RESUMO
The tropism of Sindbis virus (SB) for cells of the dendritic cell (DC) lineage and the virulence of SB in vivo are largely determined by the efficacy of alpha/beta interferon (IFN-alpha/beta)-mediated antiviral responses. These responses are essentially intact in the absence of PKR and/or RNase L (K. D. Ryman, L. J. White, R. E. Johnston, and W. B. Klimstra, Viral Immunol. 15:53-76, 2002). In the present studies, we investigated the nature of antiviral effects and identity of antiviral effectors primed by IFN-alpha/beta treatment of bone marrow-derived DCs (BMDCs) generated from mice deficient in PKR and RNase L (TD). IFN-alpha/beta priming exerted significant antiviral activity at very early stages of SB replication and most likely inhibited the initial translation of infecting genomes. The early effect targeted cap-dependent translation as protein synthesis from an SB-like and a simple RNA were inhibited by interferon treatment, but an encephalomyocarditis virus internal ribosome entry site-driven element exhibited no inhibition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 was defective after virus infection of TD cells, suggesting other mechanisms of translation inhibition. To identify components of these alternative antiviral pathway(s), we have compared global gene regulation in BMDCs derived from normal 129 Sv/Ev, IFNAR1-/-, and TD mice following infection with SB or treatment with IFN-alpha/beta. Candidate effectors of alternative antiviral pathways were those genes induced by virus infection or IFN-alpha/beta treatment in 129 Sv/Ev and TD-derived BMDC but not in virus-infected or IFN-alpha/beta-treated IFNAR1-/- cells. Statistical analyses of gene array data identified 44 genes that met these criteria which are discussed.
Assuntos
Antivirais/farmacologia , Células Dendríticas/virologia , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Sindbis virus/patogenicidade , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células da Medula Óssea , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Células Dendríticas/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/genética , Proteínas/metabolismo , Proteínas/farmacologia , Proteínas de Ligação a RNA , Sindbis virus/genética , Sindbis virus/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
The spike glycoprotein E2 of Sindbis virus (SIN) is synthesized in the infected cell as a PE2 precursor protein, which matures through cleavage by a cellular furin-like protease. Previous work has shown that SIN mutants impaired in PE2 cleavage are noninfectious on BHK-21 cells, the block in infection being localized at a step after virus-receptor interaction but prior to RNA replication. Here, we studied the membrane fusion properties of SIN PE2 cleavage mutants and observed that these viruses are impaired in their ability to form an E1 homotrimer and to fuse with liposomes at a mildly acidic pH. The block in spike rearrangement and fusion could be overridden by exposure of the mutant viruses to very low pH (<4.5). Cleavage mutants with second-site resuscitating mutations in PE2 were highly infectious for BHK-21 cells. The ability of these viruses to form E1 homotrimers and to fuse at a mildly acidic pH was completely restored despite a sustained lack of PE2 cleavage.
Assuntos
Fusão de Membrana , Glicoproteínas de Membrana/química , Precursores de Proteínas/química , Sindbis virus/patogenicidade , Proteínas do Envelope Viral/química , Animais , Linhagem Celular , Cricetinae , Dimerização , Concentração de Íons de Hidrogênio , Mutação , Proteínas do Envelope Viral/metabolismoRESUMO
Infection of adult 129 Sv/Ev mice with consensus Sindbis virus strain TR339 is subclinical due to an inherent restriction in early virus replication and viremic dissemination. By comparing the pathogenesis of TR339 in 129 Sv/Ev mice and alpha/beta interferon receptor null (IFN-alpha/betaR(-/-)) mice, we have assessed the contribution of IFN-alpha/beta in restricting virus replication and spread and in determining cell and tissue tropism. In adult 129 Sv/Ev mice, subcutaneous inoculation with 100 PFU of TR339 led to extremely low-level virus replication and viremia, with clearance under way by 96 h postinoculation (p.i.). In striking contrast, adult IFN-alpha/betaR(-/-) mice inoculated subcutaneously with 100 PFU of TR339 succumbed to the infection within 84 h. By 24 h p.i. a high-titer serum viremia had seeded infectious virus systemically, coincident with the systemic induction of the proinflammatory cytokines interleukin-12 (IL-12) p40, IFN-gamma, tumor necrosis factor alpha, and IL-6. Replicating virus was located in macrophage-dendritic cell (DC)-like cells at 24 h p.i. in the draining lymph node and in the splenic marginal zone. By 72 h p.i. virus replication was widespread in macrophage-DC-like cells in the spleen, liver, lung, thymus, and kidney and in fibroblast-connective tissue and periosteum, with sporadic neuroinvasion. IFN-alpha/beta-mediated restriction of TR339 infection was mimicked in vitro in peritoneal exudate cells from 129 Sv/Ev versus IFN-alpha/betaR(-/-) mice. Thus, IFN-alpha/beta protects the normal adult host from viral infection by rapidly conferring an antiviral state on otherwise permissive cell types, both locally and systemically. Ablation of the IFN-alpha/beta system alters the apparent cell and tissue tropism of the virus and renders macrophage-DC-lineage cells permissive to infection.
