Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties.
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A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein. (+info)
Late domain function identified in the vesicular stomatitis virus M protein by use of rhabdovirus-retrovirus chimeras.
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Little is known about the mechanisms used by enveloped viruses to separate themselves from the cell surface at the final step of budding. However, small sequences in the Gag proteins of several retroviruses (L domains) have been implicated in this process. A sequence has been identified in the M proteins of rhabdoviruses that closely resembles the PPPPY motif in the L domain of Rous sarcoma virus (RSV), an avian retrovirus. To evaluate whether the PPPY sequence in vesicular stomatitis virus (VSV) M protein has an activity analogous to that of the retroviral sequence, M-Gag chimeras were characterized. The N-terminal 74 amino acids of the VSV (Indiana) M protein, including the PPPY motif, was able to replace the L domain of RSV Gag and allow the assembly and release of virus-like particles. Alanine substitutions in the VSV PPPY motif severely compromised the budding activity of this hybrid protein but not that of another chimera which also contained the RSV PPPPY sequence. We conclude that this VSV sequence is functionally homologous to the RSV L domain in promoting virus particle release, making this the first example of such an activity in a virus other than a retrovirus. Both the RSV and VSV motifs have been shown to interact in vitro with certain cellular proteins that contain a WW interaction module, suggesting that the L domains are sites of interaction with unknown host machinery involved in virus release. (+info)
Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector.
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Adenovirus (Ad) and adeno-associated virus (AAV) have attractive and complementary properties that can be exploited for gene transfer purposes. Ad vectors are probably the most efficient vehicles to deliver foreign genes both in vitro and in vivo. AAV exhibits the unique ability to establish latency by efficiently integrating at a specific locus of human chromosome 19 (AAVS1). Two viral elements are necessary for the integration at AAVS1: Rep68/78 and the inverted terminal repeats (AAV-ITRs). In this study, we report the development of two helper-dependent adenoviral (HD) vectors, one carrying the Rep78 gene, the other an AAV-ITR-flanked transgene. Although Rep proteins have been demonstrated to interfere with Ad replication, HD Rep78 vector was successfully amplified on serial passages in 293CRE4 cells with a yield of 50-100 transducing units per cell. DNA integration at the AAVS1 site also was demonstrated in hepatoma cells coinfected with the HD-expressing Rep78 and with the second HD vector carrying a transgene flanked by AAV-ITRs. The high transduction efficiency, large cloning capacity, and high titer of the HD, combined with the site-specific integration machinery provided by AAV-derived components, make the Ad/AAV hybrid viruses a promising vehicle for gene therapy. (+info)
Viral burden and disease progression in rhesus monkeys infected with chimeric simian-human immunodeficiency viruses.
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To determine the role of viral burden in simian-human immunodeficiency virus (SHIV)-induced disease, cellular provirus and plasma viral RNA levels were measured after inoculation of rhesus monkeys with four different SHIVs. These SHIVs included SHIV-HXBc2 and SHIV-89.6, constructed with env, tat, rev, and vpu derived from either cell line-passaged or primary patient isolates of human immunodeficiency virus type 1; the viral quasispecies SHIV-89.6P derived after in vivo passage of SHIV-89.6; and a molecular clone, SHIV-KB9, derived from SHIV-89.6P. SHIV-HXBc2 and SHIV-89.6 are nonpathogenic in rhesus monkeys; SHIV-89.6P and SHIV-KB9 cause rapid CD4(+) T cell depletion and an immunodeficiency syndrome. Relative SHIV provirus levels were highest during primary infection in monkeys infected with SHIV-89.6P, the virus that caused the most rapid and dramatic CD4(+) T cell depletion. However, by 10 weeks postinoculation, provirus levels were similar in monkeys infected with the pathogenic and nonpathogenic chimeric viruses. The virus infections that resulted in the highest peak and chronic viral RNA levels were the pathogenic viruses SHIV-89.6P and SHIV-KB9. SHIV-89. 6P uniformly caused rapid and profound CD4(+) T cell depletion and immunodeficiency. Infection with the SHIV-KB9 resulted in very low CD4(+) T cell counts without seroconversion in some monkeys and a substantial but less profound CD4(+) T cell depletion and rapid seroconversion in others. Surprisingly, the level of plasma viremia did not differ between SHIV-KB9-infected animals exhibiting these contrasting outcomes, suggesting that host factors may play an important role in AIDS virus pathogenesis. (+info)
Characterization of a neutralization-escape variant of SHIVKU-1, a virus that causes acquired immune deficiency syndrome in pig-tailed macaques.
