RESUMEN
The immune system is tasked with defending against a myriad of microbial infections, and its response to a given infectious microbe may be strongly influenced by coinfection with another microbe. It was shown that infection of mice with lactate dehydrogenase-elevating virus (LDV) impairs early adaptive immune responses to Friend virus (FV) coinfection. To investigate the mechanism of this impairment, we examined LDV-induced innate immune responses and found LDV-specific induction of IFN-α and IFN-γ. LDV-induced IFN-α had little effect on FV infection or immune responses, but unexpectedly, LDV-induced IFN-γ production dampened Th1 adaptive immune responses and enhanced FV infection. Two distinct effects were identified. First, LDV-induced IFN-γ signaling indirectly modulated FV-specific CD8+ T cell responses. Second, intrinsic IFN-γ signaling in B cells promoted polyclonal B cell activation and enhanced early FV infection, despite promotion of germinal center formation and neutralizing Ab production. Results from this model reveal that IFN-γ production can have detrimental effects on early adaptive immune responses and virus control.
Asunto(s)
Inmunidad Adaptativa , Regulación hacia Abajo/inmunología , Interferón gamma/fisiología , Virus de la Leucemia Murina/inmunología , Infecciones por Retroviridae/inmunología , Inmunidad Adaptativa/genética , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Femenino , Virus de la Leucemia Murina de Friend/inmunología , Virus de la Leucemia Murina de Friend/patogenicidad , Interferón gamma/deficiencia , Interferón gamma/genética , Virus Elevador de Lactato Deshidrogenasa/inmunología , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Virus de la Leucemia Murina/patogenicidad , Leucemia Experimental/genética , Leucemia Experimental/inmunología , Leucemia Experimental/virología , Ratones , Ratones Congénicos , Ratones Endogámicos A , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/patología , Virus Formadores de Foco en el Bazo/inmunología , Virus Formadores de Foco en el Bazo/patogenicidad , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/virologíaRESUMEN
The effect of mouse infection with lactate dehydrogenase-elevating virus (LDV), a usually non-pathogenic virus, on concomitant bacterial endotoxin shock was analyzed, in terms of lethality and cytokine production. A strong enhancement of susceptibility to the shock was observed in mice acutely infected with this virus. It correlated with a sharp increase of tumor necrosis factor and leukemia inhibitory factor production and was controlled by the mouse genetic background. The viral infection led to an imbalance in the cytokine response to LPS, with an enhancement of pro-inflammatory cytokines, including IL-18 and IFN-gamma and a delayed secretion of anti-inflammatory IL-10 that could result in exacerbated macrophage activation. Enhanced IFN-gamma production was involved in the virus-induced susceptibility to shock. In sharp contrast with other viral infections, IFN-alpha/beta diminished IFN-gamma production and the resulting increased response to LPS in LDV-infected animals.
Asunto(s)
Infecciones por Arterivirus/inmunología , Endotoxinas/toxicidad , Interferones/inmunología , Virus Elevador de Lactato Deshidrogenasa/inmunología , Choque Séptico/inmunología , Animales , Infecciones por Arterivirus/virología , Citocinas/inmunología , Femenino , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBAAsunto(s)
Infecciones por Arterivirus/patología , Interferón gamma/fisiología , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Animales , Infecciones por Arterivirus/metabolismo , Enfermedades Virales del Sistema Nervioso Central/virología , Citocinas/metabolismo , Epítopos/química , Inflamación , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Replicación ViralRESUMEN
Viral infections are involved in the pathogenesis of blood autoimmune diseases such as hemolytic anemia and thrombocytopenia. Although antigenic mimicry has been proposed as a major mechanism by which viruses could trigger the development of such diseases, it is not easy to understand how widely different viruses might induce these blood autoimmune diseases by this sole mechanism. In mice infected with lactate dehydrogenase-elevating virus (LDV), or mouse hepatitis virus, and treated with anti-erythrocyte or anti-platelet monoclonal autoantibodies at a dose insufficient to induce clinical disease by themselves, the infection sharply enhances the pathogenicity of autoantibodies, leading to severe anemia or thrombocytopenia. This effect is observed only with antibodies that induce disease through phagocytosis. Moreover, the phagocytic activity of macrophages from infected mice is increased and the enhancing effect of infection on autoantibody-mediated pathogenicity is strongly suppressed by treatment of mice with clodronate-containing liposomes. Finally, the disease induced by LDV after administration of autoantibodies is largely suppressed in animals deficient for gamma-interferon receptor. Together, these observations suggest that viruses may trigger autoantibody-mediated anemia or thrombocytopenia by activating macrophages through gamma-interferon production, a mechanism that may account for the pathogenic similarities of multiple infectious agents.
Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Anemia Hemolítica Autoinmune/virología , Autoanticuerpos/toxicidad , Púrpura Trombocitopénica Idiopática/inmunología , Púrpura Trombocitopénica Idiopática/virología , Virosis/complicaciones , Animales , Modelos Animales de Enfermedad , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Macrófagos/inmunología , Ratones , Fagocitosis/inmunologíaRESUMEN
Age-dependent poliomyelitis (ADPM) or murine amyotrophic lateral sclerosis (ALS) is a murine paralytic disease triggered in immunosuppressed genetically-susceptible mice by infection with the arterivirus lactate dehydrogenase-elevating virus (LDV). This disease provides an animal model for ALS, affecting anterior horn neurons and resulting in neuroparalysis 2-3 weeks after LDV infection. We have tested the hypothesis that spinal cord apoptosis is a feature of the LDV-induced murine ALS, since apoptosis is postulated to be a causal factor in human ALS. Gene microarray analyses of spinal cords from paralyzed animals revealed upregulation of several genes associated with apoptosis. Spinal cord apoptosis was investigated further by TUNEL and activated caspase-3 assays, and was observed to emerge concurrent with paralytic symptoms in both neuronal and non-neuronal cells. Caspase-3-dependent apoptosis was also triggered in cultured macrophages by neurovirulent LDV infection. Thus, virus-induced spinal cord apoptosis is a pre-mortem feature of ADPM, which affects both neuronal and support cells, and may contribute to the pathogenesis of this ALS-like disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Apoptosis , Infecciones por Arterivirus/patología , Virus Elevador de Lactato Deshidrogenasa/fisiología , Macrófagos/virología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Infecciones por Arterivirus/fisiopatología , Infecciones por Arterivirus/virología , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Ratones , Ratones Endogámicos , Médula Espinal/patologíaRESUMEN
Lactate dehydrogenase-elevating virus (LDV) is a macrophage-tropic arterivirus which generally causes a persistent viremic infection in mice. LDV replication in vivo seems to be primarily regulated by the extent and dynamics of a virus-permissive macrophage population. Previous studies have shown that glucocorticoid treatment of chronically LDV-infected mice transiently increases viremia 10-100-fold, apparently by increasing the productive infection of macrophages. We have further investigated this phenomenon by comparing the effect of dexamethasone on the in vivo and in vitro replication of two LDV quasispecies that differ in sensitivity to immune control by the host. The single neutralizing epitope of LDV-P is flanked by two N-glycans that impair its immunogenicity and render LDV-P resistant to antibody neutralization. In contrast, replication of the neuropathogenic mutant LDV-C is suppressed by antibody neutralization because its epitope lacks the two protective N-glycans. Dexamethasone treatment of mice 16 h prior to LDV-P infection, or of chronically LDV-P infected mice, stimulated viremia 10-100-fold, which correlated with an increase of LDV permissive macrophages in the peritoneum and increased LDV infected cells in the spleen, respectively. The increase in viremia occurred in the absence of changes in total anti-LDV and neutralizing antibodies. The results indicate that increased viremia was due to increased availability of LDV permissive macrophages, and that during a chronic LDV-P infection virus replication is strictly limited by the rate of regeneration of permissive macrophages. In contrast, dexamethasone treatment had no significant effect on the level of viremia in chronically LDV-C infected mice, consistent with the view that LDV-C replication is primarily restricted by antibody neutralization and not by a lack of permissive macrophages. beta-Glucan, the receptor of which is induced on macrophages by dexamethasone treatment, had no effect on the LDV permissiveness of macrophages.
Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Virus Elevador de Lactato Deshidrogenasa/efectos de los fármacos , Virus Elevador de Lactato Deshidrogenasa/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/virología , Replicación Viral/efectos de los fármacos , Animales , Anticuerpos Antivirales/biosíntesis , Infecciones por Arterivirus/inmunología , Infecciones por Arterivirus/virología , Femenino , Virus Elevador de Lactato Deshidrogenasa/inmunología , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Ratones , Pruebas de Neutralización , Bazo/efectos de los fármacos , Bazo/virologíaRESUMEN
Maternal-to-fetal transmission of the murine lactate dehydrogenase-elevating virus (LDV) has been previously shown to be regulated by maternal immunity as well as gestational age. For the present study, the role of maternal immunity in placental and umbilical cord virus protection was studied, and virus targeting of umbilical cord and fetal macrophages was correlated with expression of the F4/80 macrophage phenotypic marker. The results showed that LDV-infected macrophages appeared in umbilical cord by 24 h post-infection of pregnant mice, and some LDV-infected macrophages displayed the F4/80 phenotype. This potential reservoir of virus for the fetus was inhibited by passive immunization of pregnant mice with IgG anti-LDV antibodies, which rapidly concentrated in the placenta and umbilical cord. Probing of umbilical cord cells with antibodies directed at MHC genetic markers demonstrated the presence of both maternal and fetal cells in umbilical cords. A strong developmental correlation was observed between fetal F4/80 expression and LDV susceptibility, at about 13.6 days of gestation. These results demonstrate immune suppression of free and cell-associated virus in umbilical cord, thus defining a potentially important mechanism for immune protection of the fetus from transplacental virus infection. The results also clarify the developmental basis for fetal susceptibility to LDV infection.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Infecciones por Arterivirus/transmisión , Sangre Fetal/virología , Transmisión Vertical de Enfermedad Infecciosa , Virus Elevador de Lactato Deshidrogenasa/inmunología , Intercambio Materno-Fetal/inmunología , Animales , Animales no Consanguíneos , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Antígenos de Diferenciación/inmunología , Infecciones por Arterivirus/inmunología , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/virología , Femenino , Sangre Fetal/inmunología , Enfermedades Fetales/inmunología , Enfermedades Fetales/virología , Inmunización Pasiva , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Virus Elevador de Lactato Deshidrogenasa/fisiología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/virología , Ratones , Ratones Endogámicos ICR , Embarazo , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/virología , Viremia/inmunologíaRESUMEN
The common quasispecies of lactate dehydrogenase-elevating virus (LDV), LDV-P and LDV-vx, are highly resistant to the humoral host immune response because the single neutralization epitope on the ectodomain of the primary envelope glycoprotein, VP-3P, carries three large N-glycans. Two laboratory mutants, LDV-C and LDV-v, have lost two of the N-glycans on the VP-3P ectodomain, thereby gaining neuropathogenicity for AKR/C58 mice but at the same time, becoming susceptible to the humoral immune response of the host. In attempts to further assess the origins and evolution of these LDVs we have determined their competitiveness by monitoring their fate in mixed infections of wild type, SCID, nude, and cyclophosphamide-treated mice by reverse transcription/polymerase chain reaction assays that distinguish between them. In mixed infections with LDV-P and LDV-vx, LDV-C and LDV-v became rapidly lost even when present initially in large excess over the former. In mixed infections of mice unable to generate neutralizing antibodies, LDV-C and LDV-v also became replaced by LDV-P and LDV-vx as predominant quasispecies but more slowly than in immunocompetent mice. The results indicate that the humoral immune response plays an important role in the displacement of LDV-C and LDV-v by LDV-P and LDV-vx but that in addition, LDV-C and LDV-v possess an impaired ability to compete with LDV-P and LDV-vx in the productive infection of the subpopulation of macrophages that represents the host for all these LDVs. In addition, LDV-v outcompeted LDV-C in mixed infections and the same was the case for neutralization escape mutants of LDV-v and LDV-C which had regained all three N-glycosylation sites on the VP-3P ectodomain. Thus a hierarchy exists in replication fitness: LDV-P/LDV-vx>LDV-v>LDV-C, which is unrelated to the number of N-glycans on the VP-3P ectodomain. The implications of the results in relation to the evolution and selection of the LDV-quasispecies is discussed. LDV-P and LDV-vx are genetically highly stable and thus seem to have achieved evolutionary stasis with optimum ability to establish viremic persistent infections of mice that are unimpeded by the host immune responses.
