Lethal Dose Of Virus Research Articles

Glycyrrhizin, an active component of licorice roots, reduces morbidity and mortality of mice infected with lethal doses of influenza virus.

The antiviral effect of glycyrrhizin (GR), an active component of licorice roots, was investigated in mice infected with influenza virus A2 (H2N2). When mice that had been exposed to 10 50% lethal doses of the virus were treated intraperitoneally with 10 mg of GR per kg of body weight 1 day before infection and 1 and 4 days postinfection, all of the mice survived over the 21-day experimental period. At the end of this period, the mean survival time (in days) for control mice treated with saline was 10.5 days, and there were no survivors. The grade of pulmonary consolidations and the virus titers in the lung tissues of infected mice treated with GR were significantly lower than those in the lung tissues of infected mice treated with saline. GR did not show any effects on the viability or replication of influenza virus A2 in vitro. When splenic T cells from GR-treated mice were adoptively transferred to mice exposed to influenza virus, 100% of the recipients survived, compared to 0% survival for recipient mice inoculated with naive T cells or splenic B cells and macrophages from GR-treated mice. In addition, the antiviral activities of GR on influenza virus infection in mice were not demonstrated when it was administered to infected mice in combination with anti-gamma interferon (anti-IFN-gamma) monoclonal antibody. These results suggest that GR may protect mice exposed to a lethal amount of influenza virus through the stimulation of IFN-gamma production by T cells, because T cells have been shown to be producer cells of IFN-gamma stimulated with the compound.

Antibody alone does not prevent experimental cytomegalovirus retinitis in mice with retrovirus-induced immunodeficiency (MAIDS).

Passive-transfer studies were performed to assess the ability of antibody alone to reduce the frequency and/or severity of necrotizing retinitis caused by murine cytomegalovirus (MCMV) in C57BL/6 mice with retrovirus-induced immunodeficiency syndrome (MAIDS). Initial experiments showed a gradual decline in the ability of mice to initiate humoral immunity during the evolution of MAIDS so that neither MCMV-specific IgM nor IgG could be detected during late-stage MAIDS. Passively administered hyperimmune MCMV immunoglobulin, however, could be detected within the serum of mice with MAIDS for at least 9 days after intraperitoneal injection and protected these animals in preliminary experiments from systemic MCMV disease and death when administered 24 h prior to intraperitoneal challenge with a lethal dose of virus. Nonetheless, passive transfer of hyperimmune MCMV serum to mice with MAIDS failed to reduce intraocular MCMV titers, frequency of retinitis, or severity of retinitis when administered 24 h prior to subretinal MCMV inoculation. Whereas whole eyes of MAIDS animals that received normal mouse serum and were injected subretinally with MCMV had an ocular MCMV titer of 4.3 log10 and a frequency of retinitis of 89% (severity score = 55%), whole eyes of antibody-treated mice with MAIDS had an ocular MCMV titer of 4.3 log10 and a frequency of retinitis of 87% (severity score = 57 %). Passive transfer of a neutralizing MCMV-specific monoclonal antibody also failed to reduce the frequency or severity of MCMV retinitis when administered to mice with MAIDS prior to subretinal MCMV inoculation. Our findings suggest that antibody immunotherapy alone will not be effective therapeutically for cytomegalovirus retinitis in patients with AIDS.

Inhibitory effects of recombinant manganese superoxide dismutase on influenza virus infections in mice.

The oxygen free-radical scavenger recombinant human manganese superoxide dismutase (MnSOD) was studied for its effects on influenza virus infections in mice when used alone and in combination with ribavirin. Mice challenged with influenza A/NWS/33 (H1N1) virus were treated parenterally in doses of 25, 50, and 100 mg/kg of body weight per day every 8 h for 5 days beginning at 48 h post-virus exposure. An increase in mean day to death, lessened decline in arterial oxygen saturation, and reduced lung consolidation and lung virus titers occurred in the treated animals. To determine the influence of viral challenge, experiments were run in which mice were infected with a 100 or 75% lethal dose of virus and were treated intravenously once daily for 5 days beginning 96 h after virus exposure. Weak inhibition of the mortality rate was seen in mice receiving the high viral challenge, whereas significant inhibition occurred in the animals infected with the lower viral challenge, indicating that MnSOD effects are virus dose dependent. To determine if treatment with small-particle aerosol would render an antiviral effect, infected mice were treated by this route for 1 h daily for 5 days beginning 72 h after virus exposure. A dose-responsive disease inhibition was seen. An infection induced by influenza B/Hong Kong/5/72 virus in mice was mildly inhibited by intravenous MnSOD treatment as seen by increased mean day to death, lessened arterial oxygen saturation decline, and lowered lung consolidation. MnSOD was well tolerated in all experiments. A combination of MnSOD and ribavirin, each administered with small-particle aerosol, resulted in a generally mild improvement of the disease induced by the influenza A virus compared with use of either material alone.

