Accumulation Of Viral Antigen Research Articles

ОТРАБОТКА РЕЖИМОВ СТАЦИОНАРНОГО КУЛЬТИВИРОВАНИЯ ШТАММА EHV-1/К ВИРУСА РИНОПНЕВМОНИИ ЛОШАДЕЙ

The purpose of these studies is to develop stationary cultivation regimes to determine the most effective ways of viral antigen accumulation while controlling the accumulation kinetics. The novelty of the conducted research is the use of modern methods for analyzing the kinetics of accumulation of the equine rhinopneumonia virus in various modes. Modern production of immunobiological preparations and diagnostic test systems relies primarily on cost-effectiveness. Traditionally, for the accumulation of virus-containing material, a stationary cultivation method is used, which has a number of advantages, primarily associated with the absence of the need for expensive equipment, consumables, and great opportunities for controlling fungal and bacterial contamination, with low staff qualifications. Virological, microbiological, technological and analytical methods of management and control of stationary cultivation of the EHV-1 strain of equine rhinopneumonia virus were used in the work. As a result of the research, a mathematical model of stationary cultivation of the EHV-1 strain was developed, based on the influence of external and internal factors on the kinetics of virus accumulation, with subsequent implementation of the results into the production process. With a standard production process, the biological activity of the virus was 5.75 lg TCID50/cm3, which, when recalculated, is 500 thousand viral particles per 1 cm3, and when the production process was modified using a mathematical model, the virus activity was raised to 6.5 lg TCID50/cm3 , which, when recalculated, is 3 million viral particles per 1 cm3.

Assessing the oncolytic potential of rotavirus on mouse myeloma cell line Sp2/0-Ag14

Introduction: Cancer is the second leading cause of death in the United States, surpassed only by cardiovascular disease. However, cancer has now overtaken cardiovascular disease as the main cause of death in 12 countries in Western Europe. The burden of cancer is posing a major challenge to health care systems worldwide and demanding improvements in methods for cancer prevention, diagnosis, and treatment. Alternative and complementary strategies for orthodox surgery, radiotherapy, and chemotherapy need to be developed.Objective: To determine the oncolytic potential of tumor cell-adapted rotavirus in terms of their ability to infect and lysate murine myeloma Sp2/0-Ag14 cells.Materials and methods: We inoculated rotaviruses Wt1-5, WWM, TRUYO, ECwt-O, and WTEW in Sp2/0-Ag14 cells and we examined their infectious effects by immunocytochemistry, immunofluorescence, flow cytometry, and DNA fragmentation assays. Results: Rotavirus infection involved the participation of some heat shock proteins, of protein disulfide isomerase (PDI), and integrin β3. We detected the accumulation of viral antigens within the virus-inoculated cells and in the culture medium in all the rotavirus isolates examined. The rotavirus-induced cell death mechanism in Sp2/0-Ag14 cells involved changes in cell membrane permeability, chromatin condensation, and DNA fragmentation, which were compatible with cytotoxicity and apoptosis.Conclusions: The ability of the rotavirus isolates Wt1-5, WWM, TRUYO, ECwt-O, and WTEW to infect and cause cell death of Sp2/0-Ag14 cells through mechanisms that are compatible with virus-induced apoptosis makes them potential candidates as oncolytic agents.

Transgenic expression of Serratia marcescens native and mutant nucleases modulates tobacco mosaic virus resistance in Nicotiana tabacum L.

Extracellular Serratia marcescens nuclease is an extremely active enzyme which non-specifically degrades RNA and DNA. Its antiviral activity was previously shown both in animals and in plants when applied exogenously. Transgenic tobacco plants (Nicotiana tabacum L cv. SR1) expressing S. marcescens chimeric, mutant, and intracellular mutant nuclease gene variants were regenerated and challenged with tobacco mosaic virus. The transgenic plants exhibited a higher level of resistance to the virus infection than the control non-transgenic plants. The resistance was evidenced by the delay of the appearance of mosaic symptoms and the retarded accumulation of viral antigen. Thus, these results reveal that modulations of both extracellular nuclease activity and intracellular RNA/DNA binding can protect plants against viral diseases.

