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PubMed2000

Phylogenetic comparison of the DEN-2 Mexican isolate with other flaviviruses.

Recent attention has focused on the geographic variation of dengue viruses, since major epidemies may follow introduction of a new virus strain into susceptible populations. We cloned and sequenced a very interesting Mexican isolate (200787/1983) which is antigenically unique by signature analysis with respect to all other dengue-2 topotype viruses. This strain is also unique in biological behavior (neurotropism) and is of epidemiological significance in Mexico. The dengue-2 Mexican isolate sequence information was compared with that of other flaviviruses, analyzing the branching structure of the phylogenetic tree reconstructed from the E gene amino acid sequences. The E glycoprotein, is target for neutralizing antibodies and T-cell responses, and defines the tropism and virulence of flaviviruses. In the phylogram, our strain was located in the position of greatest dissimilarity within serotype-2. Also, frequency analysis of amino acids revealed a very different signature pattern from that found in viral serotype-2.

Amino Acid Sequence↗
PubMed2006

Selective regimen shift and demographic growth increase associated with the emergence of high-fitness variants of canine parvovirus.

The natural evolution of Canine parvovirus (CPV) is characterized by a variety of mutations, mainly in the VP1/VP2 gene. Although positive selection has been previously reported in CPV, little is known about its overall contribution to viral adaptation in the canine population. Herein, the influences of the evolutive constraints on CPV during a period of viral adaptation into a previously uninfected population are more clearly investigated. To do this, 31 sequences of VP1/VP2 gene obtained from symptomatic domestic dogs in Brazil were used, sampled from 1980 to 2000. Marked evolutionary changes in CPV associated with a process of fine-tuning adaptation were observed. Specifically, sequences from the 1980s revealed two distinct antigenic types (i.e. 2a and 2b) cocirculating in Brazil. Moreover, analysis of the selective regimen showed that 90% of the VP2 sites were conserved (d(N)/d(S)=0). In contrast, sequences from the 1990s were composed solely of CPV-2a with 96% of VP2 sites under purifying selection (d(N)/d(S)<1) and site 297 under strong positive selection (omega=4.9). Important features regarding the demographic history of CPV in Brazil were also observed. The viral population size passed through a short period of explosive growth that declined and then stabilized into a constant rate of spread. Remarkably, the explosive growth coincided with the appearance of CPV variants that presented a unique repertoire of mutations never before seen in other worldwide strains. The analysis also showed that the estimated nucleotide substitution was similar to those commonly observed in fast evolving RNA viruses. The present results demonstrated the adaptive potential of CPV to acquire, in short interval of 10 years, key mutations in the VP1/VP 2 gene that increased viral fitness and enabled the virus to disseminate even in vaccinated dogs.

Animals↗
PubMed2003

Increase of HIV-1 pro-viral DNA per million peripheral blood mononuclear cells in patients with advanced HIV disease (CD4<200 cells/mm3) receiving interleukin 2 combined with HAART versus HAART alone (ANRS-082 trial).

OBJECTIVE: To assess the effect of interleukin (IL)-2 combined with highly active antiretroviral therapy (HAART) on HIV-1 pro-viral DNA quantification in HIV-1-infected patients with CD4<200/mm3. PATIENTS AND METHODS: Seventy patients with CD4<200 cells/mm3 and CV<1000 copies/ml were enrolled in a 6-month randomized controlled study to evaluate the subcutaneous injection of IL-2 in addition to HAART versus HAART alone. Then, in a non-comparative phase from week 28 to week 80, IL-2 was proposed to patients from both groups. The HIV-1 pro-viral DNA was quantified at baseline, week 24 and week 50 by a real-time polymerase chain reaction (PCR) assay in peripheral blood mononuclear cells (PBMC). Analysis of resistance mutations was performed at baseline and after 6-months of IL-2. RESULTS: HIV-1 DNA level in PBMC increased significantly in patients treated with 6 months of HAART combined to IL-2 (+0.24 log10, P=0.009) compared to a slight decrease in patients treated with HAART alone (-0.05 log10). Moreover, a DNA increase was observed in control group patients when receiving 6 months of IL-2 (+0.34 log10). No increase of HIV-1 DNA was seen when this measure was expressed per million CD4 cells; it was probably hidden by the intense increase of CD4 lymphocytes after 6 months of IL-2 administration. No evolution of resistance mutations in pro-viral DNA was observed during 6 months of IL-2, despite the increase of pro-viral DNA and the occurrence of transient increase of viraemia. CONCLUSION: Administration of IL-2 combined with HAART in HIV-1 infected-patients with advanced disease led to an increase of HIV-1 pro-viral DNA per million PBMC without virological failure or emergence of resistance.