Do cytotoxic T lymphocytes clear some HIV/SIV infections?

Early work on the roles of cytotoxic T lymphocytes (CTLs) in acute viral infections in animal models showed that i) the clearance of virus coincided with the increase in CTL activity rather than specific antibody levels, ii) transfer of CTLs after infection could protect from a lethal dose of virus, and iii) in primed, compared to naive, animals, CTL activity appeared 1-3 days earlier after a challenge infection. There is now a series of findings with individuals who have been exposed to HIV but are HIV-seronegative that suggest a protective role for CTLs. Usually after in vitro culture, HIV-specific CTLs have been isolated from i) infants born of infected mothers, ii) long-time partners of HIV-infected people, iii) some prostitutes in Africa, and iv). Most recently, 7/20 seronegative health care workers exposed once to HIV have been shown to possess HIV env-specific CTLs. The findings suggest that CTLs (a type I T cell response) may rapidly clear a low dose of HIV. Experiments with SIV are proposed that may provide more direct supporting data for this possibility.

Acceleration of influenza virus clearance by Th1 cells in the nasal site of mice immunized intranasally with adjuvant-combined recombinant nucleoprotein.

The protective roles of influenza viral nucleoprotein (NP), together with the cellular mechanism of the protection in the nasal site, were examined in BALB/c mice immunized intranasally with an adjuvant (cholera toxin B subunit containing 0.2% of the whole toxin)-combined A or B virus recombinant NP. The NP-immune mice, when challenged intranasally with a sublethal dose of the virus 3 wk after immunization, had accelerated virus clearance from the nasal site in both an influenza type-specific and a nonspecific manner, as shown by the protection from high morbidity from the second day after challenge. Both type-specific and nonspecific acceleration of recovery was confirmed by the increased survival rate after challenge with a lethal dose of virus in mice immunized and boosted with adjuvant-combined NP. The acceleration of nasal virus clearance was accompanied with acceleration of type-specific systemic delayed-type hypersensitivity (DTH) and with IFN-gamma production by nasal lymphocytes. The nasal lymphocytes from the immunized and challenged mice generated a significantly high level of DTH when transferred locally, but no class I MHC-restricted CTL response. Moreover, nasal CD4+ T cells, induced by NP immunization and increased in number by the subsequent challenge, were involved in the accelerated IFN-gamma production. These results suggest that nasal Th1 cells, capable of producing IFN-gamma and mediating DTH, are involved in the type-specific acceleration of recovery from influenza after challenge in mice immunized intranasally with adjuvant-combined NP, although the nonspecific mechanism of accelerated recovery remains to be solved.

Tumor necrosis factor as a mediator of inflammation in influenza A viral pneumonia

The role of tumor necrosis factor α (TNFα) in the pathogenesis of influenza A viral pneumonia was examined. CD-1 male mice were challenged intranasally with influenza A virus A/PR/8/34 (H1N1) and administered rabbit anti-mouse TNFα-specific-neutralizing antibodies intraperitoneally. The effect of treatment on virus titer, TNFα levels, morbidity, mortality, and on pathologic lung lesions were compared with sham-treated controls. The severity of gross and histologic lung lesions positively correlated with the peak bronchoalveolar TNFα levels and was ameliorated with anti-TNFα treatment. Survivorship was prolonged in mice given a lethal dose of virus by treatment with TNF-α neutralizing antibodies. Reduction of TNFα levels by treatment with TNFα-antibodies did not affect virus titers in the lung. These results suggest that TNFα is a mediator of pulmonary inflammation during influenza A viral pneumonia, but may not play a significant anti-viral role in influenza pneumonia.

Effect of methionine enkephalin on natural killer cell and cytotoxic T lymphocyte activity in mice infected with influenza A virus.