Altered lipid metabolism influences course of hepatitis C virus infection and hepatocyte cytokine production (VIR1P.1011)

Abstract Hepatitis C virus (HCV) is remarkably efficient in establishing persistent infection leading to the development of chronic inflammatory liver diseases. Lipids play a central role in regulating inflammatory responses and are necessary for the replication and assembly of HCV. Fatty acids are the building blocks of lipids and are synthesized via a rate-limiting step catalyzed by the enzyme acetyl-CoA carboxylase (ACC). To determine the role of lipid synthesis in regulating innate inflammatory responses and HCV biogenesis, we examined the effect of ACC inhibition on HCV replication and cytokine production. Inhibition of ACC decreases intracellular viral RNA in Huh7.5.1 cells infected with JFH-1, a genotype 2a strain of HCV. Surprisingly, ACC inhibition slightly decreases IL-1β transcription and results in comparable production of IL-8, IL-13, and IP-10 in HCV-infected hepatocytes relative to vehicle controls. Further studies using electron and fluorescent microscopy demonstrate a reduction in the number of lipid droplets, which was accompanied by an accumulation of viral protein. These data highlight the paradoxical immune response of HCV-infected hepatocytes, where the continued accumulation of viral antigen fails to stimulate synthesis of inflammatory cytokines. The potential role of lipids in dampening immune responses in HCV-infected hepatocytes may provide an important link between host factors driving infection and suppression of immune responses in chronic infection.

Tick-borne flaviviruses alter membrane structure and replicate in dendrites of primary mouse neuronal cultures

Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, detailed mechanisms of viral replication in the brain and features of viral pathogenesis remain poorly understood. We carried out a comparative analysis of replication of neurotropic flaviviruses: West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus (TBEV), in primary cultures of mouse brain neurons. All the flaviviruses multiplied well in primary neuronal cultures from the hippocampus, cerebral cortex and cerebellum. The distribution of viral-specific antigen in the neurons varied: TBEV infection induced accumulation of viral antigen in the neuronal dendrites to a greater extent than infection with other viruses. Viral structural proteins, non-structural proteins and dsRNA were detected in regions in which viral antigens accumulated in dendrites after TBEV replication. Replication of a TBEV replicon after infection with virus-like particles of TBEV also induced antigen accumulation, indicating that accumulated viral antigen was the result of viral RNA replication. Furthermore, electron microscopy confirmed that TBEV replication induced characteristic ultrastructural membrane alterations in the neurites: newly formed laminal membrane structures containing virion-like structures. This is the first report describing viral replication in and ultrastructural alterations of neuronal dendrites, which may cause neuronal dysfunction. These findings encourage further work aimed at understanding the molecular mechanisms of viral replication in the brain and the pathogenicity of neurotropic flaviviruses.

The Dominant-Negative Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR Increases the Efficacy of Rift Valley Fever Virus MP-12 Vaccine

Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-α) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-α nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans.

Cowpox virus serpin CrmA is necessary but not sufficient for the red pock phenotype on chicken chorioallantoic membranes

