Biological Clones Research Articles

Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant "progeny" from episomal transformants.

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.

Characterization and in vitro translation analysis of pelargonium flower break virus.

Pelargonium flower break virus (PFBV) is one of the common viruses in the glasshouses of Western Europe and has been assigned to the genus Carmovirus. A Spanish isolate obtained from nursery-grown Pelargonium zonale plants (PFBV-m) has been characterized. The molecular weight of genomic RNA and coat protein of PFBV-m were determined to be 1.36 x 10(6) (corresponding to approximately 4 kb) and 36,000, respectively. Only genomic-size RNA was encapsidated in PFBV virions; making necessary to purify double-stranded RNA from infected tissue in order to detect putative PFBV subgenomic RNAs. PFBV RNA directed the synthesis of a major polypeptide of 34 kDa and three other relevant polypeptides of estimated sizes 88-90 kDa, 42 kDa and 35-36 kDa. Antisera specific to PFBV immunoprecipitated the in vitro translated 35-36 kDa polypeptide indicating that this polypeptide is the PFBV coat protein. The PFBV in vitro translation pattern was very similar to that of CarMV although the relative levels of translated coat protein differed dramatically between the two viruses, most probably due to the lack of encapsidation of subgenomic PFBV. In vitro translation studies with a different biological clone obtained from the same PFBV-m isolate revealed a prominent additional polypeptide which is postulated to be a truncation of the 5' proximal ORF.

HIV-1 subtype C syncytium- and non-syncytium-inducing phenotypes and coreceptor usage among Ethiopian patients with AIDS.

To assess syncytium-inducing (SI) and non-syncytium-inducing (NSI) frequencies, coreceptor usage and gp120 V3 sequences of HIV-1 isolates from Ethiopian AIDS patients. Cross-sectional study on 48 hospitalized AIDS patients (CD4 T cells < 200 x 10(6) cell/l) with stage III or IV of the WHO staging system for HIV-1 infection and disease. Peripheral blood mononuclear cells (PBMC) from all 48 patients were tested by MT-2 assay to determine SI/NSI phenotypes. Lymphocyte subsets were enumerated using Coulter counting and FACScan analysis. Viral load determination used a nucleic acid sequence-based amplification assay (NASBA). Coreceptor usage of HIV-1 biological clones was measured using U87 CD4/chemokine receptor transfectants and phytohemagglutinin-stimulated PBMC of healthy donors with wild-type CCR5 and homozygous mutation CCR5delta32 (a 32 base-pair deletion in CCR5). Reverse transcriptase polymerase chain reaction sequencing was performed on the third variable region (V3) of the HIV-1 gene gp120. Sequence alignments were done manually; phylogenetic analyses used PHYLIP software packages. SI viruses were detected for 3/48 (6%) AIDS patients only. Lower mean absolute CD4 counts were determined in patients with SI virus compared with NSI (P = 0.04), but no differences in viral load were observed. All patients were found to be infected with HIV-1 subtype C, based on V3 sequencing. NSI biological clones used CCR5 as coreceptor; SI biological clones used CXCR4 and/or CCR5 and/or CCR3. Ethiopian patients with HIV-1 C-subtype AIDS harbour a remarkably low frequency of SI phenotype viruses. Coreceptor usage of these viruses correlates with their biological phenotypes.

Drastic fitness loss in human immunodeficiency virus type 1 upon serial bottleneck events.

Muller's ratchet predicts fitness losses in small populations of asexual organisms because of the irreversible accumulation of deleterious mutations and genetic drift. This effect should be enhanced if population bottlenecks intervene and fixation of mutations is not compensated by recombination. To study whether Muller's ratchet could operate in a retrovirus, 10 biological clones were derived from a human immunodeficiency virus type 1 (HIV-1) field isolate by MT-4 plaque assay. Each clone was subjected to 15 plaque-to-plaque passages. Surprisingly, genetic deterioration of viral clones was very drastic, and only 4 of the 10 initial clones were able to produce viable progeny after the serial plaque transfers. Two of the initial clones stopped forming plaques at passage 7, two others stopped at passage 13, and only four of the remaining six clones yielded infectious virus. Of these four, three displayed important fitness losses. Thus, despite virions carrying two copies of genomic RNA and the system displaying frequent recombination, HIV-1 manifested a drastic fitness loss as a result of an accentuation of Muller's ratchet effect.

