Isolation and Characterization of a Herpesvirus from Leukemic Guinea Pigs

A guinea pig herpesvirus (GPHV) has consistently been isolated from leukemic lymphoblasts of strain-2 guinea pigs. GPHV is serologically related to the guinea pig herpes-like virus isolated by Hsiung and Kaplow. The virions of GPHV consist of an icosahedral capsid containing a dense nucleoprotein core enclosed in a double-layered membrane. The average diameters of GPHV virion and capsid were 166 and 101 nm, respectively. Studies on the morphogenesis of GPHV revealed that, as in other herpesvirus infections, only the naked capsids with or without the nucleoprotein core were found in the infected cell nuclei; it was also learned that the virion acquired its envelope by budding from the nuclear membrane of the infected cells. However, GPHV-infected cell nuclei also contained dense fibrous rods, resembling nucleo-protein core outside the capsids, and tubules resembling viral core protein. The capsids were often embedded in dense granular antigen. GPHV deoxyribonucleic acid (DNA) has a density of 1.716 g/ml in cesium chloride compared to herpes simplex virus DNA (rho = 1.728 g/ml) and cellular DNA (rho = 1.700 g/ml).     

Expression of Endogenous Retroviral Genes in Leukemic Guinea Pig Cells

The expression of guinea pig retrovirus (5-bromodeoxyuridine[BUdR]-induced GPV) was studied in guinea pig L(2)C leukemic lymphoblasts by use of molecular hybridization of viral complementary DNA (cDNA) to cellular RNA. It was found that L(2)C leukemic lymphoblasts, leukemic spleen, and BUdR-induced virus-producing cells contain virus-specific RNA: 0.05% (800 to 960 copies per cell), 0.02% (360 copies per cell), and 0.3% (5,120 copies per cell), respectively. Adult normal liver and spleen, on the other hand, contain less than 0.2 copy of viral RNA per cell. Both BUdR-induced cells and L(2)C leukemic lymphoblasts contained 14S, 22S, 35S, and 70S RNA species of total and cytoplasmic virus-specific RNA as determined by sucrose velocity gradient analysis and hybridization of sucrose gradient fractions to cDNA. Virus-specific mRNA was identified in both BUdR-induced cells and L(2)C leukemic lymphoblasts by the criterion that it cosedimented with purified polyribosomes in a sucrose gradient and that it changed to a lower sedimentation value if polyribosomes were disaggregated with EDTA prior to centrifugation. Virus-specific mRNA obtained from either the polyribosome region of purified polyribosomes or the released messenger region of EDTA-disaggregated purified polyribosomes consisted of 14S, 20S, and 35S species in both BUdR-induced cells and L(2)C leukemic lymphoblasts. Hybridization of cDNA to the RNA of L(2)C leukemic lymphoblasts and BUdR-induced cells was essentially complete. Additionally, leukemic lymphoblast RNA could displace 95% of the hybridization of BUdR-induced GPV 70S RNA to guinea pig DNA. The midpoints of thermal denaturation of hybrids formed between GPV cDNA and the RNA of either L(2)C leukemic lymphoblasts or the 70S RNA of BUdR-induced GPV were both 89 degrees C in 2x concentrated 0.15 M NaCl plus 0.015 M sodium citrate. These results show that BUdR-induced GPV genes are essentially completely expressed in L(2)C leukemic lymphoblasts and that virus-specific mRNA is present, although fewer copies of RNA are present in L(2)C leukemic lymphoblasts than in BUdR-induced cells.     

Biological Activities of Guinea Pig Mitogenic Factor from Immune Lymphocytes Stimulated with Antigen

Mitogenic factor was produced by sensitized guinea pig lymph node cells stimulated with a specific antigen. Both T lymphocytes and macrophages were required for the production of this factor. The culture supernatant of lymphocytes containing the mitogenic factor exhibited a strong helping effect on the proliferative response of T lymphocytes to phytohemagglutinin (PHA). Mitogenic factor and the factor with the helping activity coeluted in the molecular weight range of 25,000-35,000 daltons in gel filtration. Furthermore the fraction containing mitogenic factor was found to support the proliferation of lymphoblasts induced by PHA or antigen, suggesting that the mitogenic factor may be the guinea pig equivalent of T cell growth factor (TCGF) reported in the mouse, rat, and human. On the other hand, the T cell-activating monokine of the guinea pig, possessing the helping activity for the proliferative response of T lymphocytes to PHA, never exhibited TCGF-like activity.     