Assuntos
Infecções por Alphavirus/prevenção & controle , Interferon Tipo I/uso terapêutico , Sindbis virus/patogenicidade , Animais , Linhagem Celular , Cricetinae , Citocinas/biossíntese , Glicoproteínas/genética , Macrófagos/virologia , Camundongos , Sindbis virus/genética , Sindbis virus/fisiologia , Tropismo , Virulência , Replicação ViralRESUMO
Binding of yellow fever virus wild-type strains Asibi and French viscerotropic virus and vaccine strains 17D and FNV to monkey brain and monkey liver cell membrane receptor preparations (MRPs) was investigated. Only FNV bound to monkey brain MRPs, while French viscerotropic virus, Asibi, and FNV all bound to monkey liver MRPs. Four monkey brain and two mouse brain MRP escape (MRP(R)) variants of FNV were selected at pH 7.6 and 6.0. Three monkey brain MRP(R) variants selected at pH 7.6 each had only one amino acid substitution in the envelope (E) protein in domain II (E-237, E-260, or E274) and were significantly attenuated in mice following intracerebral inoculation. Two of the variants were tested in monkeys and retained parental neurotropism following intracerebral inoculation at the dose tested. We speculate that this region of domain II is involved in binding of FNV E protein to monkey brain and is, in part, responsible for the enhanced neurotropism of FNV for monkeys. A monkey brain MRP(R) variant selected at pH 6.0 and two mouse brain MRP(R) variants selected at pH 7.6 were less attenuated in mice, and each had an amino acid substitution in the transmembrane region of the E protein (E-457 or E-458).
Assuntos
Encéfalo/virologia , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/metabolismo , Vírus da Febre Amarela/metabolismo , Animais , Encéfalo/metabolismo , Chlorocebus aethiops , Variação Genética , Macaca fascicularis , Camundongos , Conformação Proteica , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vacinas Virais/genética , Vírus da Febre Amarela/genéticaRESUMO
Laboratory strains of viruses may contain cell culture-adaptive mutations which result in significant quantitative and qualitative alterations in pathogenesis compared to natural virus isolates. This report suggests that this is the case with Sindbis virus strain AR339. A cDNA clone comprising a consensus sequence of Sindbis virus strain AR339 has been constructed (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). This clone (pTR339) regenerates a sequence predicted to be very close to that of the original AR339 isolate by eliminating several cell culture-adaptive mutations present in individual laboratory strains of the virus (K. L. McKnight et al., J. Virol. 70:1981-1989, 1996). It thus provides a unique reagent for study of the pathogenesis of Sindbis virus strain AR339 in mice. Neonatal mouse pathogenesis of virus (TR339) generated from the pTR339 clone was compared with that of virus from a cDNA clone of the cell culture-passaged laboratory AR339 strain, TRSB, and virus from a clone of a more highly cell culture-adapted strain, HR(sp) (Toto 50). The sequence of TRSB differs from the consensus at three coding positions, while Toto 50 differs at eight codons and one nucleotide in the 5' nontranslated region. Both cell culture-adapted strains contain mutations associated with heparan sulfate (HS)-dependent attachment to cells (W.B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). TR339 caused 100% mortality with an average survival time (AST) of 1.7 +/- 0.25 days. While TRSB also caused 100% mortality, the AST was extended to 2.9 +/- 0.52 days. The more extensively cell culture-adapted virus Toto 50 caused only 30% mortality with an AST extended to 11.0 +/- 4.8 days. TRSB and TR339 induced high serum levels of alpha/beta interferon, gamma interferon, tumor necrosis factor alpha, interleukin-6, and corticosterone and induced pathology reminiscent of lipopolysaccharide-induced endotoxic shock, a type of systemic inflammatory response syndrome. However, the reduced intensity of this response in TRSB-infected mice correlated with the increased AST. Toto 50 failed to induce the shock-like cytokine cascade. In situ hybridization studies indicated that TR339 and TRSB replicated in identical tissues, but the TRSB signal was less widespread at early times postinfection. While Toto 50 also replicated in similar tissues, the extent of replication was severely restricted and mice developed lesions characteristic of encephalitis. A single mutation in TRSB at E2 position 1 (Arg) conferred HS-dependent attachment to cells and was associated with reduced cytokine induction and extended AST in vivo.