In vivo cell and tissue tropism of SIVsmmPBj14-bcl.3.

To gain insight into the unique pathogenicity of simian immunodeficiency virus (SIV) variant PBj14, which produces an acutely lethal enteropathic syndrome in infected pigtail macaques, we investigated the cell and tissue tropisms of a highly pathogenic biologic clone (bcl.3) of SIVsmmPBj14. To compare the relative amount of viral antigen in lymphoid organs of infected macaques we used an objective semiquantitative immunohistochemistry (sQIHC) assay. We found that in all animals viral antigen load was greater in alimentary-associated lymphoid tissues (gut-associated lymphoid tissue [GALT], tonsil, mesenteric and retropharyngeal lymph nodes) than in non-alimentary-associated lymphoid tissues (spleen, thymus, inguinal and axillary lymph nodes). Moreover, in six of nine animals examined, virus load in GALT was greater than that in any other lymphoid tissue. To determine whether the acute pathogenicity and prolific replication of SIVsmmPBj14 might be explained by a broader in vivo cell tropism than is typical of SIVs, we used cell subset separation and nested PCR. We found that the primary target cells in mesenteric lymph node for SIVsmmPBj14 were CD4+ T lymphocytes. However, the virus also infected macrophages, as well as CD8+ T cells and B cells, albeit at low frequencies. These results suggest that alimentary lymphoid tissue localization rather than unusual cell phenotype tropism distinguishes the singular pathogenesis of SIVsmmPBj14.

Selective antibody neutralization prevents neuropathogenic lactate dehydrogenase-elevating virus from causing paralytic disease in immunocompetent mice.

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.

Mucosal Antibody Expression Following Rapid SIVMneDissemination in Intrarectally InfectedMacaca nemestrina

The early kinetics of antibody expression following transmucosal infection by SIV(Mne) were examined in several mucosal compartments in Macaca nemestrina. Five male-female pairs of macaques were inoculated intrarectally with SIV(Mne) E11S, a biological clone, and serially euthanized at 1, 2, 4, 8, and 12 weeks postinoculation. Plasma, tears, saliva, rectal secretions, and vaginal washes were collected serially and just prior to euthanasia. Both total and SIV-specific IgG and IgA levels were measured by immunoglobulin isotype-specific quantitative enzyme-linked immunosorbent assays (ELISAs), and were further examined by conventional and enhanced chemiluminescence (ECL) immunoblots. Virus coculture, polymerase chain reaction, and in situ hybridization assays revealed the systemic spread of virus as early as 1 week postinoculation in 8 of 10 animals. ECL immunoblots detected SIV-specific antibodies in mucosal samples collected 1 week postinoculation. The most dramatic increases in both total and SIV-specific IgA levels were detected in rectal secretion samples. In contrast, plasma and nonrectal mucosal samples from the same time points increased only slightly, suggesting that the most robust antibody response occurred at the portal of infection. Our results show that the SIV-infected macaque is an excellent model for studies designed to assess mucosal immune responses to primate lentivirus infections. Additional studies will assess the correlation between the antiviral protection afforded by candidate vaccines and mucosal antibody responses.

Genes of the Ascidian: An Annotated List as of 1997

The ascidians occupy a crucial phylogenetic position between invertebrates and vertebrates. Advances in molecular biological techniques and molecular cloning have enabled studies of molecular mechanisms to be more and more accessible in ascidians. Recently, an increasing number of ascidian genes have been cloned. In this review, we summarize the molecular features and expression profiles of all ascidian genes published to date and describe how and why they were cloned. Such information not only is valuable for understanding the recent advancements in ascidian molecular biology but also to bring out the importance of ascidians as an experimental system for a variety of fields in biology.

