified syncytia formation mediated by CDV-H proteins from dog and non-dog CDV strains in cells expressing dog, lion or cat SLAM. CDV-H proteins from dog strains produced significantly higher values with cells expressing dog SLAM than with cells expressing lion or cat SLAM. CDV-H proteins from strains of non-dog species produced similar values in all three cell types, but lower values in cells expressing dog SLAM than the values obtained for CDV-H proteins from dog strains. By experimentally changing one amino acid (Y549H) in the CDV-H protein of one dog strain we decreased expression of specialist traits and increased expression of generalist traits, thereby confirming its functional importance. A virus titer assay demonstrated that dog strains produced higher titers in cells expressing dog SLAM than cells expressing SLAM of non-dog hosts, which suggested possible fitness benefits of specialization post-cell entry. We provide in vitro evidence for the expression of specialist and generalist traits by CDV strains, and fitness trade-offs across carnivore host environments caused by antagonistic pleiotropy. These findings extend knowledge on CDV molecular epidemiology of particular relevance to wild carnivores.     

Ablation of Nectin4 Binding Compromises CD46 Usage by a Hybrid Vesicular Stomatitis Virus/Measles Virus

Measles virus (MV) immunosuppression is due to infection of SLAM-positive immune cells, whereas respiratory shedding and virus transmission are due to infection of nectin4-positive airway epithelial cells. The vaccine lineage MV strain Edmonston (MV-Edm) acquired an additional tropism for CD46 which is the basis of its oncolytic specificity. VSVFH is a vesicular stomatitis virus (VSV) encoding the MV-Edm F and H entry proteins in place of G. The virus spreads faster than MV-Edm and is highly fusogenic and a potent oncolytic. To determine whether ablating nectin4 tropism from VSVFH might prevent shedding, increasing its safety profile as an oncolytic, or might have any effect on CD46 binding, we generated VSVFH viruses with H mutations that disrupt attachment to SLAM and/or nectin4. Disruption of nectin4 binding reduced release of VSVFH from the basolateral side of differentiated airway epithelia composed of Calu-3 cells. However, because nectin4 and CD46 have substantially overlapping receptor binding surfaces on H, disruption of nectin4 binding compromised CD46 binding and greatly diminished the oncolytic potency of these viruses on human cancer cells. Thus, our results support continued preclinical development of VSVFH without ablation of nectin4 binding.     

犬瘟热病毒基因变异及其细胞受体研究进展

由犬瘟热病毒(Canine distemper virus,CDV)引起的犬瘟热(CD)一直是对世界养犬业、毛皮动物养殖业以及野生动物保护事业危害最为严重的传染病之一,甚至在广泛应用疫苗防制CD的近年,世界各地仍有犬或野生动物感染CDV并造成CD流行的报道.通过对CDV主要抗原基因--血凝基因(H基因)氨基酸序列分析,CDV可分为6个基因型.研究证实,CDV野毒株在抗原性上与疫苗株存在较大差异,因此推测H蛋白抗原变异造成了弱毒疫苗免疫效力的降低进而导致了某些地区CD的爆发.CDV作为一种宿主范围广泛的病毒,其细胞受体--信号淋巴细胞激活因子(SLAM)和硫酸乙酰肝素(HS)在哺乳动物体内的广泛存在是CDV跨物种间感染的重要因素.本文就国内外最新研究进展并结合作者的工作,对上述问题进行了综述和探讨.     

Prominent role of the Ig-like V domain in trans-interactions of nectins. Nectin3 and nectin 4 bind to the predicted C-C'-C"-D beta-strands of the nectin1 V domain

Nectins form a family of integral molecules that belong to the immunoglobulin superfamily. Their ectodomain is made of three Ig-like domains (V, C, C). This family comprises at least five members, namely nectin1, -2, -3, -4, and poliovirus receptor (PVR), that are involved in different physiological and pathological processes. (i) Nectins are adhesion molecules localized at adherens junctions in epithelial cells. (ii) Some nectins act as poliovirus or alpha-herpesvirus receptors (nectin1). (iii) Nectin1 mutations are involved in orofacial developmental abnormalities in humans. Adhesion properties of nectins are mediated by Ca(2+)-independent homophilic and heterophilic processes through ectodomain trans-interactions. We have described a nectin trans-hetero-interaction network: nectin3 binds to nectin1, nectin2, and PVR; nectin1 also binds to nectin4. In the present study we compared the affinities of the different trans-interactions mediated by nectin1. We found that the K(D) of nectin1/nectin3 and nectin1/nectin4 interactions is 1 and 100 nm, respectively, whereas the K(D) of the nectin1-mediated homophilic interaction is 1 microm. We show that nectin1/nectin3 and nectin1/nectin4 trans-hetero-interactions were mediated through trans V to V domain interactions, whereas C domains contributed to increase the affinity of the interaction. Nectin3 and nectin4 share a common binding region in the nectin1 V domain: (i) nectin3 strongly competed with nectin4 binding, (ii) nectin3 and nectin4 binding to nectin1 was reduced by a number of monoclonal antibodies directed against the nectin1 V domain, and (iii) the glycoprotein D of herpes simplex virus-1 that binds to the V domain of nectin1 reduced nectin3 and nectin4 binding. Finally, using chimeric nectin1/PVR receptors where PVR V domain beta-strands were substituted with the corresponding regions of nectin1, the nectin3 and nectin4 minimal binding region on nectin1 V domain was mapped to the C-C'-C"-D beta-strands.     