Assuntos
Infecções por Alphavirus/imunologia , Sindbis virus/imunologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Infecções por Alphavirus/patologia , Infecções por Alphavirus/virologia , Animais , Animais Recém-Nascidos , Linhagem Celular , Cricetinae , Citocinas/biossíntese , Feminino , Lipopolissacarídeos/farmacologia , Camundongos , Sindbis virus/genética , Sindbis virus/metabolismo , Sindbis virus/patogenicidade , Síndrome de Resposta Inflamatória Sistêmica/patologia , Síndrome de Resposta Inflamatória Sistêmica/virologia , Virulência , Replicação ViralRESUMO
Six passages of the mosquito-borne flavivirus yellow fever (YF) wild-type strain Asibi in HeLa cells attenuated the virus for monkeys and newborn mice and resulted in loss of mosquito competence. Attenuation after the passage in HeLa cells was not unique to YF virus strain Asibi as demonstrated by the HeLa passage attenuation of wild-type YF virus strain French viscerotropic virus and YF vaccine virus 17D-204 for newborn mice. In contrast, wild-type strain Dakar 1279 and the French neurotropic vaccine virus remained virulent for newborn mice after six passages in HeLa cells. Thus not all strains of YF virus can be attenuated by passage in HeLa cells. Attenuation of YF virus strains Asibi and French viscerotropic virus was accompanied by alterations in the antigenic and biological properties of the viruses, including changes to envelope protein epitopes. Attenuation for newborn mice was coincidental with the acquisition by the HeLa-passaged viruses of the vaccine-specific envelope protein epitope recognized by monoclonal antibody H5. This suggests that this conformational change may play a role in the attenuation process. Wild-type Dakar 1279, which remained virulent for newborn mice after passage in HeLa cells, retained its wild-type antigenic character. The genome of Asibi HeLa p6 virus differed from wild-type Asibi virus by 29 nucleotides that encoded 10 amino acid substitutions: 5 in the envelope protein, 1 in NS2A, 3 in NS4B, and 1 in NS5. The substitution at NS4B-95 is seen in three different attenuation processes of wild-type YF virus, leading us to speculate that it is involved in the attenuation of virulence of wild-type strain Asibi.
Assuntos
Células HeLa/virologia , Febre Amarela/virologia , Vírus da Febre Amarela/fisiologia , Vírus da Febre Amarela/patogenicidade , Animais , Humanos , Camundongos , Especificidade da Espécie , Proteínas do Envelope Viral/fisiologia , Virulência , Replicação ViralRESUMO
The 17D-204 vaccine manufactured in South Africa (17D-204-SA) and a large plaque variant (17D-LP) derived from it were highly virulent in adult mice. The LD50 of 17D-LP virus was 0-2 p.f.u. for mice following intracerebral inoculation. In comparison, a medium plaque variant derived from 17D-LP, termed 17D-MP virus, was found to be attenuated in adult mice following the same route of inoculation (> 10(4) p.f.u./LD50). Replication of 17D-MP virus was decreased in infected mouse brains compared to 17D-LP virus. Also, 17D-MP virus was slightly temperature sensitive at 39.5 degrees C. Compared to its parent viruses, 17D-204-SA and 17D-LP, 17D-MP virus had one unique mutation at nt 8045 in the genome which resulted in a single amino acid substitution (Pro --> Ser) at residue 137 of the NS5 protein and appeared to be the mutation responsible for the attenuation of 17D-MP virus. This is the first time that altered virulence of a flavivirus caused by mutation in a non-structural protein gene, other than NS1, has been reported.