Mechanism of the Schultz-Dale Reaction in the Denervated Diaphragmatic Muscle of the Guinea Pig

The mechanism of the contractions elicited by specific antigens in immunologically sensitized muscle tissue (Schultz-Dale responses) has been investigated on single fibers of denervated guinea pig hemidiaphragms. This preparation can be either actively or passively allergized, showing Schultz-Dale responses similar to those of visceral muscle. Specific antigens were applied with an electrically operated microtap to discrete areas of the cell surface while recording the electrical activity with intracellular microelectrodes. In this manner, a depolarizing action of the antigens on the muscle membrane was demonstrated. Brief applications of antigen gave rise to phasic potential changes (antigen potentials) similar to those elicited in the same fibers with acetylcholine-filled microtaps. However, antigen potentials occur only in denervated fibers sensitized to the specific antigen or closely related proteins; they are not seen in either innervated fibers of allergized animals or in denervated, nonallergized fibers. Repeated antigen application to the same area of the fiber causes a local irreversible desensitization. The antigen potentials are associated with a reduction in the resistance of the muscle membrane, similar to that caused by acetylcholine. It is concluded that besides causing the liberation of biogenic amines from the mast cells, antigens exert a direct action on the permeability of the muscle membrane; the molecules of antibody adsorbed to the cells appear to act as specific chemoreceptors for the antigen.     

Characterization of Bromodeoxyuridine-Induced Endogenous Guinea Pig Virus

An endogenous virus (GPV) was induced after 5-bromodeoxyuridine treatment of cultured guinea pig cells. Compared to Gross murine leukemia virus (G-MuLV) GPV has a reproducibly heterogenous density of about 1.16 to 1.18 g/ml. The virion-associated RNA is slightly larger than that in G-MuLV. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of dissociated GPV resolved five major structural proteins: I (molecular weight 70,000), II (molecular weight 36,000), III (molecular weight 24,000), IV (molecular weight 18,000), and V (molecular weight 16,000) which are similar to but distinct from G-MuLV proteins. Proteins I and II were demonstrated to be glycoproteins by incorporation of [(3)H]glucosamine. GPV and G-MuLV did not have any appreciable genetic homology or any common group-specific antigens when analyzed by immunodiffusion, radioimmunoassay, and indirect immunofluorescence. Morphogenesis of GPV also differed from that of a typical type C oncornavirus and proceeded via two pathways: (i) a majority of virus particles were formed in cytoplasmic vacuoles and were released after cellular disruption; and (ii) a minor population of particles were assembled in the cytoplasmic matrix and then migrated to the plasma membrane where they budded into the extracellular space. To date, GPV has been unable to initiate or maintain a productive replication in any cell line tested.     

A comparison of the avidin-biotin-peroxidase complex (ABC) and peroxidase-anti-peroxidase (PAP) immunocytochemical techniques for demonstrating Sendai virus infection in fixed tissue specimens

Mice were infected with Sendai virus and killed 8 days later. Lungs were removed and perfused with ethanol, 10% neutral formalin, Bouin's, B-5, or Zenker's fixatives. Tissues were dehydrated, embedded in paraffin, sectioned and stained for the presence of Sendai virus using the avidin-biotin-peroxidase-complex (ABC) and peroxidase antiperoxidase (PAP) immunocytochemical techniques. Results of these techniques were compared. The ABC technique was more sensitive than the PAP. Sendai antigen was demonstrated by the ABC technique in lung tissue fixed with any fixative, whereas antigen could be demonstrated with consistency only in ethanol-fixed lung by the PAP technique. Trypsin treatment of lung prior to immunoperoxidase treatment failed to enhance staining with either technique and actually caused a decrease in staining in ethanol, B-5 and Zenker's-fixed specimens.     

Antigen-presenting B cells: efficient uptake and presentation by activated B cells for induction of cytotoxic T lymphocytes against vesicular stomatitis virus