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A chimeric simian-human immunodeficiency virus (SHIV-4) containing the tat, rev, vpu, and env genes of HIV type 1 (HIV-1) in a genetic background of SIVmac239 was used to develop an animal model in which a primate lentivirus expressing the HIV-1 envelope glycoprotein caused acquired immune deficiency syndrome (AIDS) in macaques. An SHIV-infected pig-tailed macaque that died from AIDS at 24 weeks postinoculation experienced two waves of viremia: one extending from weeks 2-8 and the second extending from week 18 until death. Virus (SHIVKU-1) isolated during the first wave was neutralized by antibodies appearing at the end of the first viremic phase, but the virus (SHIVKU-1b) isolated during the second viremic phase was not neutralized by these antibodies. Inoculation of SHIVKU-1b into 4 pig-tailed macaques resulted in severe CD4(+) T cell loss by 2 weeks postinoculation, and all 4 macaques died from AIDS at 23-34 weeks postinoculation. Because this virus had a neutralization-resistant phenotype, we sequenced the env gene and compared these sequences with those of the env gene of SHIVKU-1 and parental SHIV-4. With reference to SHIV-4, SHIVKU-1b had 18 and 6 consensus amino acid substitutions in the gp120 and gp41 regions of Env, respectively. These compared with 10 and 3 amino acid substitutions in the gp120 and gp41 regions of SHIVKU-1. Our data suggested that SHIVKU-1 and SHIVKU-1b probably evolved from a common ancestor but that SHIVKU-1b did not evolve from SHIVKU-1. A chimeric virus, SHIVKU-1bMC17, constructed with the consensus env from the SHIVKU-1b on a background of SHIV-4, confirmed that amino acid substitutions in Env were responsible for the neutralization-resistant phenotype. These results are consistent with the hypothesis that neutralizing antibodies induced by SHIVKU-1 in pig-tailed macaque resulted in the selection of a neutralization-resistant virus that was responsible for the second wave of viremia. (+info)
T cell-tropic simian immunodeficiency virus (SIV) and simian-human immunodeficiency viruses are readily transmitted by vaginal inoculation of rhesus macaques, and Langerhans' cells of the female genital tract are infected with SIV.
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Intravaginal inoculation with T cell-tropic molecular clones of simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) or some dual-tropic strains of SIV or SHIV produced systemic infection in rhesus macaques. Vaginal inoculation with other dual-tropic molecular clones of SIV or SHIV did not infect rhesus macaques even after multiple inoculations. While in vitro measures of macrophage tropism do not predict which primate lentiviruses will produce systemic infection after intravaginal inoculation, the level to which a virus replicates in vivo after intravenous inoculation does predict the outcome of intravaginal inoculation. Another series of studies, using combined in situ hybridization and immunolabeling to simultaneously detect SIV RNA and identify the immunophenotype of infected cells, demonstrated that a large proportion (approximately 40% in some animals) of the SIV-infected cells in the vagina and cervix were Langerhans' cells. This is the first in vivo demonstration that Langerhans' cells in the genital tract are infected with SIV and that dendritic cells are significant reservoirs for lentiviruses. (+info)
Distinct pathogenic sequela in rhesus macaques infected with CCR5 or CXCR4 utilizing SHIVs.
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Infection of macaques with chimeric simian-human immunodeficiency virus (SHIV) provides an excellent in vivo model for examining the influence of envelope on HIV-1 pathogenesis. Infection with a pathogenic CCR5 (R5)-specific enveloped virus, SHIVSF162P, was compared with infection with the CXCR4 (X4)-specific SHIVSF33A.2. Despite comparable levels of viral replication, animals infected with the R5 and X4 SHIV had distinct pathogenic outcomes. SHIVSF162P caused a dramatic loss of CD4+ intestinal T cells followed by a gradual depletion in peripheral CD4+ T cells, whereas infection with SHIVSF33A.2 caused a profound loss in peripheral T cells that was not paralleled in the intestine. These results suggest a critical role of co-receptor utilization in viral pathogenesis and provide a reliable in vivo model for preclinical examination of HIV-1 vaccines and therapeutic agents in the context of the HIV-1 envelope protein. (+info)
Recombinant viruses expressing the foot-and-mouth disease virus capsid precursor polypeptide (P1) induce cellular but not humoral antiviral immunity and partial protection in pigs.
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The importance of the induction of virus neutralizing antibodies to provide protection against foot-and-mouth disease virus (FMDV) infection is well established. However, recent studies with recombinant adenovirus expressing the precursor polypeptide of the viral capsid (P1) indicate that cattle inoculated with this recombinant vector developed partial protection against FMDV infection, in the absence of a detectable specific humoral response. Other viral vectors have been widely used to induce protective immunity against many pathogens, and it has been reported that the use of different vectors for priming and boosting injections can provide a synergistic effect on this response. In this work, we determined the immunogenicity of two recombinant viruses (adenovirus and vaccinia) expressing P1-FMDV, administered either individually or sequentially, and the protection that they induced against FMDV challenge in pigs. A double immunization with the adeno-P1 virus was the most effective strategy at inducing protective immunity. In contrast to previous reports, the use of two different vectors for priming and boosting did not show a synergistic effect on the protection induced against FMD. Interestingly, immunized pigs developed FMDV-specific T cell responses but not detectable antibodies. Thus, the protection observed was likely to be mediated by a cellular immune response. (+info)