Assuntos
Mutação , Proteínas não Estruturais Virais/genética , Vacinas Virais , Vírus da Febre Amarela/patogenicidade , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/virologia , Linhagem Celular , Chlorocebus aethiops , DNA Viral , Feminino , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Vacinas Atenuadas , Células Vero , Ensaio de Placa Viral , Virulência , Vírus da Febre Amarela/genéticaRESUMO
Attachment of Sindbis virus to the cell surface glycosaminoglycan heparan sulfate (HS) and the selection of this phenotype by cell culture adaptation were investigated. Virus (TR339) was derived from a cDNA clone representing the consensus sequence of strain AR339 (K. L. McKnight, D. A. Simpson, S. C. Lin, T. A. Knott, J. M. Polo, D. F. Pence, D. B. Johannsen, H. W. Heidner, N. L. Davis, and R. E. Johnston, J. Virol. 70:1981-1989, 1996) and from mutant clones containing either one or two dominant cell culture adaptations in the E2 structural glycoprotein (Arg instead of Ser at E2 position 1 [designated TRSB]) or this mutation plus Arg for Ser at E2 114 [designated TRSB-R114]). The consensus virus, TR339, bound to baby hamster kidney (BHK) cells very poorly. The mutation in TRSB increased binding 10- to 50-fold, and the additional mutation in TRSB-R114 increased binding 3- to 5-fold over TRSB. The magnitude of binding was positively correlated with the degree of cell culture adaptation and with attenuation of these viruses in neonatal mice. HS was identified as the attachment receptor for the mutant viruses by the following experimental results. (i) Low concentrations of soluble heparin inhibited plaque formation on and binding of mutant viruses to BHK cells by >95%. In contrast, TR339 showed minimal inhibition at high concentrations. (ii) Binding and infectivity of TRSB-R114 was sensitive to digestion of cell surface HS with heparinase III, and TRSB was sensitive to both heparinase I and heparinase III. TR339 infectivity was only slightly affected by either digestion. (iii) Radiolabeled TRSB and TRSB-R114 attached efficiently to heparin-agarose beads in binding assays, while TR339 showed virtually no binding. (iv) Binding and infectivity of TRSB and TRSB-R114, but not TR339, were greatly reduced on Chinese hamster ovary cells deficient in HS specifically or all glycosaminoglycans. (v) High-multiplicity-of-infection passage of TR339 on BHK cell cultures resulted in rapid coselection of high-affinity binding to BHK cells and attachment to heparin-agarose beads. Sequencing of the passaged virus population revealed a mutation from Glu to Lys at E2 70, a mutation common to many laboratory strains of Sindbis virus. These results suggest that TR339, the most virulent virus tested, attaches to cells through a low-affinity, primarily HS-independent mechanism. Adaptive mutations, selected during cell culture growth of Sindbis virus, enhance binding and infectivity by allowing the virus to attach by an alternative mechanism that is dependent on the presence of cell surface HS.
Assuntos
Heparitina Sulfato/metabolismo , Receptores Virais/metabolismo , Sindbis virus/metabolismo , Células 3T3 , Adaptação Biológica , Infecções por Alphavirus/mortalidade , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Ligação Competitiva , Células CHO , Linhagem Celular , Cricetinae , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Heparina Liase/metabolismo , Camundongos , Dados de Sequência Molecular , Polissacarídeo-Liases/metabolismo , Sindbis virus/patogenicidade , Sindbis virus/fisiologia , VirulênciaRESUMO
The heterogeneous nature of the yellow fever (YF) 17D-204 vaccine virus population was exploited in this study to isolate virus variants able to escape neutralization by the 17D-204 vaccine-specific MAb 864. The conformational change on the virus surface that resulted in the loss of the MAb 864-defined epitope was effected in each variant by a single amino acid mutation in the envelope (E) protein at either position E-305 or E-325. Interestingly, both positions were mutated during attenuation of the 17D-204 vaccine substrain from the wildtype Asibi strain. The mutations in several of the variants represented reversion to the wildtype Asibi virus sequence consistent with loss of a 17D-204 substrain-specific epitope. The majority of the variant viruses were shown to have altered mouse neurovirulence phenotypes, ranging from complete avirulence through to increased virulence. The avirulent variants are the first flavivirus MAb-neutralization-resistant variants to be attenuated for neurovirulence in the adult mouse model. Overall, the results indicate that the E protein epitope recognized by MAb 864 defines a functionally important region that encodes major molecular determinants of YF virus pathogenesis in vivo.