Vero及Vero-dst细胞对犬瘟热病毒敏感性比较

目的为了更好地分离犬瘟热病毒（CDV）并确诊犬瘟热,本实验比较了Vero及Vero-dst细胞对此病毒的敏感性。方法将CDV标准毒株Snyder Hill株及临床犬瘟热阳性犬组织匀浆分别接种Vero及Vero-dst两种细胞,通过观察细胞病变、检测病毒滴度（TCID50）,并通过RT-PCR法进行比较,分析两种细胞对CDV的敏感性。结果接种病毒后Vero细胞盲传5代始终未见细胞病变,而Vero-dst细胞12 h出现了明显的合胞样细胞病变,且RT-PCR扩增出了CDV基因特异性片段。结论 Vero-dst细胞对CDV表现了良好的敏感性,是体外分离培养CDV的一个有效细胞系。而所本实验中使用的Vero细胞并不适于CDV的分离与培养。另外,本实验利用Vero-dst细胞从临床犬瘟热阳性病例中成功分离到了野毒株,并确定其毒力较标准毒株毒力强,可用于进一步的研究。     

Tyzzer's disease as a complication of canine distemper in a raccoon

Intercurrent canine distemper and Tyzzer's disease were diagnosed in a mature raccoon (Procyon lotor) submitted for necropsy. Clinical, gross and microscopic findings characteristic of canine distemper virus (CDV), included ataxia, dyspnea, suppurative conjunctivitis, interstitial pneumonitis and generalized lymphocytolysis. Inclusion bodies typical of CDV infection were present in many epithelial tissues. Acute multifocal hepatic necrosis and acute segmental necrotizing enteritis were attributed to the presence of Bacillus piliformis organisms in these lesions, confirmed by special stains and electron microscopy. This is apparently the first reported case of Tyzzer's disease in a raccoon.     

Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion

Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.     

Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM (CD150) and their adaptability to marmoset B95a cells

We have previously shown that canine signaling lymphocyte activation molecule (SLAM; also known as CD150) acts as a cellular receptor for canine distemper virus (CDV). In this study, we established Vero cells stably expressing canine SLAM (Vero.DogSLAMtag cells). Viruses were isolated in Vero.DogSLAMtag cells one day after inoculation with spleen samples from five out of seven dogs with distemper. By contrast, virus isolation with reportedly sensitive marmoset B95a cells was only successful from three diseased animals at 7 to 10 days after inoculation, and no virus was recovered from any dogs when Vero cells were used for isolation. The CDV strain isolated in Vero.DogSLAMtag cells did not cause cytopathic effects in B95a and human SLAM-expressing Vero cells, whereas the strain isolated in B95a cells from the same dog did so in canine or human SLAM-expressing Vero cells as well as B95a cells. There were two amino acid differences in the hemagglutinin sequence between these strains. Cell fusion analysis after expression of envelope proteins and vesicular stomatitis virus pseudotype assay showed that their hemagglutinins were responsible for the difference in cell tropism between them. Site-directed mutagenesis indicated that glutamic acid to lysine substitution at position 530 of the hemagglutinin was required for the adaptation to the usage of marmoset SLAM. Our results indicate that Vero cells stably expressing canine SLAM are highly sensitive to CDV in clinical specimens and that only a single amino acid substitution in the hemagglutinin can allow the virus to adapt to marmoset SLAM.     

Canine distemper virus infection requires cholesterol in the viral envelope

Cholesterol is known to play an important role in stabilizing particular cellular membrane structures, so-called lipid or membrane rafts. For several viruses, a dependence on cholesterol for virus entry and/or morphogenesis has been shown. Using flow cytometry and fluorescence microscopy, we demonstrate that infection of cells by canine distemper virus (CDV) was not impaired after cellular cholesterol had been depleted by the drug methyl-beta-cyclodextrin. This effect was independent of the multiplicity of infection and the cellular receptor used for infection. However, cholesterol depletion of the viral envelope significantly reduced CDV infectivity. Replenishment by addition of exogenous cholesterol restored infectivity up to 80%. Thus, we conclude that CDV entry is dependent on cholesterol in the viral envelope. Furthermore, reduced syncytium formation was observed when the cells were cholesterol depleted during the course of the infection. This may be related to the observation that CDV envelope proteins H and F partitioned into cellular detergent-resistant membranes. Therefore, a role for lipid rafts during virus assembly and release as well is suggested.     

Recombinant canine distemper virus strain Snyder Hill expressing green or red fluorescent proteins causes meningoencephalitis in the ferret

The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDV(SH)) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDV(SH) (rCDV(SH)) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV(5804P) and the prototypic wild-type CDV(R252) showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDV(SH)-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis.     

CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus.

Canine distemper virus (CDV), a lymphotropic and neurotropic negative-stranded RNA virus of the Morbillivirus genus, causes a life-threatening disease in several carnivores, including domestic dogs. To identify the cellular receptor(s) involved in the uptake of CDV by susceptible cells, we isolated a monoclonal antibody (MAb K41) which binds to the cell surface and inhibits the CDV infection of several cell lines from various species. Pretreatment of cells with MAb K41 reduces the number of infectious centers and the size of the syncytia. Using affinity chromatography with MAb K41, we purified from HeLa and Vero cell extracts a 26-kDa protein which contained the amino acid sequence TKDEPQRETLK of human CD9, a member of the tetraspan transmembrane or transmembrane 4 superfamily of cell surface proteins. Transfection of NIH 3T3 or MDBK cells with a CD9 expression plasmid rendered these cells permissive for viral infection and raised virus production by a factor of 10 to 100. The mechanism involved is still unclear, since we were unable to detect direct binding of CDV to CD9 by using immunoprecipitation and a virus overlay protein binding assay. These findings indicate that human CD9 and its homologs in other species are necessary factors for the uptake of CDV by target cells, the formation of syncytia, and the production of progeny virus.