Anti-HIV Agents↗
PubMed2002

Emergence and selection of RNA virus variants: memory and extinction.

Two features of viral quasispecies are reviewed: the presence of memory genomes as minority components of their mutant spectra, and viral extinction due to enhanced mutagenesis. Memory has been documented with several genetic markers of the important animal picornavirus foot-and-mouth disease virus (FMDV). The presence of memory genomes in viral quasispecies may accelerate their adaptive response whenever a selective constraint has already been experienced by a viral population during previous stages of its evolution. Enhanced mutagenesis has been shown to lead to losses of infectivity of a number of RNA viruses: poliovirus, vesicular stomatitis virus, human immunodeficiency virus type 1 and FMDV. These observations, based on the theoretical prediction of the existence of a copying error-threshold for maintenance of genetic information, may contribute to the development of a new antiviral strategy.

Animals↗
PubMed1996

Evolution of canine parvovirus involved loss and gain of feline host range.

Canine parvovirus (CPV) type-2 emerged as a new virus infecting dogs in 1978, and it was probably derived as a variant of feline panleukopenia virus or of a closely related virus infecting another carnivore. CPV type-2 was subsequently replaced in nature by antigenically variant viruses (CPV type-2a and CPV type-2b) which now coexist in dog populations worldwide. We show that CPV type-2 isolates did not replicate in cats, but that both CPV type-2a and CPV type-2b isolates replicated efficiently. About 10% of the viruses isolated from cats with natural parvovirus disease were antigenically indistinguishable from CPV type-2a or type-2b. The capsid protein gene sequence of a 1990 feline parvovirus isolate ("FPV-24") was essentially identical to the sequence of CPV type-2b viruses from dogs. The loss and reacquisition of the feline host range in CPV was most likely due in each case to small numbers of changes in a region of the virus capsid where three protein monomers interact.

Animals↗
PubMed1993

In vivo selection of randomly mutated retroviral genomes.

Darwinian evolution, that is the outgrowth of the fittest variants in a population, usually applies to living organisms over long periods of time. Recently, in vitro selection/amplification techniques have been developed that allow for the rapid evolution of functionally active nucleic acids from a pool of randomized sequences. We now describe a modification of the nucleic acid-evolution protocol in which selection and amplification take place inside living cells by means of a retroviral-based replication system. We have generated a library of HIV-1 DNA genomes with random sequences in particular domains of the TAR element, which is the binding site for the Tat trans-activator protein. This mixture of HIV genomes was transfected into T cells and outgrowth of the fittest viruses was observed within two weeks of viral replication. The results of this in vivo selection analysis are consistent with the notion that primary sequence elements in both TAR bulge and loop domains are critical for Tat-mediated trans-activation and viral replication.

Base Sequence↗
PubMed1986

[Viral vaccines produced by gene technology and synthesis].

The gradual evolution of vaccine development from Jenner's smallpox vaccine to recombinant genetechnology has provided the basis for prophylactic control of a lot of important infectious diseases of humans and animals. Modern biotechnology has accomplished antigen identification and isolation of antigen determinants as well as the preparation of proteins and synthetic peptides. The application of such techniques has failed, however, to develop a better vaccine in case of FMD-Virus. Present efforts are directed on the improvement of antigen presentation and immunogenicity of such protein- and peptide vaccines.

Amino Acid Sequence↗
PubMed2004

Genotypic resistance and HIV-1 subtype in Brazilian children on dual and triple combination therapy.

BACKGROUND: Antiretroviral therapy is provided by the Brazilian Ministry of Health to eligible HIV-infected individuals. Based on clinical and immunological classification, the Brazilian guidelines recommend dual or triple therapy for children. However, the development of drug-resistant strains or poor adherence to therapy could impact the efficacy of this approach. OBJECTIVES: We examined relationships between RNA levels, CD4+ T-cell counts, treatment history, and the prevalence of drug-resistant variants in a cohort of HIV-1-infected children in Rio de Janeiro, Brazil. STUDY DESIGN: Direct sequencing of reverse transcriptase and protease genes from plasma was performed. Virologic and CD4+ T-cell counts responses to therapy were assessed by changes in HIV-1 RNA levels and CD4+ T-cell counts from baseline. RESULTS: Thirty-seven patients were receiving dual therapy and 38 were on triple therapy at enrollment, segregated by antiretroviral history. Both groups had a higher increase in CD4+ T cell counts and a lower viral load in pre-treatment antiretroviral-naïve subjects. Notably, there was a direct correlation between the higher frequencies of drug-resistance mutations and cross-resistance with previous usage of antiretroviral (ARV) therapy in both groups. Non-B subtypes isolates were found in 21.3% of samples. A smaller increase in CD4+ T cell counts was found between non-B subtypes when compared to B-subtypes. CONCLUSIONS: These results suggest that less immunological recovery and a higher number of mutations related to drug resistance were associated with previous usage of ARV and consequent higher time under drug selective pressure in these HIV-infected Brazilian children. These facts suggest the preferential use of triple drug combination as first line regimen in children.