Assuntos
Epitopos de Linfócito B/genética , Mutação Puntual , Proteínas do Envelope Viral/genética , Vacinas Virais , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/patogenicidade , Animais , Encéfalo/patologia , Encéfalo/virologia , Chlorocebus aethiops , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Imuno-Histoquímica , Camundongos , Conformação Proteica , Análise de Sequência de DNA , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência , Replicação Viral , Febre Amarela/patologia , Febre Amarela/virologia , Vírus da Febre Amarela/imunologia , Vírus da Febre Amarela/fisiologiaRESUMO
We have examined and compared at the molecular level three strains of wild-type yellow fever (YF) virus isolated from Senegal in 1927, 1953 and 1965, termed French viscerotropic virus, Rendu and Dak1279 respectively. Over the structural protein genes, Rendu differed from the other two strains by 8% at the nucleotide level. Rendu also differed antigenically, possessing a 'vaccine'-specific envelope (E) protein epitope (i.e. an epitope previously shown to be found on 17D and French neurotropic vaccine viruses only and not wild-type strains of YF virus). Consequently, we propose that at least two distinct genotypes of wild-type YF virus have been present in Senegal. Since Rendu virus was isolated from a fatal case of YF, it would indicate that the vaccine-specific epitope on the E protein is not associated with attenuation of the viscerotropism of wild-type YF virus.
Assuntos
Vírus da Febre Amarela/genética , Genótipo , Senegal , Proteínas Estruturais Virais/genéticaRESUMO
Two monoclonal antibody neutralization resistant (MAbR) variants of the yellow fever (YF) 17D-204 vaccine virus strain were selected using YF type-specific MAb B39. These B39R variants were compared with the variant virus selected by Lobigs et al. (Virology 161, 474-478, 1987) using a second YF-type specific MAb (2E10) which mapped to amino acid position 71/72 in the envelope (E) protein. Neutralization assays with a panel of MAbs suggested that these two YF-type-specific epitopes are located in two discrete regions of the folded E protein. Each of the B39R variants had a single nucleotide mutation which encoded an amino acid substitution at either position E-155 or E-158. Thus, YF type-specific epitopes map to both domain I (B39) and II (2E10) of the YF virus E protein. The B39 defined epitope represents the first flavivirus neutralizing epitope localized to this region of domain I of the E protein.
Assuntos
Epitopos , Proteínas do Envelope Viral/imunologia , Vírus da Febre Amarela/imunologia , Sequência de Aminoácidos , Animais , Feminino , Camundongos , Dados de Sequência Molecular , Virulência , Vírus da Febre Amarela/patogenicidadeRESUMO
The live-attenuated yellow fever (YF) vaccine virus, strain 17D-204, has long been known to consist of a heterologous population of virions. Gould et al. (J. Gen. Virol. 70, 1889-1894 (1989)) previously demonstrated that variant viruses exhibiting a YF wild-type-specific envelope (E) protein epitope are present at low frequency in the vaccine pool and were able to isolate representative virus variants with and without this epitope, designated 17D(+wt) and 17D(-wt), respectively. These variants were employed here in an investigation of YF virus pathogenesis in the mouse model. Both the 17D-204 parent and the 17D(+wt) variant viruses were lethal for adult outbred mice by the intracerebral route of inoculation. However, the 17D(-wt) variant was significantly attenuated (18% mortality rate) and replicated to much lower titer in the brains of infected mice. A single amino acid substitution in the envelope (E) protein at E-240 (Ala-->Val) was identified as responsible for the restricted replication of the 17D(-wt) variant in vivo. The 17D(+wt) variant has an additional second-site mutation, believed to encode a reversion to the neurovirulence phenotype of the 17D-204 parent virus. The amino acid substitution in the E protein at E-173 (Thr-->Ile) of the 17D(+wt) variant which results in the appearance of the wild-type-specific epitope or nucleotide changes in the 5' and 3' noncoding regions of the virus are proposed as a candidates.
Assuntos
Variação Antigênica , Antígenos Virais/genética , Febre Amarela/virologia , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/patogenicidade , Animais , Encéfalo/patologia , Encéfalo/virologia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , RNA Viral/análise , Proteínas do Envelope Viral/genética , Virulência , Febre Amarela/patologia , Vírus da Febre Amarela/imunologiaRESUMO
Neonatal mice were infected with virus derived from a molecular clone of a laboratory strain of Sindbis virus, TRSB. The resulting acute fatal infection was typified by few if any of the classic hallmarks of encephalitis, very high levels of interferon-alpha/beta (IFNalphabeta), and lesions in the thymus and hematopoietic tissues usually associated with a severe stress response. Infection with an attenuated mutant of TRSB, which harbors a single amino acid change in the E2 surface glycoprotein (TRSBr114), was characterized by encephalitis, reduced mortality, low levels of IFNalphabeta, and no thymic pathology (J. Trgovcich, J. F. Aronson, and R. E. Johnston, 1996, Virology 224, 73-83). Here we report that infection of neonatal mice with TRSB, but not TRSBr114, resulted in induction of high levels of tumor necrosis factor-alpha as well as high and sustained levels of adrenalcorticotropin-releasing hormone and corticosterone. This syndrome of potentially toxic cytokine and stress hormone induction correlates with lethal Sindbis virus infection and constitutes a previously unrecognized aspect of Sindbis virus pathogenesis in mice.
