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.     

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.     

Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.     

Studies on the interaction between coxsackievirus A9 and HeLa cells. I. Plaque-forming ability of coxsackievirus A9 in HeLa cell cultures.

Most of the coxsackievirus A9 (CA 9 virus) including the prototype strain formed plaques in HeLa cell monolayers under agar overlay, although they showed little or no cytopathogenicity under fluid medium. These viruses were isolated or passaged in primary cynomolgus monkey kidney (MK) cell cultures, and the infectivity of any strain in terms of plaque-forming units was much higher in MK cells than in HeLa cells, even after plaque purification of the virus in HeLa cell cultures. CA 9 virus contained in the original throat swabs as well as some clones obtained by plaque purification in MK cells failed to form plaques in HeLa cells, but virus preparations obtained after several undiluted passages through MK cells included plaque-formers in HeLa cells, suggesting that such plaque (HeLa)-forming viruses may have developed at a certain rate during multiplication of the original non-plaque (HeLa)-forming virus population in MK cells. Out of four lines of HeLa cells examined, two, including a clonal line S3, failed to support plaque formation by CA 9 virus.     

Properties of influenza C virus grown in cell culture.

Influenza C virus was propagated successfully in primary chicken embryo lung (CEL) and fibroblast cells and in Madin-Darby canine kidney (MDCK) cells. In other cell lines, either no virus or only noninfectious hemagglutinin (HA) was produced. In productively infected cells (CEL), HA and infectious virus appeared by 24 h and reached a maximum by 36 to 48 h, cell-associated virus remaining at a constant low level. Infected Vero cells produced noninfective HA by 24 h which also remained predominantly cell associated until 60 to 72 h, when the cells disintegrated. Viral antigen was demonstrable on membranes of both CEL- and Vero-infected cells at 24 h; Vero cells yielded membrane vesicles containing HA, but none of the spherical or filamentous viral particles synthesized in CEL cells. Influenza C virus produced in cell culture or in eggs differed in several important respects from A and B viruses and from Newcastle diseases virus. All influenza C preparations, regardless of infectivity or source, lacked detectable neuraminidase activity, yet retained the ability specifically to inactivate receptors only for influenza C. Influenza C HA was not inhibited by soluble glycoproteins highly active against HA of A virus. A rat serum glycoprotein uniquely inhibited influenza C by binding to the surface components of virious.     

Inhibition of Herpes Virus-Induced Cell Fusion by Concanavalin A, Antisera, and 2-Deoxy-d-Glucose

Pseudorabies virus-induced cell fusion in rabbit kidney cells can be prevented by Concanavalin A added early after infection. The infected cells are not agglutinated and the infectivity of cell-free virus is not reduced. Sera from productively infected animals also inhibit polykaryocytosis, whereas a hyperimmune serum directed against virus structural components has no effect. 2-Deoxy-d-glucose reversibly disturbs virus-induced fusion and reduces significantly the virus infectivity.     

Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion

Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected.     

Priming: a Nonantiviral Function of Interferon

No interferon is made by L cells when they are infected with MM virus. However, several thousand units of interferon are produced when interferon-treated L cells are infected with MM virus. We call the conversion of cells, from nonproducers to producers, priming. The time required for cells to become fully primed is dependent on the interferon concentration with which they are incubated. Primed cells produced interferon earlier than normal cells stimulated by other inducers. Cells which were exposed to interferon in the presence of inhibitors of protein synthesis became fully primed yet developed no virus resistance. Also, primed cells produced interferon in response to low concentrations of polyriboinosinic acid . polyribocytidylic acid that did not induce interferon in normal cells. Therefore, priming appears to be a function of interferon separable from its antiviral activity. Several other picornaviruses that failed to induce interferon in L cells, human embryonic lung cells, or monkey kidney cells did induce interferon when these cells had been primed by homologous interferons.     

Growth of Murine Cytomegalovirus in Various Cell Lines

Murine cytomegalovirus (MCMV) was capable of infecting and replicating in both primary and continuous cell lines obtained from various species. In African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells, there were cytopathic effects (CPE) and virus replication upon initial exposure of cells to virus. In primary fetal sheep brain (FSB) cells, L cells, and rabbit kidney (RK-13) cells, it was necessary to subculture the infected cells one or more times before appearance of CPE and replication of virus. In the case of the infected FSB cultures, it was found that the virus effect could be induced if subculturing were accomplished by trypsinization but did not occur if cells were subcultured by scraping. FSB-grown virus replicated better in FSB than in mouse embryo fibroblast (MEF) cells. The CPE produced in all of the above cell lines was similar to that observed in MEF infected with MCMV. The virus grown in different cell lines was completely neutralized when mixed with several reference sera prepared in rabbits or mice. The populations of virions released from infected MEF and FSB cells were compared by isopycnic centrifugation in potassium tartrate, and no differences were revealed in the buoyant densities of the populations. Human embryonic brain cells, human embryonic kidney cells, a human lung fibroblast cell strain (WI-38), HeLa, and Hep-2 were not susceptible to MCMV.     

