Role of antibodies and host cells in plaque enhancement of Murray Valley encephalitis virus.

Chicken antisera to Murray Valley encephalitis (MVE) virus, when incubated with virus and assayed for plaques on chicken embryo (CE) monolayers, neutralized MVE virus at high concentrations of antibody, but caused increases in plaque counts at low concentrations of antibody. Plaque enhancement did not occur when the same virus-antibody mixtures were assayed on a continuous line of rhesus monkey kidney cells (LLC-MK2), nor when the anti-MVE antibody was of mammalian origin and the assay system was CE monolayers. Correspondingly, chicken anti-MVE did not enhance the plaque formation of MVE virus in a stable line of mouse macrophages, P-388D1, whereas rabbit and mouse anti-MVE did enhance plaque formation. This enhancing activity was associated with noncytophilic immunoglobulin G (IgG). The Fc terminus of the IgG molecule was required, as no plaque enhancement occurred with chicken anti-MVE Fab. These data indicate that there is a requirement for taxonomic complementarity between Fc termini and Fc receptors in the above systems. CE cell monolayers were found to contain approximately 2% of Fc receptor-bearing cells among the fibroblast-like cells. Fc receptor-bearing cells in CE monolayers were isolated and found to be of the mononuclear phagocytic lineage. These mononuclear phagocytes, which originate in lymphoid tissues and blood associated with CE tissue fragments, are integrated into primary CE monolayers and form infectious centers in the presence of virus and low dilutions of antibody.     

Nectin4 is an epithelial cell receptor for canine distemper virus and involved in neurovirulence

Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.     

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.     

Antibody-mediated growth of influenza A NWS virus in macrophagelike cell line P388D1

We investigated the internalization and growth of influenza A NWS virus in macrophagelike P388D1 cells. Flow cytometric analysis using fluorescein isothiocyanate-labeled virus showed that the attachment of normal rabbit serum-exposed virus (NS-V) to neuraminidase (NA)-treated cells was noticeably limited compared with that to untreated cells. However, rabbit antiserum-exposed virus (AS-V) could attach equally well to both cells. Virus coated with Fab prepared from antiviral immunoglobulin G could not attach. These data suggest that the NWS virus can infect P388D1 cells in one of two ways, via viral or via Fc receptors, depending on the presence of antibodies. The NS-V could grow in the untreated cells, but not in the NA-treated cells. The highest growth of AS-V in the NA-treated cells was observed at an antibody concentration showing 50% plaque reduction titer. Growth was exponentially decreased toward the lower and higher dilutions of antibodies. By using three different immunoglobulin G subclasses of monoclonal antibodies against hemagglutinin, it was demonstrated that both Fc receptors I and II could take part in this phenomenon. The presence of 20 mM NH4Cl inhibited the growth of both AS-V and NS-V, suggesting that the intracellular pathways after internalization via Fc or viral receptors are similar. These data indicate that the concentration of antibodies has a critical role on the antibody-mediated growth of influenza virus in macrophages.     

Monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages.

Antibody-dependent enhancement of virus infection is a process whereby virus-antibody complexes initiate infection of cells via Fc receptor-mediated endocytosis. We sought to investigate antibody-dependent enhancement of feline infectious peritonitis virus infection of primary feline peritoneal macrophages in vitro. Enhancement of infection was assessed, after indirect immunofluorescent-antibody labelling of infected cells, by determining the ratio between the number of cells infected in the presence and absence of virus-specific antibody. Infection enhancement was initially demonstrated by using heat-inactivated, virus-specific feline antiserum. Functional compatibility between murine immunoglobulin molecules and feline Fc receptors was demonstrated by using murine anti-sheep erythrocyte serum and an antibody-coated sheep erythrocyte phagocytosis assay. Thirty-seven murine monoclonal antibodies specific for the nucleocapsid, membrane, or spike proteins of feline infectious peritonitis virus or transmissible gastroenteritis virus were assayed for their ability to enhance the infectivity of feline infectious peritonitis virus. Infection enhancement was mediated by a subset of spike protein-specific monoclonal antibodies. A distinct correlation was seen between the ability of a monoclonal antibody to cause virus neutralization in a routine cell culture neutralization assay and its ability to mediate infection enhancement of macrophages. Infection enhancement was shown to be Fc receptor mediated by blockade of antibody-Fc receptor interaction using staphylococcal protein A. Our results are consistent with the hypothesis that antibody-dependent enhancement of feline infectious peritonitis virus infectivity is mediated by antibody directed against specific sites on the spike protein.     