Adolescent↗
PubMed1994

Comparative analysis of NS3 sequences of temporally separated dengue 3 virus strains isolated from southeast Asia.

By a combination of PCR and direct-cycle sequencing using consensus primers, we analyzed approximately 400-bp fragments within the NS3 genes of twenty-one dengue virus type 3 strains isolated from five neighboring Southeast Asian countries at different time intervals from 1956 to 1992. The majority of base disparities were silent mutations, with few predicted amino acid substitutions, thus emphasizing the strict conservation of the NS3 gene. Phylogenetic trees constructed on the basis of these nucleotide differences revealed distinct but related clusters of strains from the Philippines, Indonesia, and strains from Singapore and Malaysia of the 1970s and early 1980s, while the Thai cluster was relatively more distant. This genetic relationship was compatible with that proposed by other workers who have studied other dengue 3 virus genes such as E, M and prM. However, we observed that the more recent, epidemic-associated dengue 3 strains from Singapore and Malaysia of the late 1980s and early 1990s were more closely related to the Thai cluster, implying their evolution from the latter, and emphasizing the importance of viral spread via increasing travel within the Southeast Asian area and elsewhere. Nucleotide sequence analysis of the NS3 genes of dengue viruses can serve to advance the understanding of the epidemiology and evolution of these viruses.

Amino Acid Sequence↗
PubMed2006

Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.

Phage-mediated lysis has been an essential laboratory tool for rapidly identifying Bacillus anthracis for more than 40 years, relying on the gamma phage derivative of a Bacillus cereus prophage called W. The complete genomic sequences of the temperate W phage, referred to as Wbeta, and its lytic variant gamma were determined and found to encode 53 open reading frames each, spanning 40,864 bp and 37,373 bp, respectively. Direct comparison of the genomes showed that gamma evolved through mutations at key loci controlling host recognition, lysogenic growth, and possibly host phenotypic modification. Included are a cluster of point mutations at the gp14 tail fiber locus of gamma, encoding a protein that, when fused to green fluorescent protein, binds specifically to B. anthracis. A large 2,003-bp deletion was also identified at the gamma lysogeny module, explaining its shift from a temperate to a lytic lifestyle. Finally, evidence of recombination was observed at a dicistronic Wbeta locus, encoding putative bacterial cell surface-modifying proteins, replaced in gamma with a locus, likely obtained from a B. anthracis prophage, encoding demonstrable fosfomycin resistance. Reverse transcriptase PCR analysis confirmed strong induction at the dicistronic Wbeta locus and at four other phage loci in B. anthracis and/or B. cereus lysogens. In all, this study represents the first genomic and functional description of two historically important phages and is part of a broader investigation into contributions of phage to the B. anthracis life cycle. Initial findings suggest that lysogeny of B. anthracis promotes ecological adaptation, rather than virulence, as with other gram-positive pathogens.

Amino Acid Sequence↗
PubMed2004

Persistent HIV type 1 infection in semen and blood compartments in patients after long-term potent antiretroviral therapy.