Methionine enkephalin (Met-Enk) was evaluated for efficacy as an immune activator and potential therapeutic agent in influenza A/NWS/33 (H1N1) viral infections in female BALB/C mice. Influenza infection was induced intranasally with an approximate 90% lethal dose of virus and mice were treated intraperitoneally with doses of 10, 3 and 1 mg/kg/day, with treatments given 24 h pre-, 24 h post- and 72 h post-virus exposure. Splenocytes were assayed for natural killer cell (NK) and cytotoxic T lymphocyte (CTL) activity at time periods 76, 96 and 120 h post virus exposure. The 10 mg/kg dosage level significantly increased both CTL and NK activity at all time periods assayed. Other treatment schedules included single doses of 20, 10 and 3 mg/kg/day Met-Enk at either 24 h post- or 72 h post-virus exposure, with highly significant increases in NK and CTL activity noted after the latter treatment. The results of this study demonstrate the immunomodulatory effects of Met-Enk on NK and CTL in influenza infected mice and suggest a potential for therapeutic applications.

Effects of influenza virus-specific cytotoxic T-lymphocyte responses induced by a synthetic nucleoprotein peptide on the survival of mice challenged with a lethal dose of virus

Even though virus-induced cytotoxic T lymphocytes (CTLs) recognize antigens as peptides presented on infected cells, short synthetic peptides without any modifications are generally considered unsuitable for inducing antigen-specific CTLs in vivo. Our results demonstrate rapid induction of influenza virus-specific CTLs in Balb/c mice by an unmodified core protein peptide known to be a dominant H-2d-restricted CTL epitope. Additionally, the immunization procedure we employed in these studies produced significant influenza virus-specific CTLs in lymph nodes, spleen and lungs. When challenged with a lethal dose of influenza virus, a statistically significant delay in the day of death was observed in peptide-immunized mice. However, viral clearance was only slightly different from that in control mice. While these results are encouraging, they suggest a requirement for multiple CTL-inducing peptides, helper T cell-inducing peptides and/or virus-specific IgA responses in order to achieve protection from influenza infection.

Strict peptide length is not required for the induction of cytotoxic T lymphocyte-mediated antiviral protection by peptide vaccination.

Sendai virus nuclear protein peptides of different lengths were titrated in peptide vaccination experiments. We observed that peptide length was not important in inducing cytotoxic T lymphocyte-mediated protective immunity in vivo against a challenge with a lethal dose of virus. These results suggest that long peptides are trimmed in vivo to peptides that fit into the groove of major histocompatibility complex class I molecules. In addition several adjuvants were screened for their effectiveness in peptide vaccination protocols. Incomplete Freund's adjuvant and Titermax turned out to be useful, whereas alum was much less effective.

Calix (4) arene-polyoles as anti-ageing stabilizers for elastomers

Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.

Immunization of Cats Against Feline Infectious Peritonitis with Anti-Idiotypic Antibodies

Anti-idiotypic antibodies (Ab2s) generated against neutralizing antibodies (Ab1s) specific for feline infectious peritonitis virus (FIPV) were shown to be specific for paratope-associated idiotopes of the Ab1s and not against isotypic determinants. In a study to determine the efficacy of an anti-idiotypic vaccine against feline infectious peritonitis (FIP), cats that were immunized with a pool of monoclonal Ab2s developed Ab3s that recognized the variable regions of the Ab2s as well as the natural antigen. In cats challenged with a lethal dose of virus the control group followed a predictable course of infection ultimately succumbing to FIP. Two immunized cats survived virus challenge and a third cat lived twice as long as the controls. The fourth immunized cat showed no evidence of protection. The ability to induce levels of protection against FIP lends support to the concept of using anti-idiotypic antibodies as a prophylactic vaccine.

Protection against lethal Sendai virus infection by in vivo priming of virus-specific cytotoxic T lymphocytes with a free synthetic peptide.

The only peptide of Sendai virus that is recognized by cytotoxic T lymphocytes (CTL) in B6 mice was found with (i) the use of recombinant vaccinia virus constructs containing separate genes of Sendai virus and (ii) a set of overlapping peptides completely spanning the identified nucleoprotein (NP) gene product. This immunodominant NP peptide is recognized by Sendai virus-specific CTL that are known to have therapeutic effects in vivo. By subcutaneous immunization, this peptide induced Sendai virus and NP peptide-specific CTL memory responses in vivo. Most importantly, mice that had been immunized with this peptide were protected against a lethal virus dose, indicating that viral peptides can be used as antiviral T-cell vaccines. The induction of T-cell memory by free peptide immunization potentially has wide applicability in biology and medicine, including protection against infectious disease.