Antiviral antibodies stimulate production of reactive oxygen species in cultured canine brain cells infected with canine distemper virus.

Canine distemper is characterized mainly by respiratory, enteric, and nervous symptoms. Infection of the central nervous system results in demyelination, to which inflammation has been shown to contribute significantly. It has been proposed that macrophages play a major role as effector cells in this process. We report that cultured dog brain cells contain a population of macrophages capable of producing reactive oxygen species as measured by luminol-dependent chemiluminescence. In cultures infected with canine distemper virus, a burst of reactive oxygen is triggered by antiviral antibody. This response depends on the presence of viral antigens on the surfaces of infected cells and is mediated by the interaction of antigen-bound antibody with Fc receptors on the macrophages. Since there is no evidence in vitro or in vivo that oligodendrocytes, the cells forming myelin, are infected, our observation supports the hypothesis that "innocent bystander killing" is important in demyelination caused by canine distemper virus. Reactive oxygen species released from macrophages may contribute to destruction of myelin.     

Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.     

Serologic survey for antibodies to canine distemper virus in collared peccary (Tayassu tajacu) populations in Arizona

In 1989, a disease outbreak was observed among collared peccaries (javelina, Tayassu tajacu) in southern Arizona (USA) and canine distemper virus (CDV) was isolated from affected animals. Subsequently, 364 sera were collected from hunter-harvested javelina over a 4 yr period (1993-96) and were tested for antibody to CDV. Neutralizing antibody to CDV was detected in 58% of the serum samples suggesting that CDV infection is probably enzootic in the collared peccary populations of southern Arizona.     

A new plaque system for canine distemper: characteristics of the green strain of canine distemper virus.

A Vero cell adapted Green strain of canine distemper virus (CDV) was tested for its plaque-forming capacity in different cell lines. Plaque formation was observed in HEp-2, BS-C-1, and HeLa cells but not in Vero or dog kidney cells even though replication and cytopathology were observed in the latter cell types. In the cells in which the virus was capable of producing plaques, the plaques were observed within 24 h post infection and continued to increase in size with subsequent cellular destruction such that by 72 h postinfection the size of the plaques approached 0.5 mm. With the use of the plaquing technique, it was possible to demonstrate the thermal lability of the virus as well as the kinetics of adsorption. Thus, it was shown that the half-life of the virus was 125 min at 25 degrees C, 75 min at 35 degrees C, and 65 min at 37 degrees C. The rate of adsorption of CDV to HEp-2 cells was 17.2% in 30 min at 37 degrees C and continued slowly for 4 h before completion. Application of this rapid plaque-forming assay to plaque-reduction tests for CDV antibody and for CDV-infected cells by the infectious center assay are described.     

Canine distemper virus infects canine keratinocytes and immune cells by using overlapping and distinct regions located on one side of the attachment protein

The morbilliviruses measles virus (MeV) and canine distemper virus (CDV) both rely on two surface glycoproteins, the attachment (H) and fusion proteins, to promote fusion activity for viral cell entry. Growing evidence suggests that morbilliviruses infect multiple cell types by binding to distinct host cell surface receptors. Currently, the only known in vivo receptor used by morbilliviruses is CD150/SLAM, a molecule expressed in certain immune cells. Here we investigated the usage of multiple receptors by the highly virulent and demyelinating CDV strain A75/17. We based our study on the assumption that CDV-H may interact with receptors similar to those for MeV, and we conducted systematic alanine-scanning mutagenesis on CDV-H throughout one side of the β-propeller documented in MeV-H to contain multiple receptor-binding sites. Functional and biochemical assays performed with SLAM-expressing cells and primary canine epithelial keratinocytes identified 11 residues mutation of which selectively abrogated fusion in keratinocytes. Among these, four were identical to amino acids identified in MeV-H as residues contacting a putative receptor expressed in polarized epithelial cells. Strikingly, when mapped on a CDV-H structural model, all residues clustered in or around a recessed groove located on one side of CDV-H. In contrast, reported CDV-H mutants with SLAM-dependent fusion deficiencies were characterized by additional impairments to the promotion of fusion in keratinocytes. Furthermore, upon transfer of residues that selectively impaired fusion induction in keratinocytes into the CDV-H of the vaccine strain, fusion remained largely unaltered. Taken together, our results suggest that a restricted region on one side of CDV-H contains distinct and overlapping sites that control functional interaction with multiple receptors.     