HIV-1 RNA levels in semen and blood compartments decrease below detection limits during highly active antiretroviral therapy. Despite these therapeutic effects, it is clear that persistent, latent HIV-1 reservoirs are capable of rebounding in the absence of drug treatment or by evolution of escape mutants remain. The current study was designed to examine the presence of latent virus in semen and blood compartments and its evolution following potent combination therapy with indinavir (protease inhibitor) and efavirenz [nonnucleoside reverse transcriptase (RT) inhibitor]. Using an ultrasensitive in situ hybridization assay HIV-1 mRNA was detected in cultured seminal and blood mononuclear cells in all patients up to 1789 days posttherapy. Higher levels of HIV-1 mRNA were consistently detected in seminal mononuclear cells as compared to peripheral blood mononuclear cells (PBMC) in all time points analyzed posttherapy. Analysis of viral RNA from cultured PBMC before and after therapy displayed no evidence of therapy-induced drug resistance in the viral polymerase gene in the majority of patients. However, distinct envelope populations were detected in these viral RNA populations following therapy, indicating possible selection of quasispecies. The observed ongoing replication and evolution in the PBMC viral envelope sequences likely occurred in the seminal compartment HIV populations, given that the seminal cells showed the ability to express HIV-1 mRNA following cultivation. This together with our previous studies (Gupta P, et al.: J Infect Dis 2000;182:79-87) suggest that the genital and blood compartments likely serve as distinct reservoirs harboring latent HIV-1 during prolonged drug therapy.

Alkynes↗
PubMed1984

The late functions of Drosophila sigma virus.

Several criteria can be used to characterize the temperature-sensitive mutants of sigma virus: the lability of infectious centers initiated by the efficient viral particles following the inoculation; the localization of the temperature-sensitive period; the hereditary transmission of infection in a maternal Drosophila line at high temperature; the evolution of the CO2 sensitivity symptom and of the viral titer per fly for hereditarily infected flies kept at a permissive temperature and shifted to a restrictive one. Experimental data concerning the last two criteria are shown and discussed here. From all the criteria, three classes of ts mutants can be defined: the first class (prototype ts4) are the mutants blocked in hereditary transmission at high temperature. These mutants are affected during the whole viral cycle but the viral maturation is not directly affected. The second class (ts9, haP27) are the mutants directly affected in viral maturation. For these latter, the hereditary transmission in a maternal line is efficient at high temperature, and only a late temperature sensitive period is observed. The third class (prototype haP7) are the mutants for which only an early temperature-sensitive period is observed. In fact, for these mutants, the viral information is strongly heat-sensitive at the beginning of viral cycle. This defect is rapidly corrected, and the hereditary transmission, late viral functions and viral maturation are as efficient as those of wild type virus, at high temperature. Using temperature-sensitive mutants similar to the wild type virus at permissive temperature, a formal description of the viral cycle at 20 degrees C in the standard flies can be obtained. The long period necessary to perform an entire viral cycle (about 60 hours) is a result of the restrictive action on late viral functions at 20 degrees C of the Oe allele of the ref(3)O host gene present in the standard flies.

Animals↗
PubMed2004

Virological and immunological response to antiretroviral therapy in HIV-1 infected children: genotypic and phenotypic assays in monitoring virological failure.

Children differ from adults in both natural history of HIV-1 infection and their response to anti-retroviral therapy (ART). ART appears to be less successful in children than in adults at reducing HIV-1 RNA to below the level of detection of current assays. Nonetheless, children receiving ART frequently experience rises in CD4 cell counts, even in the absence of full virological suppression in plasma (discordant response). This immune repopulation in the presence of viral replication may increase the risk of emergence of drug-resistant viral variants. While the rationale for resistance testing is to optimise therapy, particularly when drugs are being changed following virological failure, it should be pointed out that currently available genotypic and phenotypic assays fail to detect drug resistance in about one third of viremic ART-treated children. This cannot be fully explained by the limitation of assays in detecting minor variants; factors other than resistance might be involved in the failure of therapy. Reduced fitness of drug-resistant viral variants is also unlikely to fully explain the discordant response to therapy, since immunological recovery is often observed even in the absence of detectable drug resistance. Rather, restoration of thymic function and higher thymic output may play a critical role in sustaining peripheral CD4 cell increases despite the persistence of viral replication. Such immune recovery might also drive the evolution of the replicating viruses.

Anti-HIV Agents↗
PubMed2002

Hemagglutinin sequence clusters and the antigenic evolution of influenza A virus.

Continual mutations to the hemagglutinin (HA) gene of influenza A virus generate novel antigenic strains that cause annual epidemics. Using a database of 560 viral RNA sequences, we study the structure and tempo of HA evolution over the past two decades. We detect a critical length scale, in amino acid space, at which HA sequences aggregate into clusters, or swarms. We investigate the spatio-temporal distribution of viral swarms and compare it to the time series of the influenza vaccines recommended by the World Health Organization. We introduce a method for predicting future dominant HA amino acid sequences and discuss its potential relevance to vaccine choice. We also investigate the relationship between cluster structure and the primary antibody-combining regions of the HA protein.

Antibodies↗
PubMed2005

Phylodynamic analysis of human immunodeficiency virus type 1 in distinct brain compartments provides a model for the neuropathogenesis of AIDS.