It was previously reported that cowpox virus (CPXV) strain Brighton Red (BR) causes red pocks upon inoculation of chorioallantoic membranes (CAMs) of embryonated chicken eggs. Red pocks are characterized by hemorrhage and reduced numbers of inflammatory cells while white pocks induced by other members of the genus Orthopoxvirus lack hemorrhage and have higher numbers of infiltrating heterophils. Analyses of CPXV BR white pock variants identified the cytokine response modifier A (CrmA) as the factor responsible for the differences in pock phenotype through induction of hemorrhage and inhibition of chemotaxis. In the present study CPXV crmA deletion mutants were generated based on a full-length bacterial artificial chromosome clone of CPXV BR (pBR). Deletion of the first crmA start codon was sufficient to abolish protein expression, whereas modification of a potential second start codon had no impact on CrmA production as shown by Western blot analysis. Immunohistochemistry of CAMs inoculated with crmA-positive BR viruses showed accumulation of viral antigen in endothelial cells, which was consistent with the red pock phenotype. On the other hand, crmA-negative mutants were characterized by the induction of white pocks and the absence of CPXV antigen in endothelia. The introduction of the complete CPXV BR crmA gene into the homologous genome region of the attenuated vaccinia virus strain MVA (modified vaccinia virus Ankara), however, resulted in CrmA production but not the red pock phenotype. We therefore conclude that (i) CPXV CrmA is associated with increased accumulation of virus in endothelial cells and (ii) the poxvirus-encoded serpin is necessary but not sufficient for the red pock phenotype and the anti-chemotactic capabilities on CAMs.

Progression of Pathogenic Events in Cynomolgus Macaques Infected with Variola Virus

Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections – an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions.

Effect of the disodium salt of cyclopentene β,β′-triketone on the development of infections induced by TMV in the leaves of hypersensitive and susceptible tobacco plants

We show here that the disodium salt of 2-acetyl-5-chloro-4- hydroxycarbonylmethylthiocyclopent-4-ene-1,3-dione (salt 2) inhibits tobacco mosaic virus (TMV)-induced infections in detached leaves of TMV-resistant (Xanthi-nc) and -susceptible (Samsun) varieties of Nicotiana tabacum L. Inoculation of Xanthi-nc leaves with TMV mixed with salt 2 (2 mg/ml) resulted in a significant decrease (78%) in the number of local lesions as compared to leaves inoculated with TMV alone. By applying the salt immediately before or after infection, the inhibitory effect on the number of lesions decreased. The infectivity and content of viral antigen in Samsun leaves inoculated with a mixture of TMV and salt 2 were considerably less early in infection compared to leaves inoculated with TMV only. As the infection progressed, the inhibitory action of salt 2 decreased. Applying the salt 24 h before infection enhanced the number of both local and heat-induced lesions in Xanthi-nc and Samsun leaves, respectively, that is increased the number of “infectious centers” produced on the leaf surface, which probably promoted virus penetration into the host cells. At the same time, pretreatment decreased the infectivity in Xanthi-nc leaf lesions and infectivity and viral antigen accumulation in Samsun leaves. Therefore, the antiviral effects of salt 2 are not because of diminished penetration of the virus into cells, but seem to be caused by salt 2-mediated inhibition of virus replication.

Suppression of mRNA accumulation by the duck hepatitis B virus reverse transcriptase

Hepadnaviruses establish chronic liver infections, but the mechanisms of persistence and immune evasion are poorly understood. We previously found that the duck hepatitis B virus (DHBV) and hepatitis B virus reverse transcriptases (P protein) unexpectedly accumulate in the cytoplasm where they could affect function(s) beyond viral DNA synthesis, such as gene expression. Therefore, we measured effects of DHBV P on gene expression from reporter constructs and the viral genome. P reduced reporter expression at the mRNA level to ∼30–40%, independent of reporter tested. Accumulation of the viral pregenomic RNA from its native promoter was suppressed three-to four-fold by P, and accumulation of the capsid protein and intracellular core particles was similarly suppressed because the pregenomic RNA encodes the capsid protein. Therefore, suppression of the pregenomic RNA by DHBV P creates a negative feedback loop to limit viral antigen accumulation and replication, possibly contributing to maintenance of chronic infection.