Sequence and Functional Analysis of the Intergenic Regions Separating Babesial Rhoptry-Associated Protein-1 (rap-1)Genes

Suarez, C. E., Palmer, G. H., Hötzel, I., Hines, S. A., and McElwain, T. F. 1998. Sequence and functional analysis of the intergenic regions separating babesial rhoptry-associated protein-1 (rap-1) genes.Experimental Parasitology90, 189–194. The rhoptry-associated protein 1 (RAP-1) expressed by all babesial parasites is encoded by tandemly arranged genes separated by discrete intergenic (IG) regions. We hypothesize that these IG regions regulaterap-1gene expression. InBabesia bovistwo identicalrap-1gene copies are separated by a 1.0-kb noncoding region which is also exactly conserved 5′ to therap-1gene 1. In contrast, the complexB. bigemina rap-1locus contains at least 5 polymorphicrap-1agenes separated by uncharacterized 3.38-kb regions. A genomic clone encoding the 3′ sequence ofrap-1gene copy 1, the 1 kb IG region, and the 5′ sequence of gene copy 2 was obtained by PCR amplification of DNA from the Mo7 biological clone ofB. bovisand sequenced. This was follow by amplification and sequence analysis of the 3.38-kb region separating twoB. bigemina rap-1agenes, revealing the presence of two different IG regions denominated IG-1 (0.7 kb) and IG-2 (1.3 kb), flanking a newly identifiedrap-1borf. Sequence analysis and comparison among babesialrap-1IG regions fromB. bovis, B. bigemina, B. canis, andB. ovisrevealed conservation of at least three putative regulatory boxes consistently positioned 5′ of the start of therap-1orfs. To determine whetherrap-1IG regions contained a functional promoter, the entire 1-kb IG region fromB. boviswas cloned into pCAT, a promoterless plasmid containing thecatgene. The IG region in the 5′ → 3′ orientation strongly promoted transcriptionin vitroby homologousB. bovisRNA polymerases. The presence of conserved regions 5′ to eachrap-1gene copy and among other babesialrap-1IG regions and thein vitropromoter function in the 5′ → 3′ orientation support a role for the IG region inrap-1gene regulation.

Multiple concurrent reverse transcriptase and protease mutations and multidrug resistance of HIV-1 isolates from heavily treated patients.

Drug resistance of HIV-1 is an obstacle to the long-term efficacy of antiretroviral therapy. To characterize reverse transcriptase and protease genes of multidrug-resistant HIV-1 isolates. Descriptive case series. Academic medical center. Four consecutive patients with HIV-1 infection were selected because they had previously received many antiretroviral drugs and had not achieved plasma HIV-1 RNA suppression despite treatment with several three-drug combinations. Reverse transcriptase sequencing, protease sequencing, and drug susceptibility testing of HIV-1. Isolates of HIV-1 from the four patients shared seven protease mutations and eight reverse transcriptase mutations. These mutations were present in biological clones and at three time points in three of the patients. Susceptibility testing showed high-level resistance (30-fold to >100-fold) to zidovudine, lamivudine, saquinavir, indinavir, and nelfinavir and lower-level resistance (3-fold to 5-fold) to didanosine, zalcitabine, and stavudine. Simultaneous resistance to almost all available antiretroviral drugs may occur in HIV-1. The concordance and persistence of mutations in drug-resistant HIV-1 isolates suggest that some combinations of reverse transcriptase and protease mutations give the virus a selective advantage in the presence of various drug combinations.

Evolution of syncytium-inducing and non-syncytium-inducing biological virus clones in relation to replication kinetics during the course of human immunodeficiency virus type 1 infection.

To investigate the temporal relationship between human immunodeficiency virus type 1 (HIV-1) replicative capacity and syncytium-inducing (SI) phenotype, biological and genetic characteristics of longitudinally obtained virus clones from two HIV-1-infected individuals who developed SI variants were studied. In one individual, the emergence of rapidly replicating SI and non-syncytium-inducing (NSI) variants was accompanied by a loss of the slowly replicating NSI variants. In the other subject, NSI variants were always slowly replicating, while the coexisting SI variants showed an increase in the rate of replication. Irrespective their replicative capacity, the NSI variants remained present throughout the infection in both individuals. Phylogenetic analysis of the V3 region showed early branching of the SI variants from the NSI tree. Successful SI conversion seemed a unique event since no SI variants were found among later-stage NSI variants. This was also confirmed by the increasing evolutionary distance between the two subpopulations. At any time point during the course of the infection, the variation within the coexisting SI and NSI populations did not exceed 2%, indicating continuous competition within each viral subpopulation.