"Phylodynamic" analysis combines various statistical procedures that can be used to correlate the epidemiological and evolutionary behavior of viral pathogens with the immune system of the host. We utilized this approach to examine human immunodeficiency virus type 1 (HIV-1) gp120 envelope DNA sequences (V1, V2, and V3) isolated from different brain compartments of a T-cell-depleted patient diagnosed with severe HIV-associated dementia at the time of death. In agreement with previous reports, phylogenetic analysis showed distinct virodemes but also revealed a significant amount of viral gene flow among different brain compartments. Local-molecular-clock analysis showed that HIV-1 meninges and temporal lobe subpopulations evolve about 30 and 100 times faster, respectively, than the other viral populations in the brain. However, maximum likelihood codon-based substitution models did not detect any site under significant positive selective pressure, and the main cause of HIV-1 genetic variation appeared to be random genetic drift. Therefore, the higher evolutionary rate in the meninges and temporal lobe could be due to an enhanced infection/expansion rate of macrophages as a consequence of the immune system failure. In conclusion, in this case study, viral infection in the brain progressed with a nonspecific genetic evolution, recurrent migration events, and an expansion of macrophage-tropic sequences. The data suggest that after immune failure newly produced viral variants, which would be rapidly cleared under normal conditions, begin to productively infect macrophages in a "self-amplifying" cycle of infection/inflammatory response that could be at the origin of HIV-associated dementia.

AIDS Dementia Complex↗
PubMed1978

Type D primate retroviruses: a review.

The prototype virus of the type D retroviruses is the Mason-Pfizer monkey virus (MPMV). MPMV was originally isolated from a breast carcinoma of a female rhesus monkey (an Old World monkey). MPMV is of obvious importance in that it is the only retrovirus thus far isolated from a mammary tumor of a primate and has been shown to have transforming potential for primate cells in vitro. Subsequent to the isolation of MPMV viruses morphologically and immunologically indistinguishable from MPMV have been isolated from normal placenta and lactating mammary glands of other rhesus monkeys in captivity. Recently, viruses morphologically resembling MPMV have been isolated from a langur monkey (another Old World monkey) and from squirrel monkeys (a New World monkey). Based on nucleic acid hybridization studies, the latter 2 viruses represent endogenous viruses in their species of origin, whereas MPMV appears to be a horizontally related to the langur monkey isolate. Studies on the immunological relatedness of the type D retroviruses have demonstrated interspecies cross-reactivities between the major internal and external proteins of the viruses. Furthermore, these viruses also share cross-reactivity of their major external glycoproteins with those of the type C baboon endogenous virus. These interspecies reactivities can also be demonstrated in natural sera from both imported and laboratory-bred monkeys. The demonstration of these interspecies cross-reactivities shared by distantly related primate retroviruses provides a means for detecting determinants that are representative of all primate retroviruses presently known and yet to be isolated and may provide new assays for detection of a human retrovirus.

Animals↗
PubMed1996

Positive selection and rates of evolution in immunodeficiency viruses from humans and chimpanzees.

Evolutionary theory predicts the recent spread of primate immunodeficiency viruses (PIVs) to new human populations to be accompanied by positive selection in response to new host environments and/or by random genetic drift. I assess evidence for positive selection in human and chimpanzee PIVs type I (PIV1s), using ratios of synonymous to nonsynonymous nucleotide change based on branch lengths and outgroup rooting. Ratios are smaller for PIV1s from humans than for PIV1 from a chimpanzee for the pol, gag, and env glycoprotein 120 (gp120) regions, indicating greater effects of positive selection in PIV1s from humans. Parsimony-based relative rate tests for amino acid changes showed significant differences between PIV1s from humans and chimpanzees in 18 of 48 pairwise comparisons, with all 18 showing faster rates of change in PIV1s from humans. This study indicates that in some instances, the recent evolution of human PIV1s follows a speciational pattern, in which increased diversification of taxa is correlated with greater amounts of character change appearing and being maintained through time. This extends the generality of the speciational pattern to a group of organisms (viruses) having the fastest known rates of anagenetic change for nucleotide characters and indicates that comprehensive understanding of PIV1 evolution requires consideration of both anagenetic change within viral lineages and the relative historical success of different viral clades. Phylogenetic analyses show that neither PIV1s infecting humans nor those infecting chimpanzees represent monophyletic groups and suggest multiple host-species shifts for PIV1s.

Animals↗

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