We have evaluated the efficacy of mitogen (LPS/DxSO4)-activated B cells (B lymphoblasts) to function as antigen-presenting cells (APC) for vesicular stomatitis virus (VSV). Our studies revealed that B lymphoblasts induced potent cytotoxic thymus (T)-derived lymphocyte (CTL) activity in VSV-immune splenic T cells depleted of adherent accessory cells. Dose-response curves indicated that B lymphoblasts were approximately 15-20 times more efficient APC than spleen cells for CTL induction against VSV. There was little evidence of reprocessing of viral antigens by the responder population because only CTL activity restricted to the parental haplotype of the B lymphoblast was generated following stimulation of VSV-immune F1 T cells. B lymphoblasts activated VSV-specific memory CTL which expressed the Lyt-1-23+, AsGM1+ phenotype without activating natural killer and/or lymphokine-activated killer cells. The ability of B lymphoblasts to function as efficient APC was not related to enhanced viral replication in these cells because potent VSV-specific proliferative and class I-restricted CTL responses were induced by B lymphoblasts infected with VSV rendered noninfectious by exposure to ultraviolet (uv) light. This indicates that activated B cells can efficiently process and present input virion protein. Purified splenic B cells that were not activated by mitogen stimulation did not function as APC for VSV even at high multiplicities of infection. The failure of B cells to function as APC for VSV was related to inefficient uptake of VSV and their inability to provide accessory cell signals required for T-cell proliferation; both these functions developed following mitogen stimulation. These data suggest that activated B cells may function as a potent APC population for virus independent of the specificity of their immunoglobulin antigen receptor.     

Replication and persistence of measles virus in defined subpopulations of human leukocytes.

Replication of Edmonston strain measles virus was studied in several human lymphoblast lines, as well as in defined subpopulations of circulating human leukocytes. It was found that measles virus can productively infect T cells, B cells, and monocytes from human blood. These conclusions were derived from infectious center studies on segregated cell populations, as well as from ultrastructural analyses on cells labeled with specific markers. In contrast, mature polymorphonuclear cells failed to synthesize measles virus nucleocapsids even after infection at a relatively high multiplicity of infection. Measles virus replicated more efficiently in lymphocytes stimulated with mitogens than in unstimulated cells. However, both phytohemagglutinin and pokeweed mitogen had a negligible stimulatory effect on viral synthesis in purified populations of monocytes. In all instances the efficiency of measles virus replication by monocytes was appreciably less than that of mitogenically stimulated lymphocytes or of continuously culture lymphoblasts. Under standard conditions of infection, all of the surveyed lymphoblast lines produced equivalent amounts of measles virus regardless of the major histocompatibility (HL-A) haplotype. Hence, no evidence was found that the HL-A3,7 haplotype conferred either an advantage or disadvantage with respect to measles virus synthesis in an immunologically neutral environment. A persistent infection with measles virus could be established in both T and B lymphoblasts. The release of infectious virus from such persistently infected cells was stable over a period of several weeks and was approximately 100-fold less than peak viral titers obtained in each respective line after acute infection.     

Effect of Ultraviolet Irradiation on the Survival of Simian Virus 40 Functions in Human and Mouse Cells

The relative sensitivities to ultraviolet light of various simian virus 40 (SV40) functions were studied in human and mouse cells. Transformation appeared to be less ultraviolet (UV)-sensitive than either T or V antigen when all functions were compared in the same cell. However, the time course of both T- and V-antigen appearance was delayed with UV-irradiated virus, so that the survival curves of these functions changed with time. Mouse and human cells which were transformed by UV-SV40 all contained SV40 T antigen. Infectious virus could be recovered from many more transformants than would be expected from the infectivity in African green monkey kidney cells of the irradiated virus. The results suggest that human and mouse cells are capable of reactivating UV-damaged SV40.     

Nonproductive Infection of Guinea Pig Cells with Human Cytomegalovirus

Human cytomegalovirus was capable of adsorbing to and penetrating guinea pig cells, but was unable to replicate new virus. Cultures infected with virus inoculum of high titer showed a cytopathic effect (CPE) characterized by cell rounding. This CPE depended upon the presence of infectious virus, and its extent was directly related to the multiplicity of infection. Staining by indirect immunofluorescence by using human convalescent sera was positive as early as 4 h postinfection. Maximal fluorescence was observed 24 h postinfection when 50% of the cells contained fluorescent antigens both in nuclei and cytoplasm. No evidence for viral replication was found, and no defective particles were detected by electron microscopy. Treatment with actinomycin D or with cycloheximide strongly inhibited both the fluorescent antigens and the CPE, whereas 5-fluorodeoxyuridine and bromodeoxyuridine were ineffective.     