Cooperation between cytotoxic and helper T lymphocytes in protection against lethal Sendai virus infection. Protection by T cells is MHC-restricted and MHC-regulated; a model for MHC-disease associations.

The in vivo importance of class I MHC regulation of the Tc response to a natural pathogenic agent of high virulence was studied on the basis of our previous demonstration of a major difference in the capacity to generate a Sendai virus-specific Tc response between C57BL/6 (B6, H-2b) mice and H-2Kb mutant B6.C-H-2bm1 (bm 1) mice. These two mouse strains differ from each other only in three amino acids in the crucial H-2Kb restriction element for this response. bm 1 mice, in contrast to B6 mice, are Tc nonresponders against this virus, but show Sendai-specific T cell proliferation, antibody production, and DTH reactions, as well as NK cell activity, equal to those of B6 mice. B6, Sendai Tc-deficient bm 1 and T cell-deficient B6 nu/nu mice differ from each other in susceptibility to lethal pneumonia induced by i.n. inoculation of virulent Sendai virus. The lethal dose (LD50) in B6 mice averaged 152 TCID50, in bm 1 mice, 14 TCID50 and in B6 nu/nu mice 0.5 TCID50. The importance of Tc was also shown by the complete protection of B6 nu/nu mice against infection with a lethal virus dose by i.v. injection of a Sendai virus-specific, IL-2-dependent and H-2Kb-restricted B6 Tc clone. In vivo protection by this Tc clone was H-2Kb-restricted. Apart from Tc, an important role for virus-specific Th cells is evident from the difference in susceptibility between bm 1 and B6 nu/nu mice. This conclusion was supported by the demonstration that the mean survival time of B6 nu/nu and bm 1 nu/nu mice could be significantly prolonged, in an I-Ab-restricted manner, by the injection of in vitro-propagated, Sendai-specific B6 or bm 1 Th clones after a lethal dose of Sendai virus, and by the demonstration that inoculation of these Th clones provided help to virus-specific Tc by means of IL-2 production. Strikingly, Th and Tc cooperate in anti-Sendai virus immunity, since permanent survival of lethally infected nu/nu mice was only achieved by inoculation of a mixture of Tc and Th clones or a mixture of a Tc clone and rIL-2. This study provides a unique model for the study of MHC-disease associations.

Protection against lethal challenge of BALB/c mice by passive transfer of monoclonal antibodies to five glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies secreted by six hybridomas and recognizing antigenic sites on glycoproteins gC, gAB, gD, gE, and gF of herpes simplex virus type 2 were examined for their ability to protect BALB/c mice from lethal infection by the virus. Administration of monoclonal antibodies to individual glycoproteins intraperitoneally 3 h before footpad challenge with 10 times the 50% lethal dose of virus protected between 35 and 75% of the mice, except for one of two monoclonal antibodies recognizing antigens on gC. The antibodies did not neutralize virus in vitro and protected A/J mice deficient in the fifth component of complement as efficiently as complement-sufficient BALB/c mice. A good correlation was found between protection and titers of monoclonal antibodies assessed by antibody-dependent cell-mediated cytolysis. The results indicate that any of the glycoproteins can serve as antigens for a protective immune response. In addition, the data are compatible with protection being mediated by an antibody-dependent cell-mediated cytolysis mechanism.

Inactivation of rabies virus in reagents used for the fluorescent rabies antibody test.

Procedures for inactivating rabies virus in reagents used for the fluorescent rabies antibody test are described. Mouse brain adsorbing suspensions containing greater than or equal to 10(9) 50% lethal doses of virus per ml were rendered noninfectious by treatment with 0.1% beta-propiolactone or by heating at 56 degrees for greater than or equal to 30 min. Viable virus in tissue impression smears was inactivated by acetone fixation at 50 degrees C for greater than or equal to 30 min or by immersion in 0.1% beta-propiolactone at 37 degrees C for 2 h. Inactivated reagents gave specific and sensitive reactions in the fluorescent rabies antibody test.

Cells mediating delayed-type hypersensitivity in the lungs of mice infected with an influenza A virus.