Asunto(s)
Infecciones por Arterivirus/virología , Virus Elevador de Lactato Deshidrogenasa/fisiología , Replicación Viral , Secuencia de Aminoácidos , Animales , Infecciones por Arterivirus/sangre , Infecciones por Arterivirus/inmunología , Ciclofosfamida/administración & dosificación , Inmunosupresores/administración & dosificación , Virus Elevador de Lactato Deshidrogenasa/genética , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Factores de Tiempo , Proteínas del Envoltorio Viral/genéticaRESUMEN
The complexity of cytokine regulation and the imbalance of helper T (Th)1 and Th2 subsets in systemic lupus erythematosus (SLE) animal models and human SLE are well recognized. In this study in NZBxNZWF(1)mice, the effects of lactic dehydrogenase virus (LDV) infection on the production of interferon (IFN)-gamma in the serum and the development of autoimmune disease were examined. The progress of the disease (the development of glomerulonephritis, formation of glomerular IgG and C3 deposits, increase in the blood urea nitrogen values, and mortality) was parallel with an increase in serum IFN-gamma in uninfected NZBxNZWF(1)mice. These changes were inhibited in LDV-infected NZBxNZWF(1)mice. Our findings suggest that increase in serum IFN-gamma may be associated with the active disease in NZBxNZWF(1)mice.
Asunto(s)
Infecciones por Arterivirus/complicaciones , Virus Elevador de Lactato Deshidrogenasa/fisiología , Lupus Eritematoso Sistémico/virología , Animales , Infecciones por Arterivirus/mortalidad , Infecciones por Arterivirus/patología , Nitrógeno de la Urea Sanguínea , Complemento C3/metabolismo , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Técnica del Anticuerpo Fluorescente Directa , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Glomerulonefritis/virología , Inmunoglobulina E/sangre , Inmunoglobulina G/metabolismo , Interferón gamma/sangre , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Lupus Eritematoso Sistémico/mortalidad , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos NZB , Tasa de SupervivenciaRESUMEN
Development of polioencephalomyelitis in mice infected with lactate dehydrogenase-elevating virus (LDV) requires expression of N-tropic ecotropic MuLV retroviruses. 129/Sv mice are resistant to N-tropic MuLV expression and therefore do not develop LDV-induced polioencephalomyelitis. The Fv1 gene determines the susceptibility to retrovirus replication. We sequenced the open reading frame of the Fv1nr allele of 129/Sv mice. It differs by only one nucleotide, modifying one amino acid in the encoded protein, from the Fv1n allele of susceptible AKR and C58 animals. We excluded that the resistance of 129/Sv mice to LDV-induced polioencephalomyelitis resulted from the absence of endogenous N-tropic retrovirus, by infecting (129/Sv x C58/J) F1 animals. Therefore it is possible that the amino acid that defines the Fv1nr allele is responsible for resistance of 129/Sv mice to N-tropic MuLV expression and to LDV-induced polioencephalomyelitis.
Asunto(s)
Alelos , Infecciones por Arterivirus/genética , Proteínas de Ciclo Celular , Predisposición Genética a la Enfermedad , Virus Elevador de Lactato Deshidrogenasa/genética , Proteínas de Neoplasias , Poliomielitis/etiología , Proteínas/genética , Animales , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/patogenicidad , Ratones , Ratones Endogámicos , Datos de Secuencia MolecularRESUMEN
Common strains of lactate dehydrogenase-elevating virus (LDV, an arterivirus), such as LDV-P and LDV-vx, are highly resistant to antibody neutralization and invariably establish a viremic, persistent, yet asymptomatic, infection in mice. Other LDV strains, LDV-C and LDV-v, have been identified that, in contrast, are highly susceptible to antibody neutralization and are incapable of a high viremic persistent infection, but at the same time have gained the ability to cause paralytic disease in immunosuppressed C58 and AKR mice. Our present results further indicate that these phenotypic differences represent linked properties that correlate with the number of N-glycosylation sites associated with the single neutralization epitope on the short ectodomain of the primary envelope glycoprotein, VP-3P. The VP-3P ectodomains of LDV-P/vx possess three N-glycosylation sites, whereas those of LDV-C/v lack the two N-terminal sites. We have now isolated four independent neutralization escape variants of neuropathogenic LDV-C and LDV-v on the basis of their ability to establish a high viremic persistent infection in mice. The VP-3P ectodomains of all four variants had specifically regained two N-glycosylation sites concomitant with decreased immunogenicity of the neutralization eptitope and decreased sensitivity to antibody neutralization as well as loss of neuropathogenicity.