The Correlation between Severity of Disease Symptoms and the Accumulation of Viral Antigen and Acidic Pathogenesis‐related Proteins in the Leaves of Thorn‐apple Plants Infected with Different Isolates of Potato Virus X

AbstractThe isolates of potato virus X causing disease symptoms of different severity in thorn‐apple plants (Datura stramonium L.) can be categorized in terms of the increase in antigen accumulation, as follows: strong, medium and weak. Extracts from leaves of healthy plants and of those infected with separate virus isolate did not differ in set of acidic pathogenesis‐related (PR) proteins as shown by discontinuous non‐denaturating polyacrylamide gel electrophoresis. Within 2 weeks after inoculation the total content of PR‐proteins in leaves of plants infected with weak or medium isolates was higher than in the healthy ones but after 3 weeks these differences became negligible. The plants infected with the strong isolate had higher total content of PR‐proteins than healthy plants and plants infected with weak or medium isolates during 3 weeks. It is proposed that the greater the damage to the plant caused by virus infection, the more PR‐proteins are accumulated.

Pirital virus (Arenaviridae) infection in the syrian golden hamster, Mesocricetus auratus: a new animal model for arenaviral hemorrhagic fever.

Adult Syrian golden hamsters inoculated intraperitoneally with Pirital virus, a recently discovered member of the Tacaribe complex of New World arenaviruses, developed a progressively severe, fatal illness with many of the pathologic features observed in fatal human cases of Lassa fever and other arenaviral hemorrhagic fevers. Most of the animals became moribund by Day 5 and were dead by Day 7 after inoculation. The most consistent histopathologic changes included interstitial pneumonitis, splenic lymphoid depletion and necrosis, and multifocal hepatic necrosis without significant inflammatory cell infiltration. The liver changes ranged from single cell death by apoptosis to coagulative necrosis of clusters of hepatocytes. Immunohistochemical studies of the liver demonstrated the presence and accumulation ot Pirital virus antigen within hepatocytes as well as Kupffer cells. An in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay showed progressively increasing apoptotic activity in the liver of infected hamsters. A human hepatoblastoma cell line (Hep G2/C3A) inoculated with Pirital virus also developed progressive cell destruction and accumulation of viral antigen, as demonstrated by immunofluorescence. Results of this pilot study suggest that the Pirital virus-hamster model is a very promising new small animal model for studying the pathogenesis of arenavirus infections, particularly, the mechanism of direct virus-induced hepatic injury. It may also be useful for testingantiviral agents for treatment of arenaviral hemorrhagic fevers.

Appearance of T Cells in the Bursa of Fabricius and Cecal Tonsils during the Acute Phase of Infectious Bursal Disease Virus Infection in Chickens

Groups of 3-wk-old specific-pathogen-free chickens were inoculated intraocularly with two virulent and one vaccine strain of infectious bursal disease virus (IBDV). During the acute phase of infection, the bursa of Fabricius and cecal tonsil were examined by immunohistochemistry for IBDV antigen and T cell. With all three viruses examined, the infection resulted in the appearance of viral antigen in the bursa accompanied by the presence of CD3+ cells. The CD3+ cells were predominantly TCR2+ and appeared at the site where viral antigens were present. The CD3+ cells continued to persist in the bursa after most of the IgM+ cells and IBDV antigen-positive cells had disappeared. Similar appearance of CD3+ cells was noted following viral antigen accumulation in the germinal centers of cecal tonsils in virulent IBDV-infected chickens. These results demonstrated that infection with IBDV resulted in extensive viral replication in the bursa and the cecal tonsils with an associated accumulation of T cells. The role these T cells may play in the pathogenesis of IBDV infection remains to be established.