Coreceptor usage of human immunodeficiency virus type 2 primary isolates and biological clones is broad and does not correlate with their syncytium-inducing capacities.

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by binding of the surface envelope glycoprotein to the CD4 molecule. Interaction of the resulting CD4-glycoprotein complex with alpha- or beta-chemokine receptors, depending on the biological phenotype of the virus, then initiates the fusion process. Here, we show that primary HIV-2 isolates and biological clones, in contrast to those of HIV-1, may use a broad range of coreceptors, including CCR-1, CCR-3, CCR-5, and CXCR-4. The syncytium-inducing capacity of these viruses did not correlate with the ability to infect via CXCR-4 or any other coreceptor. One cell-free passage of the intermediate isolates in mitogen-stimulated, CD8+ cell-depleted peripheral blood mononuclear cells resulted in the outgrowth of variants with CCR-5 only, whereas the coreceptor usage of late and early isolates did not change. Since HIV-2 is less pathogenic in vivo than HIV-1, these data suggest that HIV pathogenicity in vivo is not directly related to the spectrum of coreceptors used in in vitro systems.

Analysis of the temporal relationship between human immunodeficiency virus type 1 quasispecies in sequential blood samples and various organs obtained at autopsy.

We studied the temporal relationship between human immunodeficiency type 1 (HIV-1) quasispecies in tissues and in peripheral blood mononuclear cells (PBMC) of infected individuals. Sequential PBMC and tissue samples from various organs obtained at autopsy from three patients who died of AIDS-related complications were available for analysis. Biological HIV-1 clones were isolated from PBMC samples, and cellular tropism and syncytium-inducing (SI) capacity were determined. Genomic DNA was isolated from 1 cm3 of organ tissue, and proviral DNA was amplified by means of PCR and cloned with the PGEM-T vector system. A 185-bp region encompassing the third variable domain of the virus envelope, known to influence HIV-1 biological properties, was sequenced. HIV-1 could be amplified from all PBMC and organ samples, except from liver tissue for two patients. Both SI and non-syncytium-inducing (NSI) genotypes could be detected in the different tissues. Tissue-specific quasispecies were observed in brain, lung, and testis. Lymphoid tissues, such as bone marrow, lymph node, and spleen, harbored several different variants similar to those detected in blood in the last PBMC samples. In general, only tissues in which macrophages are likely to be the main target cell for HIV-1 harbored NSI HIV-1 sequences that clustered separately. Both SI and NSI sequences that clustered with sequences from late-stage PBMC were present in other tissues, which may indicate that the presence of HIV-1 in those tissues is secondary to lymphocyte infiltration rather than to tissue tropism of HIV-1 itself. These data suggest that the viral reservoir may be limited, which will have important implications for the success of HIV-1 eradication.

Novel and dynamic evolution of equine infectious anemia virus genomic quasispecies associated with sequential disease cycles in an experimentally infected pony

We have investigated the genetic evolution of three functionally distinct regions of the equine infectious anemia virus (EIAV) genome (env, rev, and long terminal repeat) during recurring febrile episodes in a pony experimentally infected with a well-characterized reference biological clone designated EIAV(PV). Viral populations present in the plasma of an EIAV(PV)-infected pony during sequential febrile episodes (18, 34, 80, 106, and 337 days postinfection) were amplified from viral RNA, analyzed, and compared to the inoculated strain. The comparison of the viral quasispecies showed that the inoculated EIAV(PV) quasispecies were all represented during the first febrile episode, but entirely replaced at the time of the second febrile episode, and that new predominant quasispecies were associated with each subsequent cycle of disease. One of the more surprising results was the in vivo generation of large deletion (up to 15 amino acids) in the principal neutralizing domain (PND) of gp90 during the third febrile episode. This deletion did not alter the competence for in vitro replication as shown by the analysis of a env chimeric clone with a partially deleted PND and did not altered the fitness of the virus in vivo, since this partially deleted envelope became the major population during the fourth febrile episode. Finally, we showed that the amino acid mutations were not randomly distributed but delineated eight variables regions, V1 to V8, with V3 containing the PND region. These studies provide the first detailed description of the evolution of EIAV genomic quasispecies during persistent infection and reveal new insights into the genetics and potential mechanisms of lentivirus genomic variation.