Asunto(s)
Amino Azúcares/química , Infecciones por Arterivirus/virología , Virus Elevador de Lactato Deshidrogenasa/inmunología , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Neuronas/virología , Polisacáridos/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Infecciones por Arterivirus/patología , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Virus Elevador de Lactato Deshidrogenasa/genética , Glicoproteínas de Membrana , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Viremia , Replicación ViralRESUMEN
Neuropathogenic lactate dehydrogenase-elevating viruses (LDV) cytocidally infect anterior horn neurons in C58 and AKR mice via interaction with endogenous murine retroviruses to cause a paralytic disease, age-dependent poliomyelitis (ADPM). The induction of ADPM requires a suppressed host immune system as a result of old age, genetic defects (such as nude mice) or any immunosuppressive treatment. Previous results have shown that the infection of anterior horn neurons by neuropathogenic LDV isolates and the subsequent development of ADPM are prevented by anti-LDV antibodies either induced actively during infection or when passively administered. However, the mechanism of protection was unclear since both neutralizing and non-neutralizing polyclonal antibodies seemed protective, whereas only neutralizing monoclonal antibodies were protective. Furthermore, the protection of motor neurons from infection occurred in the absence of any apparent effect on LDV replication in a subpopulation of macrophages known to be the primary permissive host cells. These paradoxes have now been resolved. We have recently reported that the neuropathogenic LDV isolates contain both neuropathogenic and non-neuropathogenic quasispecies that differ in their ability to establish a high viremia persistent infection. Using biological clones of both neuropathogenic and non-neuropathogenic quasispecies, we now demonstrate that both replicate in the same subpopulation of permissive macrophages, but that the neuropathogenic quasispecies are about 100 times more susceptible to in vitro antibody neutralization than the non-neuropathogenic ones, and that antibodies that neutralize the neuropathogenic but not the non-neuropathogenic quasispecies develop as soon as 7 days after infection with neuropathogenic LDVs and selectively suppress the replication of the neuropathogenic LDVs in vivo in FVB, BALB/c, C57 BL/6 and C58 mice. The previously observed lack of neutralizing effect of early polyclonal anti-LDV antibodies and the apparent ineffective antibody control of LDV replication in macrophages were due to outgrowth of the non-neuropathogenic quasispecies that are also present in the neuropathogenic LDV inoculum and are highly resistant to antibody neutralization. Using cloned neuropathogenic LDV quasispecies, we demonstrate a clear relationship in the development of neutralizing antibodies, replication suppression of the neuropathogenic LDVs and the prevention of ADPM in C58 mice. Our results therefore establish an inseparable relationship between the neuron-protective effect of an antibody and its neutralization of the neuropathogenic LDV quasispecies and explain why neuropathogenic LDVs cause paralytic disease only in immunosuppressed mice.
Asunto(s)
Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Parálisis/virología , Sobreinfección/virología , Factores de Edad , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/metabolismo , Especificidad de Anticuerpos , Técnica del Anticuerpo Fluorescente , Virus Elevador de Lactato Deshidrogenasa/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Parálisis/prevención & control , Factores de Tiempo , Viremia/tratamiento farmacológico , Replicación Viral/inmunologíaRESUMEN
On the basis of genome nucleotide differences between a nonneuropathogenic and a neuropathogenic lactate dehydrogenase-elevating virus (LDV) quasispecies (LDV-P and LDV-C, respectively), we have designed sets of primers for polymerase chain reaction (PCR) amplification that can detect recombinants between them in a 1276-nt-long segment ranging from ORF 5 to ORF 7. Mice were infected with large amounts of both LDVs and bled at various times postinfection (p.i.). RNA was extracted from plasma samples and reverse transcribed and the first-strand products were PCR amplified with four sets of sense and antisense primers that discriminate between parental (P/P and C/C) and recombinant (P/C and C/P) genomic segments. Both P/C and C/P recombinants were detected in plasma from six different mice at 1 day p.i. No recombinant products were generated with in vitro mixtures of LDV-P and LDV-C. End-point dilution experiments indicated that the generation of P/C and C/P recombinants varied between mice but that in some mice the frequency of recombination in the 1276-nt-long genome segment was as high as 5%. Sequence analyses of clones of some recombinants indicated that recombination had occurred at 26- to 43-nt-long stretches of homology between the LDV-P and the LDV-C genomes. Sequence analyses of the 3157-nt-long 3' end of the genomes of the neuropathogenic LDV-v and of a newly discovered nonneuropathogenic quasispecies, LDV-vx, showed that LDV-v is a natural recombinant of LDV-vx that has specifically acquired by a double recombination about 400 nt of the 5' end of ORF 5 of the neuropathogenic LDV-C and thereby the unique properties of LDV-C, neuropathogenicity and high sensitivity to antibody neutralization. In dual infections of mice with LDV-P and LDV-C all genetic recombinants, like the LDV-C parent itself, had been lost by 7 days p.i., and only LDV-P persisted. The results further support the view that LDV-P and LDV-vx have evolved to a highly stable relationship with their host, the mouse.