Diagnosis of Naturally Occurring Bovine Viral Diarrhea Virus Infections in Ruminants Using Monoclonal Antibody-based Immunohistochemistry

Formalin-fixed paraffin-embedded tissues from 50 spontaneous cases (39 bovine, nine ovine, two caprine) of bovine viral diarrhea virus (BVDV) infection diagnosed by virus isolation were retrospectively examined for BVDV antigen by immunohistochemistry using anti-BVDV gp-43 monoclonal antibody (Mab 15C5). The cases were separated into enteric disease syndrome, respiratory disease syndrome, and abortion/weak calf syndrome based upon clinical disease. The purposes of the study were to 1) compare routine virus isolation with immunohistochemistry in determining BVDV infection and 2) define tissue and cellular distribution of BVDV in various clinical manifestations of infection. In bovids, there was 100% concordance of virus isolation and immunohistochemistry using Mab 15C5 in cases of enteric disease (mucosal disease, acute and chronic diarrhea, neonatal diarrhea), respiratory disease, and abortion. When laboratory tests were restricted to gastrointestinal tissue and/or feces, virus isolation detected BVDV in only 65% of cattle, whereas immunohistochemistry detected BVDV antigen in 100% of cattle. Immunohistochemical detection of pestivirus was poor in cases of ovine abortion, ovine hairy shaker syndrome, and caprine abortion. The tissue distribution of BVDV antigen was widespread in individual cattle with all clinical forms of BVDV infection. Viral antigen accumulation was spatially correlated with tissue lesions (in the absence of other pathogens) only in the gastrointestinal tract, lymphoid tissue, lung, placenta, and eye. This study demonstrates the utility of immunohistochemistry using Mab 15C5 to diagnose BVDV infections in cattle with a broad spectrum of clinical disease.

Immunological and virological changes associated with decline in CD4/CD8 ratios in lymphoid organs of SIV-infected macaques.

The decline in CD4/CD8 ratios in lymph nodes (LNs) of SIV macaques and HIV-infected individuals occurs later than that in blood. In a previous study, long-term SIV-infected macaques were delineated into two groups: (1) those whose LNs had normal CD4/CD8 ratios and (2) those whose LNs had low CD4/CD8 ratios. In the present investigation, LNs, spleens, and blood from these groups have been further analyzed to ascertain the cellular and virological events, particularly those involving CD8+ cells, that occur concomitantly with LN CD4% decline. An increase in the percent of CD69-, IL-2R(p75)-, CD45RA1o CD8+ cells was the most constant event observed in lymphoid tissue from mid- to late-stage SIV-infected monkeys. Such cells were sometimes observed in LNs prior to any other immunological or morphological changes. However, decline in LN CD4/CD8 ratios and the associated degeneration of follicular dendritic cells (FDCs) in the germinal centers (GCs) of these nodes were observed only when both CD8+ cell infiltration of GCs and accumulation of viral antigens within the FDC network could be demonstrated. These dramatic changes were also associated with significantly reduced responsiveness to mitogens throughout the lymphoid compartment. In terms of viral burden, immunological and structural collapse of LNs was not always associated with increased viral DNA levels. Despite the CD4+ cell decline in blood during HIV and SIV infections, the immunological and architectural collapse of the lymphoid compartment, which comprises the bulk of the lymphocytes in the body, appears to be a critical event leading to the onset of AIDS. The present findings suggest that increased CD8+ cell activity as well as decrease in CD4+ cell function both contribute to this process.

Studies on an inhibitor of TMV viral antigen accumulation released by cultured cells of Nicotiana tabacum

An activity that inhibited the accumulation of TMV antigen in tobacco leaf disks was observed in the growth medium of uninfected tobacco cell suspension cultures. Levels of inhibitory activity were highest in medium from actively growing cells. The activity was partially heat-labile and protease-sensitive, and appeared in the high molecular weight fractions following ultrafiltration and dialysis. Growth medium was analysed by SDS-PAGE and found to contain two main protein bands corresponding to 14 and 40 kDa. These proteins were partially purified by ammonium sulphate precipitation and column chromatography. Both purified protein fractions had inhibitory activity and contained carbohydrate. The proteins were associated with the protoplast and not with cell wall material. Both proteins effluxed from protoplasts into the surrounding medium in the absence of bursting, indicating that they may normally be secreted. Incorporation of radiolabels into both viral coat protein and RNA was inhibited by growth medium. A polysaccharide fraction from growth medium also had inhibitory activity.