Generation of cytopathogenic subgenomic RNA of classical swine fever virus in persistently infected porcine cell lines

Two biological clones (A.1 and B.2) of the classical swine fever virus strain Alfort/187 and the recombinant virus vA187-1, derived from a cDNA clone of Alfort/187, were used to establish persistently infected cultures oft he swine kidney cell lines SK-6 and PK-41. It was found that 100% of the cells in the passaged cultures were positive for viral antigen throughout the course of the experiment. Additionally, supernatants collected upon passaging of the cells continuously contained high titers of infectious virus. In six separate cultures persistently infected with either the biological clones or the recombinant virus, a cytopathic effect occurred spontaneously between passage 8 and 94. The cytopathogenic agent in the supernatants of these cultures could be passaged repeatedly, suggesting the generation of a mutant virus. Analysis of RNA from such cultures revealed the presence of a subgenomic viral RNA of approximately 8 kilobases (kb). In all six cases, this RNA had an identical internal deletion of 4764 nucleotides, including the region coding for all structural proteins. The subgenomic RNA replicated and was packaged in the presence of wild-type virus. Cells infected with cytopathogenic virus contained increased amounts of the viral protein NS3 thought to be involved in pestivirus cytopathogenicity.

Phenotypic switch in a Spanish HIV type 1 isolate on serial passage on MT-4 cells.

A biological clone (F0) of a syncytium-inducing (SI) isolate (S61) was unable to produce syncytia in MT-4 cells. On serial passage on MT-4 cells this virus [F15(-3)] became capable of inducing syncytia (Sánchez-Palomino S, et al.: J Virol 1993;67:2938). After sequencing different regions of the env gene including V1-V2, V3, and the fusion domain of both viruses, we have found only an asparagine (N)-to-isoleucine (I) change in position 7 of the V3 loop. By mutagenesis and in vitro recombination, using infectious molecular clones, we have identified this amino acid change as the only one responsible for the syncytial phenotypic switch. However, this cytopathic change was not accompanied by a change in the replication rate, indicating that these two properties are not linked genotypic traits. Thus serial passaging of an HIV-1 isolate on MT-4 cells has produced a nonsyncytial-to-syncytial switch through a point mutation in position 7 of the V3 loop.

A zidovudine-resistant simian immunodeficiency virus mutant with a Q151M mutation in reverse transcriptase causes AIDS in newborn macaques.

The simian immunodeficiency virus (SIV)-newborn rhesus macaque model of AIDS can be used to study directly the virulence of viral mutants which are resistant to antiviral drugs. A viral mutant called SIVmac79A6.1, isolated from an SIV-infected macaque after prolonged zidovudine treatment, was found to have a double-base-pair change at codon 151 of reverse transcriptase, resulting in a glutamine to methionine substitution (Q151M). This mutation was associated with more than 100-fold increased resistance to zidovudine and low-level cross-resistance to other dideoxynucleoside analogs. To determine whether this Q151M mutation affects viral virulence, four newborn macaques were inoculated intravenously with a biological clone of this drug-resistant SIVmac79A6.1 mutant; two of these animals were also treated orally with zidovudine. All four animals showed persistent viremia, and two of the four animals developed fatal immunodeficiency at 3 and 8 months of age, respectively. The remaining two animals had CD4+ T-cell depletion and clinical symptoms of AIDS at 22 months. No phenotypic or genotypic reversion of virus to the wild type could be detected in any of the four animals. These results demonstrate that the Q151M mutation in SIV reverse transcriptase does not reduce viral virulence.

Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry.