Assuntos
Infecções por Alphavirus/fisiopatologia , Sindbis virus , Estresse Fisiológico/virologia , Hormônio Adrenocorticotrópico/biossíntese , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/mortalidade , Infecções por Alphavirus/virologia , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Corticosterona/biossíntese , Camundongos , Mutação Puntual , Choque Séptico/imunologia , Choque Séptico/fisiopatologia , Choque Séptico/virologia , Sindbis virus/genética , Estresse Fisiológico/imunologia , Timo/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Of four wild-type strains (Nakayama-original, SA14, 826309 and Beijing-1) of Japanese encephalitis (JE) virus that were passaged six times in HeLa cells (HeLa p6), two (Nakayama-original and 826309) became attenuated for mice. In the case of strain Nakayama-original, the virulence for mice was markedly reduced and attenuation was retained on passage in primary chicken embryo fibroblast, LLC-MK2 and C6/36 cells. The binding of non-HeLa-passaged Nakayama virus to mouse brain membrane receptor preparations could be differentiated from binding by Nakayama HeLa p6 virus, suggesting that the envelope (E) protein is involved in the attenuated phenotype. Both of the attenuated viruses can be distinguished from the virulent non-HeLa-passaged parental viruses by examination with E protein reactive vaccine and wild-type-specific monoclonal antibodies (MAbs). The vaccine-specific MAb V23, which is only reactive with the SA14 series of live vaccine viruses, recognized the HeLa cell-attenuated Nakayama-original and 826309 viruses, whereas two wild-type-specific MAbs (MAbs K13 and K39) lost reactivity. Comparison of the nucleotide sequences of the structural protein genes of the 826309 and Nakayama-original virulent parent and attenuated HeLa p6 viruses revealed that the viruses differed by 37 and 46 nucleotides coding for eight and nine amino acid mutations, respectively. However, other than one amino acid in the E protein, the membrane and E protein amino acid sequences of the two attenuated HeLa p6 viruses were identical.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Genes Virais , Animais , Anticorpos Monoclonais/imunologia , Encéfalo/virologia , Linhagem Celular , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/imunologia , Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Células HeLa , Hemaglutinação por Vírus , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Ligação Proteica , Receptores Virais , Inoculações Seriadas , Proteínas do Envelope Viral/genética , Proteínas Estruturais Virais/genética , Vacinas Virais/imunologia , VirulênciaRESUMO
The French neurotropic vaccine, or FNV, was used extensively in Africa to control yellow fever (YF). Although efficacious, the vaccine caused an unacceptable rate of post-vaccinal complications in children and was subsequently replaced by the 17D vaccine. Here we report that the genomes of the wild-type YF virus French viscerotropic virus and its attenuated vaccine derivative, FNV virus from the Institut Pasteur, Paris, (FNV-IP) differ by 77 nucleotides encoding 35 amino acid substitutions. Comparison of FNV-IP and three other isolates of FNV with other YF vaccine strains (17D-204 and 17DD derived from wild-type strain Asibi) revealed that during the two attenuation processes two common nucleotide changes arose that encode two amino acid substitutions: one is in the membrane protein at amino acid 35 (M-35), the other in non-structural (NS) protein 4B at NS4B-95. These common substitutions may be important in the process of attenuation of viscerotropic disease for humans and monkeys, and/or may be involved in loss of mosquito competence of the vaccine viruses.