Effects of temperature on virus-host relationships and on activity of the noninclusion virus of citrus red mites, Panonychus citri

The nonoccluded virus of citrus red mite retained full infectivity when exposed to 40.5°C for 24 hr within intact mite bodies but was inactivated at 46°C for 6 hr and 60°C for 1 hr. Exposures to 38°C for 28 days failed to destroy infectivity. Virus inoculated mites exposed to different temperature regimens had shortened periods of lethal infection at high temperatures and greatly lengthened periods of lethal infection at cool temperatures suggesting that failures in mite control by virus in the early spring and late fall may be due to previously unrecognized temperature relationships.     

Translation of adenovirus 2 late mRNAs microinjected into cultured African green monkey kidney cells.

Adenovirus 2-infected monkey cells fail to synthesize fiber, a 62,000 Mr virion polypeptide expressed at late times in productively infected cells. Yet these cells contain fiber mRNA that, after isolation, can be translated in vitro. The reason for the failure of monkey cells to translate fiber mRNA has been approached by microinjecting adenovirus mRNA into the cytoplasm of cultured monkey cells. Late adenovirus 2 mRNA, isolated from infected HeLa cells, was efficiently expressed when microinjected into the African green monkey kidney cell line CV-C. Expressed viral proteins identified by immunoprecipitation included the adenovirus fiber polypeptide. This result demonstrates that the monkey cell translational apparatus is capable of recognizing and expressing functional adenovirus fiber mRNA. Microinjection of late virus mRNA into cells previously infected with wild-type adenovirus 2 failed to increase significantly the yield of infectious virus.     

Simultaneous Use of Contrasting Fluorochromes to Separate Measles and Canine Distemper Viruses in a Common System

Measles and canine distemper viruses were grown together in a Vero monkey kidney cell line. Each virus could be identified and individually titrated by using the color contrast produced by the reddish tetra-methyl rhodamine isothiocyanate-tagged antimeasles conjugate and the green fluorescein isothiocyanate-tagged antidistemper conjugate. Both blue light and green light were used for the excitation of the fluorochromes. Incident light was transmitted to the specimen by a vertical illuminator of the Ploem type.     

Establishment of a persistent mutant of canine distemper virus

We produced a B95a lymphoid cell line persistently infected with canine distemper virus (CDV), in which virus-specific antigens were present in nearly 100% of cells without causing cytopathic effect. The virus recovered from this cell line was able to infect fresh B95a cells persistently, indicating that a persistent CDV was established.     

Morbilliviruses use signaling lymphocyte activation molecules (CD150) as cellular receptors

Morbilliviruses comprise measles virus, canine distemper virus, rinderpest virus, and several other viruses that cause devastating human and animal diseases accompanied by severe immunosuppression and lymphopenia. Recently, we have shown that human signaling lymphocyte activation molecule (SLAM) is a cellular receptor for measles virus. In this study, we examined whether canine distemper and rinderpest viruses also use canine and bovine SLAMs, respectively, as cellular receptors. The Onderstepoort vaccine strain and two B95a (marmoset B cell line)-isolated strains of canine distemper virus caused extensive cytopathic effects in normally resistant CHO (Chinese hamster ovary) cells after expression of canine SLAM. The Ako vaccine strain of rinderpest virus produced strong cytopathic effects in bovine SLAM-expressing CHO cells. The data on entry with vesicular stomatitis virus pseudotypes bearing measles, canine distemper, or rinderpest virus envelope proteins were consistent with development of cytopathic effects in SLAM-expressing CHO cell clones after infection with the respective viruses, confirming that SLAM acts at the virus entry step (as a cellular receptor). Furthermore, most measles, canine distemper, and rinderpest virus strains examined could any use of the human, canine, and bovine SLAMs to infect cells. Our findings suggest that the use of SLAM as a cellular receptor may be a property common to most, if not all, morbilliviruses and explain the lymphotropism and immunosuppressive nature of morbilliviruses.     

Isolation of canine rotavirus and study of its immunobiological properties]

Canine rotavirus was isolated in MA104 roller culture of rhesus macaque cells. Two passages in gnotobiotic puppies and two in colostrum-free puppies resulted in isolation of strain P of canine rotavirus. After 20 passages in MA104 culture the virus was adapted to MDCK culture. Optimal conditions for accumulation of canine rotavirus and its antigen (9.01 g TCD50/ml) in MDCK culture are trypsin pretreatment of the virus inoculate in the final concentration of 50 mcg/ml for 30 min at 37 degrees C, presence of trypsin (10 mcg/ml) in the maintenance medium, multiplicity of infection 0.1 TCD50/ml, and incubation in roller culture at 37 degrees C during 24-30 h. After 60 passages in cell culture, canine rotavirus completely lost its virulence for colostrum-free puppies but retained antigenic activity and induced manifest seroconversion in infected.     