Phagocytosis in rat Kupffer cells in vitro

Phagocytosis was studied in rat Kupffer cells in vitro by using opsonized sheep red cells as objects and inducing attachment and ingestion through the Fc and C₃ receptors. The Fc receptors functioned by and large in the same manner as in the peritoneal macrophages. When the red cells were opsonized with IgM and complement, there was attachment but little ingestion in a serum-free medium. Newborn calf serum was found to trigger ingestion. Our experiments provided no conclusive evidence as to the nature of this triggering mechanism. The limiting factor in phagocytosis was the cytoplasmic volume of the phagocyte rather than the availability of surface receptors. The expression of surface receptors on cells in culture depended on length of culture and degree of spreading. We confirmed the available information on the energy requirements of phagocytosis as studied in peritoneal macrophages. As judged by isotope release, digestion of the red cells was in process shortly after ingestion. However, morphological examination failed to detect any changes in appearance prior to 4 h. After a blocking dose of sheep red cells, a rather long period (40 h) was required before cells fully recovered their phagocytic capacity.     

Mice immunized with measles virus develop antibodies to a cell surface receptor for binding virus.

Mice were immunized with measles virus to determine whether an auto-anti-idiotypic antireceptor response could be generated as a probe for measles virus receptors. Mice initially responded to viral antigens (days 11 to 18) and subsequently developed antibodies to a putative measles virus receptor (peak at day 30 to 35) by three criteria: the sera (1) agglutinated erythrocytes which virus agglutinates, (2) reacted with Vero cells, and (3) inhibited virus attachment to Vero cells. Additionally, select sera inhibited virus infection of Vero cells. The cell-reactive activity was identified as immunoglobulin G antibody and was neutralized by sera reacting with virus (idiotype). The application of this anti-idiotypic antibody to identify measles virus-binding sites on Vero cells was revealed by the ability of sera to immunoprecipitate 20- and 30.5-kilodalton proteins from metabolically labeled ([35S]methionine) Vero cells.     

犬瘟热病毒（CDV）ELISA检测方法的建立与应用

目的建立犬CDV抗体ELISA检测方法。方法培养vero细胞,接种CDV病毒,制备vero正常抗原和CDV特异抗原,滴定酶结合物和抗原最佳工作浓度,并进行精密性、敏感性、稳定性、特异性实验。结果正常、特异抗原和酶结合物最佳工作浓度分别为1∶32 000、10μg/mL和1∶8 000;正常、特异抗原批内变异系数分别为9.1%和5.8%,批间平均变异系数分别为8.8%和6.6%;检测灵敏度为1∶2 560;与犬细小病毒（CPV）、犬肝炎病毒（ICHV）均无交叉反应。稳定性试验相对偏差小于25%。结论建立的ELISA方法重复性、稳定性好,特异性、敏感性强。可用于犬CDV抗体的检测。     

Modulation of lentivirus replication by antibodies: Fc portion of immunoglobulin molecule is essential for enhancement of binding, internalization, and neutralization of visna virus in macrophages.

Antibodies to visna virus neutralized the virus in fibroblasts and macrophages but specifically enhanced the binding, penetration, and uncoating of the virus in the latter cells. F(ab')2 fragments of the immune antibody neutralized the virus in fibroblasts but did not enhance the early stages of the virus life cycle in macrophages. Furthermore, these fragments did not neutralize infectivity in macrophages but delayed the appearance of infectious virus in cells after the inoculation of preincubated virus-F(ab')2 complexes.     