Assuntos
Genes Virais , Genoma Viral , Proteínas Estruturais Virais/genética , Vacinas Virais/genética , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/imunologia , Aminoácidos/análise , Animais , Chlorocebus aethiops , DNA Viral/análise , França , Haplorrinos , Camundongos , Dados de Sequência Molecular , Nucleotídeos/análise , Vacinas Atenuadas/genética , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vacinas Virais/isolamento & purificação , Vírus da Febre Amarela/isolamento & purificaçãoRESUMO
This study evaluates the release of atrial natriuretic factor (ANF) during maximal exercise in 7 patients with untreated moderate to severe hypertension with invasive monitoring of hemodynamic characteristics in relation to sympathetic activity. Peripheral venous ANF (fmol/ml) was determined at rest and maximal exercise producing a respiratory exchange ratio of 1.14 +/- 0.10. In 5 of 7 patients, simultaneous right ventricular and peripheral venous ANF samples could be obtained to assess exercise myocardial secretion of ANF. With exercise, mean blood pressure increased from 150 +/- 26 to 192 +/- 29 mm Hg (p less than 0.001), pulmonary wedge pressure increased from 7 +/- 3 to 18 +/- 10 mm Hg (p less than 0.05) and ANF increased from 11.7 +/- 8.2 to 25.7 +/- 15.9 (p less than 0.02). This ANF response correlated weakly with pulmonary wedge pressure (r = 0.497, p = not significant), since patients without an increase in pulmonary wedge pressure had no increase in ANF. However, changes in pulmonary wedge pressure and plasma norepinephrine during exercise were inversely correlated (r = -0.811, p less than 0.02), with the greatest increase in norepinephrine occurring with a minimal increase in pulmonary wedge pressure. Similarly, ANF and plasma norepinephrine were inversely correlated during exercise (r = -0.869, p less than 0.05). A significant increase in right ventricular ANF indicated myocardial secretion. Plasma ANF therefore increased because of active myocardial production during exercise in patients with moderate to severe hypertension. These findings further suggest a directionally opposing relation between ANF release resulting from increased atrial tension and sympathetic nervous system influence on cardiac performance during exercise.
Assuntos
Fator Natriurético Atrial/metabolismo , Exercício Físico/fisiologia , Hipertensão/sangue , Adulto , Idoso , Fator Natriurético Atrial/sangue , GMP Cíclico/sangue , Feminino , Coração/fisiologia , Hemodinâmica , Humanos , Hipertensão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Norepinefrina/sangue , Sistema Nervoso Simpático/fisiologiaRESUMO
In 10 patients with moderate to severe hypertension, the hemodynamic effects of ergometric exercise and nicardipine, a dihydropyridine calcium channel antagonist, were characterized under basal conditions and after 1 week of therapy. The responses of plasma renin activity and catecholamines were also assessed. Nicardipine induced significant reductions of systolic, diastolic and mean blood pressure under conditions of rest and peak exercise (p less than 0.001), mediated by reversal of vasoconstriction (p less than 0.001). Overall, cardiac index and stroke volume index responses were not significantly altered by nicardipine. Although rest pulmonary wedge pressure was unchanged (6 +/- 3 to 5 +/- 4 mm Hg), peak exercise pulmonary wedge pressure decreased from 24 +/- 22 to 7 +/- 5 mm Hg (p less than 0.001) with nicardipine therapy. This effect of nicardipine on pulmonary wedge pressure was present across all work loads studied, and was accompanied by reduction of peak exercise pulmonary artery pressure from 43 +/- 10 to 25 +/- 7 mm Hg (p less than 0.001). Oxygen consumption was unchanged, associated with reduction of arteriovenous oxygen difference (p less than 0.02). Both plasma renin activity (p less than 0.05) and norepinephrine (p less than 0.005) were significantly increased with nicardipine therapy. Thus, nicardipine produced significant blood pressure reduction by reversal of vasoconstriction in patients with essential hypertension. The preservation of cardiac output, with markedly reduced pulmonary wedge pressure, indicated that nicardipine improved ventricular performance in response to reversal of vasoconstriction.
Assuntos
Hemodinâmica/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Nicardipino/uso terapêutico , Esforço Físico , Circulação Pulmonar/efeitos dos fármacos , Adulto , Idoso , Feminino , Hormônios/sangue , Humanos , Hipertensão/sangue , Hipertensão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Nicardipino/efeitos adversos , Troca Gasosa Pulmonar/efeitos dos fármacosRESUMO
We evaluated the immediate hemodynamic response to nicardipine, administered as an intravenous bolus and 30-minute sustaining infusion, in 10 patients with systemic hypertension (HTN) and 10 patients with chronic congestive heart failure (CHF). Baseline systemic vascular resistance and the response to nicardipine for HTN (1968 +/- 657 to 905 +/- 256 dynes X sec X cm-5) and CHF (2002 +/- 988 to 1089 +/- 216 +/- 99 dynes X sec X cm-5) were virtually identical (P less than 0.01). In both groups there was a significant increase of cardiac index in response to afterload reduction: HTN, 2.70 +/- 0.46 to 4.47 +/- 1.01; CHF, 1.90 +/- 0.33 to 2.88 +/- 0.80 L/min/m2 (P less than 0.01), so that a negative inotropic effect was not evident. In HTN the increase in cardiac index was primarily the result of reflex increase of heart rate (67 +/- 16 to 95 +/- 25 bpm [P less than 0.05]), associated with norepinephrine increase from 402 +/- 243 to 744 +/- 364 pg/ml (P less than 0.001). In CHF the cardiac index increase was caused by a stroke volume index increase (23 +/- 4 to 33 +/- 9 ml/min; P less than 0.01), with no significant change in heart rate or norepinephrine. Thus nicardipine, by means of calcium channel antagonism, induced reversal of vasoconstriction. Differences in the cardiac response identify the importance of characterizing the pathophysiologic status of cardiac impairment to more accurately interpret pharmacologic interventions.
Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Hemodinâmica/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Nicardipino/uso terapêutico , Vasodilatação/efeitos dos fármacos , Adulto , Idoso , Feminino , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertensão/fisiopatologia , Masculino , Pessoa de Meia-IdadeRESUMO
We investigated atrial natriuretic factor (ANF) in humans, measuring plasma immunoreactive (ir) ANF (in femtomoles per milliliter), and renal, hormonal, and hemodynamic responses to ANF infusion, in normal subjects (NL) and congestive heart failure patients (CHF). Plasma irANF was 11 +/- 0.9 fmol/ml in NL and 71 +/- 9.9 in CHF (P less than 0.01); the latter with twofold right ventricular increment (P less than 0.05). In NL, ANF infusion of 0.10 microgram/kg per min (40 pmol/kg per min) induced increases (P less than 0.05) of absolute (from 160 +/- 23 to 725 +/- 198 mueq/min) and fractional (1-4%) sodium excretion, urine flow rate (from 10 +/- 1.6 to 20 +/- 2.6 ml/min), osmolar (from 3.2 +/- 0.6 to 6.8 +/- 1.2 ml/min) and free water (from 6.8 +/- 1.6 to 13.6 +/- 1.6 ml/min) clearances, and filtration fraction (from 20 +/- 1 to 26 +/- 2%). Plasma renin and aldosterone decreased 33% and 40%, respectively (P less than 0.01). Systolic blood pressure fell (from 112 +/- 3 to 104 +/- 5 mmHg, P less than 0.05) in seated NL; but in supine NL, the only hemodynamic response was decreased pulmonary wedge pressure (from 11 +/- 1 to 7 +/- 1 mmHg, P less than 0.05). In CHF, ANF induced changes in aldosterone and pulmonary wedge pressure, cardiac index, and systemic vascular resistance (all P less than 0.05); however, responses of renin and renal excretion were attenuated. ANF infusion increased hematocrit and serum protein concentration by 5-7% in NL (P less than 0.05) but not in CHF.
Assuntos
Fator Natriurético Atrial/sangue , Insuficiência Cardíaca/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Adulto , Aldosterona/sangue , Pressão Sanguínea/efeitos dos fármacos , Eletrocardiografia , Eletrólitos/sangue , Insuficiência Cardíaca/sangue , Frequência Cardíaca/efeitos dos fármacos , Humanos , Rim/fisiologia , Rim/fisiopatologia , Pessoa de Meia-Idade , Valores de Referência , Renina/sangue , Resistência Vascular/efeitos dos fármacosRESUMO
The hemodynamic response to vasodilation induced by the new calcium channel antagonist nicardipine was studied in 10 patients with severe, chronic congestive heart failure. Rest and exercise hemodynamics were evaluated in the baseline state and after 1 week of oral nicardipine therapy (30 mg 3 times daily). In addition, respiratory gas exchange and arteriovenous oxygen difference were measured to assess changes in oxygen utilization. The responses of the sympathetic nervous system were evaluated by measuring plasma norepinephrine concentrations at rest and during maximal exercise. At rest, nicardipine administration was associated with significant reductions in mean systemic arterial pressure, systemic vascular resistance, pulmonary artery wedge pressure and pulmonary arterial pressure, and significant increases in cardiac index and stroke volume index. These effects were maintained during exercise. In contrast to findings with other calcium channel antagonists, no negative inotropic effect of nicardipine was identified. Nicardipine administration was associated with reduction of arteriovenous oxygen difference. Nicardipine had no effect on plasma norepinephrine concentrations, suggesting absence of reflex sympathetic nervous activation. Thus, nicardipine-mediated vasodilation leads to significant improvements in both rest and exercise cardiac performance.