Asunto(s)
Evolución Molecular , Variación Genética , Virus Elevador de Lactato Deshidrogenasa/genética , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Recombinación Genética , Animales , Secuencia de Bases , ADN Viral , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia MolecularRESUMEN
Neuropathogenic isolates of lactate dehydrogenase virus (LDV) differ from non-neuropathogenic isolates in their unique ability to cause a paralytic disease (age-dependent poliomyelitis, ADPM) in immunosuppressed C58 and AKR mice by cytocidally infecting their anterior horn neurons. We have recently reported that an original neuropathogenic LDV isolate, LDV-C-BR, contained a low level of a coexisting non-neuropathogenic LDV which, in a mixed infection of mice, rapidly outcompeted the former resulting in apparent loss of neuropathogenicity of the reisolated LDV. This correlated with an impaired ability of the neuropathogenic LDV to establish a viremic persistent infection. In the present study we identified the presence of three different quasispecies in another original neuropathogenic LDV by sequence analysis of cDNA clones of ORF 5 (encoding the primary envelope glycoprotein VP-3P) obtained from the isolate. Successful development of differential reverse transcription-polymerase chain reaction assays allowed us to biologically clone all three quasispecies through repeated end point dilutions. Only one of the quasispecies (LDV-v) was neuropathogenic. The other two, LDV-vP (probably the same as LDV-P) and LDV-vx (a novel LDV quasispecies that had not been previously identified), were non-neuropathogenic and found to be the common LDV quasispecies associated with almost all LDVs originally isolated from mice carrying various other transplantable tumors. The neuropathogenic LDV-v became selectively amplified in the spinal cords of paralyzed mice, but possessed an impaired ability to establish a persistent viremic infection and was rapidly out-competed by LDV-vP and LDV-vx in mixed infections, just as reported previously for LDV-C-BR. The results further support our hypothesis that neuropathogenicity and impaired capability for viremic persistence of LDV are determined by the same molecular feature. The only consistent and biologically relevant molecular difference we have observed between neuropathogenic and non-neuropathogenic LDVs is the number of polylactosaminoglycan chains associated with the ectodomain of VP-3P.