Effector cells that demonstrate delayed-type hypersensitivity (DTH) on transfer with antigen to naive mice can be recovered from the lungs of mice inoculated intranasally 6 days earlier with a lethal dose (usually 5x10(1)EID50) of influenza A virus. The activity recovered was proportional to the dose of virus instilled intranasally and the extent of lung consolidation observed. Active cells could also be recovered from the draining lymph nodes and from the peripheral blood. The effector cells were identified as T lymphocytes of Ly 1 phenotype and required I-region sharing between donor and recipient for activity to be elicited. They were cross-reactive within the A group of influenza viruses. Two experiments are reported in which immune cell preparations that expressed DTH activity but had very little cytotoxic T cell activity were transferred to mice inoculated 1 or 2 days earlier with a lethal dose of virus. The mice were not protected from death, and in both experiments, the recipient mice died more rapidly than the controls. These results contrast with earlier results in which cell preparations with high cytotoxic T-cell activity were shown to protect recipient infected mice from death.

Vector competence of colonized Culiseta melanura (Diptera: Culicidae) for Western equine encephalomyelitis virus.

The susceptibility of Culiseta melanura to infection with strains of Western equine encephalomyelitis virus isolated in the eastern United States was determined by intrathoracic inoculation, by feeding on blood-virus mixtures through a membrane, and by feeding on viremic chicks. To infect 50% of the Cs. melanura by the inoculation route, 0.05 baby mouse median lethal doses of virus were necessary. Cs. melanura was the most sensitive of 8 mosquito species compared by this technique. By membrane feeding and by feeding on viremic chicks. ca 3.7 logs and ca 1.5 logs, respectively, of virus were necessary to initiate infection in 50% of the Cs. melanura . This species was capable of transmitting virus as early as 6 days following an infectious blood meal, and of the infected females held for 14 or more days at 27°C that refed on chicks, 86% successfully transmitted virus. The kinetics of virus replication in Cs. melanura following inoculation was studied at 18° and 27°C.

Cytotoxic T Cells in the Lungs of Mice Infected with an Influenza A Virus

Cytotoxic T cells are present in the lungs and the bronchoalveolar washings of mice infected intravenously (i.v.) or intranasally (i.n.) with live influenza A/WSN virus. After i.v. injection, cytotoxic T cell activity in both spleens and lungs reaches a peak at 6 days when the level of infectious virus recovered from the lungs falls sharply and the mice do not die. If a lethal dose of virus is given intranasally, very high levels of virus appear rapidly in the lungs, and the development of lung consolidation follows slightly behind the appearance of cytotoxic T cells there. When a non-lethal dose of virus is given intranasally, lower levels of virus are found in the lung and the appearance of cytotoxic T cells is delayed. These results suggest that the cytotoxic T cells play a protective role if the level of virus in the lungs does not reach very high levels. After injection of antithymocyte serum, the subsequent level of cytotoxic T cell activity in the lungs was greatly reduced, suggesting that the T cells recovered in lungs had at an earlier stage been circulating cells. However, splenectomized mice develop high levels of cytotoxic T cell activity, after intranasal infection of mice, indicating that the spleen did not contribute substantially to the T cells recovered in the lungs.

Maternal vaccination with formalin-inactivated Gross lymphoma virus in rats and transfer of immunity to offspring.

SummaryNewborn rats are highly susceptible to viral induction of lymphoma, and become naturally resistant after 2 wk of age. Protection during this period can be conferred by transfer of maternal immunity which occurs pre- and postnatally, by transplacental and milk passage.Vaccination of mothers was effectively achieved with formalin-inactivated Gross lymphoma virus (GLV), resulting in passive immunization of the progeny. Only 5% of the offspring of vaccinated mothers had lymphoma when injected with a lethal dose of virus, as compared to 90% of the offspring of nonvaccinated mothers. Complete neutralization of GLV in vitro with serum of vaccinated mothers was demonstrated by subsequent bioassays.Vaccination of females with an inactivated oncogenic virus represents a model which may also be applicable to the prevention of oncogenesis in other animal systems.

Requirement for theta-bearing cells in lymphocytic choriomeningitis virus-induced central nervous system disease.

THE immunopathological basis of the acutely fatal convulsive central nervous system (CNS) disease produced in adult mice following cerebral infection with lymphocytic Choriomeningitis (LCM) virus is well established1. Thus, a variety of immuno-suppressive treatments, given to adult mice inoculated intracerebrally with a potentially lethal dose of virus, produce a transient or permanent ablation of both the histological and clinical correlates of LCM. Permanently protected mice usually maintain high virus concentrations in their brains and blood for life2,3, presenting a picture which, on virological grounds, is strikingly similar to the lifelong virus carrier state arising in mice inoculated with LCM virus shortly after birth3,4.