Immunogenicity of Nanogram to Milligram Quantities of Inactivated Foot-and-Mouth Disease Virus. I. Relative Virus-neutralizing Potency of Guinea Pig Sera

Quantitative antigen dose-neutralizing antibody response curves were established in guinea pigs for purified foot-and-mouth disease virus (FMDV), type A, strain 119, inactivated for 48 hr with N-acetylethyleneimine (AEI). Inactivation of FMDV by 0.05% AEI at 25 C occurred without virus degradation and followed first-order kinetics over a 10(8)-fold decrease in plaque-forming units (PFU) extrapolating to 10(-5) PFU/ml at 48 hr. The AEI-treated virus was administered in doses ranging from 10 ng to 2.62 mg, alone or emulsified in oil adjuvant. Sigmoidal dose-response curves were obtained with 160 ng as the minimum effective dose. The maximum effective dose was 163 mug and 2.62 mg or more at 6 and 28 through 84 days postinoculation, respectively. Oil adjuvant had little effect at 6 days postinoculation, but its use markedly increased the amount of neutralizing antibody obtained at the later testing periods.     

Acetylcholine Releases ATP from Varicosities Isolated from Guinea Pig Myenteric Plexus

The effects of cholinergic agonists and antagonists on the release of ATP from isolated myenteric varicosities were studied using a firefly luciferin-luciferase technique. In a previous study, acetylcholine and nicotine released ATP from isolated myenteric varicosities, whereas the muscarinic agonist bethanechol did not. In the present study, release of ATP by acetylcholine was shown to be Ca2+ dependent. d-Tubocurarine competitively antagonized the release of ATP by either acetylcholine or nicotine. Maximal release of ATP by acetylcholine (10(-3) M) was approximately 24% that observed with the depolarizing drug veratridine (5 X 10(-5) M), suggesting either that not all of the varicosities capable of releasing ATP possess nicotinic receptors or that acetylcholine does not depolarize the varicosities to the degree that veratridine does. Tetrodotoxin slightly but significantly reduced ATP release induced by acetylcholine or nicotine, indicating some involvement of Na+ channels in the release process. Finally, 6-hydroxydopamine pretreatment produced a 48% reduction in the acetylcholine-evoked release of ATP, suggesting that much, but possibly not all, of the ATP release occurs from noradrenergic varicosities present in the preparation.     

H-2-specific antibodies induced by injection of syngeneic leukemia cells

AKR/J mice immunized with several syngeneic leukemia cells contained antibodies in their sera which reacted with certain AKR leukemia cell lines, depending on their H-2 expression, and precipitated H-2K antigens from lysates of leukemia cells. Precipitation of H-2K was not due to virus-specific antibodies: it could not be blocked by prior absorption with H-2-negative leukemias, but was blocked by certain allogeneic lymphocytes. Tumor-specific H-2K antibodies did not react with H-2K from normal AKR lymphocytes either on the cell surface or after detergent solubilization; however, they did react with H-2K from mitogen-activated AKR and BALB.K lymphoblasts. Since both these latter cells were also lysed by AKR-Gross/MuLV-specific and H-2K^k-restricted cytotoxic T lymphocytes, we consider the possibility that antibodies detecting conformational alterations induced in H-2K^k molecules by viral association may be present in syngeneic AKR antileukemia sera.Abbreviations used in this paper GCSA Gross-virus-induced cell-surface antigen - MCF mink cell focus-forming virus - MuLV murine leukemia virus - Th T helper     

Temperature-sensitive Steps in the Biosynthesis of Venezuelan Equine Encephalitis Virus

In contrast to Eastern equine encephalitis virus, the replication of Venezuelan equine encephalitis (VEE) virus was strongly inhibited at 44 C in chick embryo cells. The inhibited steps were analyzed by shifting the incubating temperatures up or down, and by determining during the shifts the rate and extent of infectious ribonucleic acid (RNA) synthesis, intact virus synthesis, and formation of complement-fixing antigen or of antigen detectable by a direct fluorescent-antibody technique. The inhibition appeared to be due to two temperature-sensitive steps involved in the synthesis of VEE virus in chick embryo cells. The first step of inhibition at 44 C occurred early in virus replication and could be completely reversed simply by transferring cultures to 37 C. The inhibition appeared to take place at some point between the time when the virus entered the cell and was uncoated and the beginning of viral RNA synthesis. The second temperature-sensitive step in VEE virus synthesis was irreversible; it occurred at a point after the synthesis of viral RNA, and before the formation of virus protein measured as complement-fixing antigen or as antigen that could be stained with fluorescent antibody.     

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.     

The production of heterologous antibodies to the human lymphakine: leukocyte migration inhibitory factor (LIF).