Heparan Sulfate as a Mediator of Herpes Simplex Virus Binding to Basement Membrane

Explants of human lip and oral mucosa were infected with herpes simplex virus (HSV) in vitro and the expression of viral antigen was investigated by immunofluorescent antibody staining. Viral antigen was demonstrated in the cells of basal cell layer and lower prickle cell layers. Moreover, an accumulation of viral antigen in the epithelial-mesenchymal junction was observed. To examine the possibility that the basement membrane has an affinity for HSV, the interaction between HSV and major basement membrane components including type IV collagen, laminin, fibronectin, and heparan sulfate was investigated. When tested by a plaque-reduction assay, only heparan sulfate inhibited HSV plaque formation by competing for the virus adsorption to HEp-2 cells. The inhibitory effects of heparan sulfate and heparin were not affected by pre-incubation of these glycosaminoglycans with antithrombin III, whereas de-N-sulfation resulted in a significant reduction of their inhibitory activity. These findings suggest that heparan sulfate is involved in the binding of HSV to the basement membrane and that N-sulfated glucosamine residues of heparan sulfate are essential for HSV binding. The basement membrane may act as a reservoir of HSV in muco-cutaneous tissues.

Inhibition by conditioned growth medium of accumulation of viral antigen in plant cell suspension cultures inoculated with TMV

Conditioned medium from suspension-cultured cells of tobacco or carrot infected with TMV contains an activity that results in a reduction of the TMV viral antigen content in cultured cells and tobacco leaf disks. The maximum inhibition of antigen accumulation was observed with medium from 6-day-old tobacco cells and 7-day-old carrot cells. This timing corresponds to the period of fastest growth of the cells and so it is suggested that production if the inhibitor(s) may be cell division- or growth-related. No inhibition was seen in the case of medium from uninfected cells. The inhibitory activity showed specificity in that conditioned medium resulted in twice the inhibition when applied to cells of the same species as used to condition the medium compared with the other species used. Conditioned medium from tobacco cells also reduced the accumulation of TMV antigen in persistently-infected tobacco cells, indicating that replication rather than infection events are inhibited.

Acquired Resistance in Hypersensitive Tobacco Operates by Limiting Cell-to-Cell Spread of Virus Infection without Affecting Virus Multiplication

Localized and systemic acquired resistance against tobacco mosaic virus (TMV) or tobacco necrosis virus was induced when local lesions were produced in leaves of Xanthi-nc tobacco by mechanical inoculation with TMV. Both types of resistance were characterized by reduction in the size of lesions produced by the challenging viruses, whereas accumulation of viral antigen in lesions was slightly increased. These results, confirming previous findings relative to other hypersensitive plant virus combinations, do not support the view that an inhibitor of virus replication operates in the resistant tissues, but indicate that both types of resistance operate only against cell-to-cell spread of virus.

Systemic Acquired Resistance to Virus Infections and Ethylene Biosynthesis in Asparagus Bean

Systemic acquired resistance (SAR) was induced in asparagus bean following inoculation with tobacco necrosis virus (TNV) or tobacco rattle virus (TRV), viruses that produce a hypersensitive reaction in this plant. SAR was expressed against challenge by TNV as reduction in lesion size, but not as inhibition of viral antigen accumulation. Systemic stimulation of ethylene-forming enzyme (EFA) activity, in the absence of any ethylene increase or 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation, was associated with SAR. Formation of local necrotic lesions was necessary for both induction of SAR and stimulation of EFA, because early removal of inducer leaves prevented both events. SAR was expressed at rather constant level between 7 and 12 days after inducing infection. EFA stimulation declined with time and was no longer detected 7 days after inducing infection. SAR was not expressed against cucumber mosaic virus, that infect asparagus bean systemically. Prior inoculation with TNV or TRV was ineffective to reduce CMV antigen content or to minimize the pathogenic effect of this virus in systemically infected leaves.