Powassan Virus: Morphology and Cytopathology

Powassan virus, a North American tickborne group B arbovirus, multiplied after simultaneous inoculation into bottles or tubes of virus and trypsinized suspension of continuous-line cultures of rhesus monkey kidney cells, strain LLC-MK2. Cytopathic effects comprising cell rounding and cytoplasmic vacuolation were first observed five days after inoculation. Mixture of Powassan antiserum with virus before inoculation into tissue cultures inhibited the appearance of cytopathic effects. Hemagglutinins for rooster erythrocytes, optimally at pH 6.4 and 22° C., first appeared in tissue culture supernatant fluids four days after inoculation.Electron microscopic observation of thin sections of infected tissue culture cells showed virus particles 360-380 A.U. along outer cell membranes and edges of cytoplasmic vacuoles. In phosphotungstic acid negatively stained preparations, intact virus particles, 400-450 A.U. total diameter, were observed inside infected cells. In particles in which the peripheral layer became discontinuous, geometrically arranged subunits compatible with cubic symmetry were observed.     

Synthesis and Cleavage of Influenza Virus Proteins

The NWS strain of influenza virus grows rapidly in and kills the MDCK dog kidney cell strain. Within 1 to 2 hr, the virus inhibits host cell protein synthesis and for 3 to 4 hr more it directs the synthesis of influenza virus proteins at a rate about twice that of uninfected cell synthesis. The rates of virus ribonucleic acid (RNA) and protein synthesis reach a maximum within the first few hours after infection and then drop. Plaque assays exhibit a linear dose-response, indicating that only one virion is necessary for productive infection. We have confirmed earlier reports regarding the fragmented nature of the RNA genome of purified influenza virions. However, high resolution gel electrophoresis indicated that each size class of viral RNA is heterogenous, so that there are at least 10 and probably more fragment sizes of RNA in these virions. Repeated attempts to detect infectivity in preparations of extracted viral RNA were completely negative (over a 10(8)-fold loss of infectivity after extraction). Even infection of the "infectious" RNA-treated cells with intact, related, influenza viruses failed to support infectivity of the isolated RNA or to rescue a host range genetic marker of the RNA. Purified influenza virions exhibit only three major protein peaks based on separation according to molecular weights. These three major virion proteins are the only major virion proteins synthesized in infected cells. This is true throughout the infectious cycle from several hours after infection until the cells are dying. However, the molecular weight of these virion proteins differs slightly depending upon the cell type in which the virus is grown. No host membrane proteins are incorporated into the virions as they bud through the cell membrane. Pulse-chase labeling early after infection or prolonged chase experiments indicate that influenza virus proteins are cleaved from one or more precursor polypeptides. In fact, each of the three major peaks seems to be a heterogeneous mixture of polypeptides in various stages of cleavage. Peptide analysis confirms that the three major peaks share common peptides, but the exact precursor product relationships are not clear. There may be one or several precursor proteins. Also there could be overlapping messenger RNA molecules of varying length giving rise to polypeptides of various sizes and overlapping sequences. Late in infection, amino acid labeling shows a preponderance of internal nucleocapsid protein synthesis, indicating that either this protein is much more stable to cleavage in infection or it is made from a more stable messenger. There is no obvious relationship between virion RNA fragments and viral protein sizes, so these fragments may be artifacts.     

Mechanism of murine leukemia virus-induced fusion in rat myoblasts defective in differentiation.

fu-1 cells, a line of rat myoblasts defective in differentiation, can be fused into multinucleate syncytia by Moloney murine leukemia virus. The effects of treating the virus with specific antibody, UV irradiation, and elevated temperature and the requirements for cellular RNA and protein synthesis have been studied as they relate to this virus-induced fusion. The results indicate that intact, but not necessarily infectious, virions are required to promote fusion of fu-1 cells. Neither actinomycin D nor cycloheximide altered the formation of syncytia; thus, neither viral nor cellular RNA or protein synthesis is required for fusion. fu-1 cells infected with the ts3 temperature-sensitive mutant of Moloney murine leukemia virus accumlate large amounts of budding virus on their cell membrane; however, this membrane-associated virus failed to induce syncytia. Upon release of the virus at the permissive temperature, fusion did occur. We conclude that contact or attachment of the immature virus to the cell membrane is not sufficient to promote murine leukemia virus-induced cell fusion; complete virions are required. From these data, we propose that adsorption and penetration of the virus may induce a change in the cell membrane that subsequently promotes the fusion of susceptible cells.