Human leukocyte migration inhibitory factor (LIF) produced by concanavalin A-stimulated lymphocytes was partially purified by Sephadex G-100 chromatography and immunosorption of protein contaminants. This material was injected into two rabbits, and the IgG-IgA fractions of the resulting antisera (anti-LIF) neutralized LIF induced by antigen (PPD tuberculin) with as equal efficiency as that of LIF induced by mitogen. Anti-LIF activity was neither removed by absorption with control supernatant or normal human serum nor was it suppressed by absorption with lymphocytes or lymphoblasts. On the other hand, antibodies against human lymphoid cells (ALG) did not reduce LIF activity, indicating the difference between anti-LIF and classical ALG. In support of this, anti-LIF, in contrast to ALG, was not cytotoxic to lymphocytes, and it did not inhibit spontaneous T-rosette formation with sheep erythrocytes. In crossed immunoelectrophoresis using a conventional proteinstaining technique, only three precipitates appeared. None of these contained LIF. However, a protein migrating in the prealbumin region appeared to be specific for lymphocyte stimulation. The nature and significance of this product is unknown.     

The predominant virus antigen burden is present in macrophages in Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus (TMEV) produces a persistent central nervous system infection and chronic, inflammatory demyelinating disease in susceptible mice. TMEV antigen(s) and RNA genome have been detected in astrocytes, oligodendrocytes, and macrophages during persistence. Whether there is a predominant cell type in which TMEV persists has not been resolved. Since TMEV-induced demyelinating lesions are infiltrated with macrophages and a number of other persistent viruses show near-exclusive tropism for these phagocytic cells, we used two-color immunofluorescent staining with conventional and confocal microscopy to colocalize TMEV to cells that stain with monoclonal antibodies (MOMA-2) [unknown antigen], Mac-1 [CD11b], FA-11 [CD66], and 2F8 [scavenger receptor]) to macrophages in BeAn-infected SJL mice. A predominant virus antigen burden within macrophages infiltrating demyelinating lesions was seen. A dichotomy of cells staining for virus antigen(s) was found with infected cells containing either a large or small virus antigen load. Ninety percent of cells with a large virus antigen load were large phagocytes (20 to 50 microns) that were readily detected at low power (5x objective). Cells with smaller amounts of virus antigen(s) turned out to be either these same large phagocytic cells or much smaller cells, approximately equal to 10 microns in diameter. Forty percent of cells with a small virus antigen load were macrophages. The unidentified approximately equal to 10-microns cells that are virus antigen positive and macrophage negative in this study could still be macrophages, or they may be oligodendrocytes. The fact that virus was detected in the cytoplasm and not phagolysosomes of macrophages and the sheer mass of fluorescently stained virus proteins in some macrophages suggest that TMEV persists in these phagocytic cells by active virus replication.     

Activated lymphocytes trigger lymphoblast extravasation.

Intraperitoneal stimulation of adoptively sensitized rats with bacterial antigen promotes the localization of lymphoblasts at the site of antigen deposition. Lymphoblast extravasation activity (LEA) is generated only when specifically immune donor lymphocytes and the recipients of these cells share at least on Ag-B haplotype. However, if the specificity criteria for its formation are satisfied, LEA promotes the local development of lymphoblasts of all available specificities and irrespective of their Ag-B genotype. Allogeneic lymphoblasts do not participate actively in the delayed inflammatory reaction even when they are passively recruited into exudates. The results suggest that LEA is a T cell-derived mediator that amplifies the delayed type hypersensitivity reaction by directing recently activated lymphocytes into lesions.     

Effects of monoclonal antibodies against lymphocyte surface antigens on interleukin 2 excretion by Epstein-Barr virus-specific human T-cell hybridomas

Monoclonal antibodies were used as probes to study the role of cell surface antigens in the response of Epstein-Barr virus (EBV)-specific human T-T hybridomas to autologous EBV-infected B lymphoblasts. Somatic cell hybrids were generated by fusing EBV-primed peripheral blood T lymphocytes with a mutant clone of the JM human T-lymphoblastoid-cell line. When exposed to autologous EBV-infected B lymphoblasts, the resulting hybrid clones released Interleukin 2 into the culture medium. Incubation of the EBV-infected B cells with two monoclonal antibodies against human Ia-like molecules blocked their ability to trigger the hybridomas. Under the same conditions, monoclonal antibodies against beta 2-microglobulin, and a 45,000 MW surface antigen common to EBV-infected B lymphoblasts, did not alter the capacity of the B cells to stimulate the hybridomas. None of four monoclonal antibodies against surface antigens on the T-cell hybridomas impaired their responsiveness to EBV-infected B lymphoblasts. These results suggest the possibility that naturally occurring or exogenously administered antibodies against Ia molecules might interfere with T-cell regulation of EBV-induced B-cell activation.