Receptor (SLAM [CD150]) recognition and the V protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by a morbillivirus

Experimental infections of ferrets with canine distemper virus (CDV) recapitulate many hallmarks of measles: rash, high fever, viremia, depression of delayed-type hypersensitivity responses, lowered leukocyte counts, and reduced lymphocyte proliferation activity. To understand how a morbillivirus invades the host and causes immunosuppression, we generated CDV either unable to recognize one of the receptors or incapable of expressing either one or both of the candidate interferon antagonist proteins V and C. Variants of these viruses expressing green fluorescent protein were also generated. Striking similarities between CDV infection of ferrets and human immunodeficiency virus host invasion were documented: first, massive early replication in the gut-associated lymphatic tissue, including intestinal Peyer's patches, followed by extensive infection of lymphatic organs, including thymus and circulating lymphocytes. Moreover, T cells were selectively depleted. Thus, CDV takes advantage of mucosal surfaces for host invasion and lymphocytes for swift dissemination. A CDV unable to recognize the signaling lymphocytic activation molecule (SLAM [CD150]) that is expressed in lymphocytes and other immune cells did not spread. A V-defective CDV multiplied with reduced efficiency in lymphocytes and did not inhibit the interferon and cytokine responses. Protein C affected the severity of rash and digestive symptoms elicited by V-defective CDV, but it was dispensable for the invasion of the lymphatic organs. These findings prove formally that SLAM recognition is necessary for morbillivirus virulence. They also reveal how two viral proteins affect pathogenesis: V sustains the swift lymphocyte-based invasion of mucosal tissue and lymphatic organs, whereas C sustains subsequent infection phases.     

Morbillivirus ecology in polar bears (Ursus maritimus)

Polar bear (Ursus maritimus) morbillivirus infection was initially reported by Follmann and co-workers in 1996, based upon serologic results using canine distemper virus (CDV). The impetus for the evaluation of polar bear populations for morbillivirus infections was prompted by epidemics of canine distemper-like disease in seal populations in the north Atlantic regions of Greenland, Europe, and Russia. Since marine morbilliviruses have been further characterized into three major species, phocine distemper virus (PDV), dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV), it was of value to determine the origin of the polar bear infection. One hundred serum samples were selected from a group of sera collected from regions of Alaska and Russia and tested by differential serum neutralization assay against the three major marine morbilliviruses and CDV, to determine the predominant virus infecting the polar bear. Polar bears had higher serum antibody titers to CDV than they did to PDV, DMV, and PMV. These data suggest that polar bears are being infected with a morbillivirus of terrestrial origin. Furthermore, based on the high serum antibody prevalence in the population, the virus may be indigenous to the polar bear and not necessarily the result of interspecies transmission from other arctic mammals susceptible to CDV and/or marine morbilliviruses. Accepted: 20 December 1999     

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.     

Cutting Edge: The murine high-affinity IgG receptor FcγRIV is sufficient for autoantibody-induced arthritis

K/BxN serum-induced passive arthritis was reported to depend on the activation of mast cells, triggered by the activating IgG receptor FcγRIIIA, when engaged by IgG1 autoantibodies present in K/BxN serum. This view is challenged by the fact that FcγRIIIA-deficient mice still develop K/BxN arthritis and because FcγRIIIA is the only activating IgG receptor expressed by mast cells. We investigated the contribution of IgG receptors, IgG subclasses, and cells in K/BxN arthritis. We found that the activating IgG2 receptor FcγRIV, expressed only by monocytes/macrophages and neutrophils, was sufficient to induce disease. K/BxN arthritis occurred not only in mast cell-deficient W(sh) mice, but also in mice whose mast cells express no activating IgG receptors. We propose that at least two autoantibody isotypes, IgG1 and IgG2, and two activating IgG receptors, FcγRIIIA and FcγRIV, contribute to K/BxN arthritis, which requires at least two cell types other than mast cells, monocytes/macrophages, and neutrophils.     

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.