Asunto(s)
Infecciones por Arterivirus/virología , Variación Genética , Virus Elevador de Lactato Deshidrogenasa/genética , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Enfermedades del Sistema Nervioso/virología , Viremia , Secuencia de Aminoácidos , Animales , Infecciones por Arterivirus/patología , Secuencia de Bases , Clonación Molecular , Glicosilación , Ratones , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Proteínas Virales/genéticaRESUMEN
Neuropathogenic isolates of lactate dehydrogenase-elevating virus (LDV) differ from nonneuropathogenic isolates in their unique ability to infect anterior horn neurons of immunosuppressed C58 and AKR mice and cause paralytic disease (age-dependent poliomyelitis [ADPM]). However, we and others have found that neuropathogenic LDVs fail to retain their neuropathogenicity during persistent infections of both ADPM-susceptible and nonsusceptible mice. On the basis of a segment in open reading frame 2 that differs about 60% between the neuropathogenic LDV-C and the nonneuropathogenic LDV-P, we have developed a reverse transcription-PCR assay that distinguishes between the genomes of the two LDVs and detects as little as 10 50% infectious doses (ID50) of LDV. With this assay, we found that LDV-P and LDV-C coexist in most available pools of LDV-C and LDV-P. For example, various plasma pools of 10(9.5) ID50 of LDV-C/ml contained about 10(5) ID50 of LDV-P/ml. Injection of such an LDV-C pool into mice of various strains resulted in the rapid displacement in the circulation of LDV-C by LDV-P as the predominant LDV, but LDV-C also persisted in the mice at a low level along with LDV-P. We have freed LDV-C of LDV-P by endpoint dilution (LDV-C-EPD). LDV-C-EPD infected mice as efficiently as did LDV-P, but its level of viremia during the persistent phase was only 1/10,000 that observed for LDV-P. LDV-permissive macrophages accumulated and supported the efficient replication of superinfecting LDV-P. Therefore, although neuropathogenic LDVs possess the unique ability to infect anterior horn neurons of ADPM-susceptible mice, they exhibit a reduced ability to establish a persistent infection in peripheral tissues of mice regardless of the strain. The specific suppression of LDV-C replication in persistently infected mice is probably due in part to a more efficient neutralization of LDV-C than LDV-P by antibodies to the primary envelope glycoprotein, VP-3P. Both neuropathogenicity and the higher sensitivity to antibody neutralization correlated with the absence of two of three N-linked polylactosaminoglycan chains on the ca. 30-amino-acid ectodomain of VP-3P, which seems to carry the neutralization epitope(s) and forms part of the virus receptor attachment site.
Asunto(s)
Infecciones por Arterivirus/virología , Variación Genética , Virus Elevador de Lactato Deshidrogenasa/genética , Latencia del Virus , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Viral , Femenino , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Virus Elevador de Lactato Deshidrogenasa/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neuronas/virologíaRESUMEN
ORF 5 encoding the primary envelope glycoprotein, VP-3P, of a highly neuropathogenic isolate of lactate dehydrogenase-elevating virus (LDV-v) has been sequenced. It exhibits 92% nucleotide identity with the ORF 5 of an LDV isolate that lacks neuropathogenicity, LDV-P, and the amino acid identities of the predicted VP-3Ps of the two strains is 90%. Most striking, however, is the absence in the ectodomain of LDV-v VP-3P of two out of three potential N-glycosylation sites present in the ectodomain of VP-3P of LDV-P. The ectodomain of VP-3P has been implicated to play an important role in host receptor interaction. VP-3P of another neuropathogenic LDV strain, LDV-C, lacks the same two N-glycosylation sites (Godeny et al., 1993). In vitro transcription/translation of the ORFs 5 of LDV-P and LDV-v indicated that all three N-glycosylation sites in the ectodomain of LDV-P VP-3P became glycosylated when synthesized in the presence of microsomal membranes, whereas the glycosylation of the ORF 5 proteins of LDV-v and LDV-C was consistent with glycosylation at a single site. No other biological differences between the neuropathogenic and non-neuropathogenic strains have been detected. They replicate with equal efficiency in mice and in primary macrophage cultures.
Asunto(s)
Virus Elevador de Lactato Deshidrogenasa/química , Glicoproteínas de Membrana/química , Proteínas del Envoltorio Viral/química , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , ADN Viral , Perros , Glicosilación , Virus Elevador de Lactato Deshidrogenasa/aislamiento & purificación , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Disulfide bonds were found to link the nonglycosylated envelope protein VP-2/M (19 kDa), encoded by open reading frame 6, and the major envelope glycoprotein VP-3 (25 to 42 kDa), encoded by open reading frame 5, of lactate dehydrogenase-elevating virus (LDV). The two proteins comigrated in a complex of 45 to 55 kDa when the virion proteins were electrophoresed under nonreducing conditions but dissociated under reducing conditions. Furthermore, VP-2/M was quantitatively precipitated along with VP-3 in this complex by three neutralizing monoclonal antibodies to VP-3. The infectivity of LDV was rapidly and irreversibly lost during incubation with 5 to 10 mM dithiothreitol (> 99% in 6 h at room temperature), which is known to reduce disulfide bonds. LDV inactivation correlated with dissociation of VP-2/M and VP-3. The results suggest that disulfide bonds between VP-2/M and VP-3 are important for LDV infectivity. Hydrophobic moment analyses of the predicted proteins suggest that VP-2/M and VP-3 both possess three adjacent transmembrane segments and only very short ectodomains (10 and 32 amino acids, respectively) with one and two cysteines, respectively. Inactivation of LDV by dithiothreitol and dissociation of the two envelope proteins were not associated with alterations in LDV's density or sedimentation coefficient.