A panel of primary syncytium-inducing (SI) human immunodeficiency virus type 1 isolates that infected several CD4+ T-cell lines, including MT-2 and C8166, were tested for infection of blood-derived macrophages. Infectivity titers for C8166 cells and macrophages demonstrated that primary SI strains infected macrophages much more efficiently than T-cell line-adapted HIV-1 strains such as LAI and RF. These primary SI strains were therefore dual-tropic. Nine biological clones of two SI strains, prepared by limiting dilution, had macrophage/C8166 infectivity ratios similar to those of their parental viruses, indicating that the dual-tropic phenotype was not due to a mixture of non-SI/macrophage-tropic and SI/T-cell tropic viruses. We tested whether the primary SI strains used either Lestr (fusin) or CCR5 as coreceptors. Infection of cat CCC/CD4 cells transiently expressing Lestr supported infection by T-cell line-adapted strains including LAI, whereas CCC/CD4 cells expressing CCR5 were sensitive to primary non-SI strains as well as to the molecularly cloned strains SF-162 and JR-CSF. Several primary SI strains, as well as the molecularly cloned dual-tropic viruses 89.6 and GUN-1, infected both Lestr+ and CCR5+ CCC/CD4 cells. Thus, these viruses can choose between Lestr and CCR5 for entry into cells. Interestingly, some dual-tropic primary SI strains that infected Lestr+ cells failed to infect CCR5+ cells, suggesting that these viruses may use an alternative coreceptor for infection of macrophages. Alternatively, CCR5 may be processed or presented differently on cat cells so that entry of some primary SI strains but not others is affected.

In VitroCharacterization of HIV Type 1 Biological Clones from Asymptomatic and Symptomatic Pediatric Patients

To investigate the mechanisms of HIV-1 cytopathogenicity, functional biological HIV-1 clones were isolated from two infected children with high viral loads in vivo. Clone HC4 was isolated from a symptomatic child and clone GC6 8-4 was isolated from an asymptomatic child. These clones were characterized for their ability to induce syncytia, and to replicate and induce single-cell death in peripheral blood-derived normal CD4 T cell cultures containing anti-CD4 antibody. Despite similar viral loads as determined by p24 antigen production or viral RNA expression, GC6 8-4 was noncytopathogenic and HC4 was cytopathogenic. Since we had demonstrated that mitochondrial dysfunction correlated with HIV-1-induced cell death, we determined whether the cytopathogenic HC4 clone decreased mitochondrial viability using a mitochondrial-specific dye, rhodamine-123. Following infection, mitochondrial viability decreased in cells infected with HC4 by day 4 and continued to decline through day 7 when compared to uninfected cells. By day 7 postinfection, greater than 80% of the cells in culture were dead. Similar analyses on CD4 T cells infected with the noncytopathogenic GC6 8-4 demonstrated that mitochondria remained functionally viable and > 90% of the cells excluded trypan blue. These studies describe a cell culture system to study single-cell death in the absence of syncytia and secondary infection. Results with two patient-derived HIV-1 biological clones suggest that loss of mitochondrial viability may play a role in HIV-1-induced cytopathogenicity.

Genetic exchange as a possible source of genomic diversity in sylvatic populations of Trypanosoma cruzi.

Thirty six stocks of Trypanosoma cruzi isolated from sylvatic mammals (32 Didelphis marsupialis and one Philander opossum) and triatomine bugs (Rhodnius robustus and one unidentified bug) in the Amazonian forest by Carajas, Brazil were characterized by isoenzyme and random amplified polymorphic DNA (RAPD) analysis as belonging to principal zymodeme '1 (Z1). Two different homozygous phenotypes and the corresponding heterozygous phenotype were found for phosphoglucomutase with an observed frequency almost identical with that predicted by the theoretical Hardy-Weinberg distribution. Parental and hybrid profiles were also suggested by RAPD analysis, which allowed exclusion of mixed parental strains from the hybrids: isoenzyme and RAPD profiles of biological clones were also indistinguishable from those of uncloned stocks. Trypanosoma cruzi stocks from widely separated geographic origins in Central and South America gave similar RAPD profiles that allowed them to be recognized as being Z1. These results indicate that genetic exchange could contribute to the generation of genetic diversity during the sylvatic cycle of T. cruzi, and this may have epidemiologic and taxonomic implications.