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Disulfuros/química , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Células Cultivadas , Virus Elevador de Lactato Deshidrogenasa/metabolismo , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas del Envoltorio Viral/fisiologíaAsunto(s)
Equartevirus , Virus Elevador de Lactato Deshidrogenasa , Infecciones por Togaviridae/microbiología , Togaviridae , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Equartevirus/patogenicidad , Equartevirus/fisiología , Humanos , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Virus Elevador de Lactato Deshidrogenasa/fisiología , Datos de Secuencia Molecular , ARN Viral , ARN Polimerasa Dependiente del ARN/genética , Togaviridae/patogenicidad , Togaviridae/fisiologíaRESUMEN
Although the majority of mouse strains infected with lactate dehydrogenase-elevating virus (LDV) do not show any particular symptoms, the virus is able to induce acute poliomyelitis in C58 or AKR mice. Murine leukaemia virus (MuLV) has been detected at a high titre in the spinal cord of affected mice. In this study, we have analysed the possible role of MuLV in the induction of neurological disease by LDV. Immunofluorescent staining, autoradiography and an infectivity assay of virus yield have shown that LDV replicated in continuous mouse and rat cell lines that had been infected with an ecotropic MuLV isolated from C58 mice, but did not replicate in cells not infected with MuLV. No significant differences in infection were observed among the various ecotropic MuLVs employed, except for Friend leukaemia virus which rendered the cells susceptible to LDV least efficiently. The infectivity of the neurovirulent strain, LDV-C, to MuLV-infected cells was 50- to 100-fold greater than that of the avirulent strains (LDV-N, -Nu, -R and -P). The infectivity to macrophages was almost the same for virulent and avirulent strains. Adsorption studies using a radiolabelled virus revealed that LDV-C was adsorbed to MuLV-infected cells more efficiently than the avirulent strain, LDV-N. The difference in infectivity to these cells, therefore, may be due in part to the difference in adsorption rate. This may suggest differences in the interaction of the viral proteins with MuLV-infected cells from those with macrophages at the initiation of virus infection. These results may be relevant to the mechanisms of paralytic disease caused by LDV infection in C58 mice.
Asunto(s)
Virus Elevador de Lactato Deshidrogenasa/fisiología , Virus de la Leucemia Murina/fisiología , Replicación Viral , Adsorción , Animales , Línea Celular , Femenino , Técnica del Anticuerpo Fluorescente , Virus Elevador de Lactato Deshidrogenasa/patogenicidad , Macrófagos/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Parálisis/microbiología , Ratas , Interferencia Viral , Virosis/microbiología , Virosis/fisiopatologíaRESUMEN
Over 90% of cyclophosphamide-treated, 6- to 7-month-old C58/M mice developed fatal paralytic disease after infection with a virulent strain of lactate dehydrogenase-elevating virus (LDV), with a mean onset of paralysis of about 16 days. Passive immunization with polyclonal antibodies or with a group of anti-LDV monoclonal antibodies (MAbs) with single-epitope specificity 1 day before or at the time of LDV infection prevented the development of paralytic disease without interfering with the replication of LDV in permissive macrophages, the primary host cells of LDV. In situ hybridization of spinal cord sections with an LDV-specific cDNA probe indicated that the MAb specifically prevented the cytocidal infection of motor neurons by LDV without blocking the infection of smaller nonneuronal cells in the spinal cord. The protective antibodies recognize at least two different epitopes on the glycoprotein of LDV, VP-3. Passive immunizations with other anti-LDV MAbs, which recognize at least three other epitopes on VP-3 of LDV, afforded no protection. In contrast to the protective effect of anti-LDV MAb injection before or at the time of LDV infection, their administration postinfection exerted relatively little protection, though it delayed the appearance of paralytic symptoms. However, repeated injections of MAbs until at least 7 days postinfection also afforded a high degree of protection. The results indicate that protective MAbs may interfere with two stages in the development of LDV-induced paralytic disease. When administered at the time of LDV infection, they prevent the initial infection of spinal cord motor neurons. After this initial event, repeated injections of MAb are required to inhibit the spread of LDV between neurons until the endogenous production of protective anti-LDV antibodies in these mice.