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Value of a ligase chain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirus.
Human cytomegalovirus (HCMV) isolates resistant to ganciclovir were found in patients undergoing therapy. Therefore, we have developed a new specific and sensitive method--a ligase chain reaction (LCR) assay--for detection of frequently encountered 594 mutated codon in ganciclovir (GCV) resistant virus. Previous studies characterized an alanine to valine change on codon 594 in resistant strains. A novel substitution in 594, alanine to glycine, is described which is also capable of conferring ganciclovir resistance. LCR products were analyzed on polyacrylamide gel- and the mutant was detected using a non radioactive method. The LCR product detection was then adapted to a microtitre plate format with a colorimetric detection. This method allowed the distinction of mutated GCV-resistant strains from sensitive strains with a high sensitivity, and the detection of a low percentage of mutated DNA in virus load. This assay could be useful in following the evolution of mutated DNA compared to viral infection.
Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada.
Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.
Genome analysis of dengue type-1 virus isolated between 1990 and 2001 in Brazil reveals a remarkable conservation of the structural proteins but amino acid differences in the non-structural proteins.
We have investigated the genetic diversity of dengue type-1 (DEN-1) virus in Brazil. The full nucleotide sequences of three DEN-1 virus isolated from DEN fever (DF) and DEN hemorrhagic fever patients in northeastern Brazil in 1997 (BR/97) and one from a DF patient in the south of Brazil in 2001 (BR/01) were compared to that of the reference strain BR/90 obtained in the city of Rio de Janeiro in 1990. Sequence analysis showed that the structural proteins were remarkably conserved between all isolates. A total of 27 amino acid changes occurred throughout the non-structural proteins. Among them, nine amino acid substitutions were specific of BR/97 and BR/01 isolates, indicating that in situ evolution of these strains had occurred. Within the BR/97 and BR/01 samples, some amino acid substitutions have been previously identified in DEN-1 virus strains sequenced so far, suggesting that recombination events might have occurred.
Sequence variation and evolution of Cryphonectria hypovirus 1 (CHV-1) in Europe.
Cryphonectria hypovirus 1 (CHV-1) acts as a naturally occurring biological control agent for chestnut blight, a destructive fungal disease of chestnut trees, which has been introduced into Europe in the 1930s. We have determined partial nucleotide and deduced amino acid sequences of the ORF A of 47 CHV-1 isolates collected in Europe over a period of 28 years. Phylogenetic analysis revealed the presence of four groups or single viruses, which showed sequence divergences ranging from 11 to 19%. These results confirm the previous subtype classification based on RFLP markers, with the exception of the two CHV-1 subtypes E and D, which appear to be related closer than anticipated previously. Dates of divergences between CHV-1 subtypes, calculated from nucleotide substitution rates, indicate that the CHV-1 subtypes diverged several hundreds years ago. Our results suggest that the genetic variation among CHV-1 subtypes did not evolve in Europe and support the hypothesis of multiple introductions of CHV-1 into Europe.
Evidence for the assignment of two strains of SPLV to the genus Potyvirus based on coat protein and 3' non-coding region sequence data.
The use of potyvirus-specific primers and subsequent application of the RACE procedure allowed the cloning of the 3' terminal 1088 nucleotides of the genomic RNA of the Taiwan isolate of sweetpotato latent virus (SPLV-T) and the 3' genomic 1085 nucleotides of a SPLV-like virus from China (SPLV-CH). The sequence of an internal part of the presumptive nuclear inclusion b gene was also determined for both isolates. Detailed sequence analyses revealed the presence of consensus motifs which indicated that SPLV-CH and SPLV-T should be regarded as members of the genus Potyvirus. Multiple sequence alignments and phylogenetic analyses were also performed and unambiguously assessed these isolates as strains of a distinct Potyvirus. SPLV was not related to other potyviruses infecting sweetpotato nor to any other sequenced virus. From the presence of the DAG box, SPLV-CH is expected to be a typical aphid transmitted Potyvirus whereas a conceivable explanation is proposed for the non-aphid transmission of SPLV-T.
Characterization of arenaviruses using a family-specific primer set for RT-PCR amplification and RFLP analysis. Its potential use for detection of uncharacterized arenaviruses.
Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.
Molecular characterization of the Japanese encephalitis virus representative immunotype strain JaGAr 01.
We determined the full genomic sequence of the Japanese encephalitis virus JaGAr 01 strain and its predicted amino acid sequence. Nucleotide sequence comparison with ten fully sequenced JE strains shows a homology range from 89.62 to 99.49%. Amino acid sequence homologies range from 96.85 to 99.74%. Comparison of amino acid sequences shows a unique amino acid, arginine, for JaGAr 01 at position 123 of the E-protein, while the eight other strains contained serine. Secondary structure prediction by free energy minimization shows a unique structure for JaGAr 01 that includes an RNA segment that is conserved for all flaviviruses. Speculation is made about the role these results may play in the replication and antigenic characteristics of JaGAr 01. Phylogenetic analyses of the E-protein of JaGAr 01 together with 35 other JE strains showed diversity in amino acid characteristics between the prototype strains Nakayama, JaGAr 01 and Beijing-1. Phylogenetic trees computed by neighbor joining and Fitch Margoliash analysis of nucleic acid and protein sequences showed Nakayama and Beijing in one cluster different from JaGAr 01.
HIV, 'an evolving species'. Roles of cellular activation and co-infections.
Each small variation of the genome of a species can be preserved if it is useful for the survival of the species in a given environment. Within this framework, the finality of the biological cycle of HIV consists in a search for harmony (biological coherence) with man, which is to say a stable condition. Cellular activation appears to be the strategy developed by HIV in order to achieve this coherence. The price of this strategy is the AIDS. The first contact between HIV and immune system appears to determine the subsequent clinical outcome and the future of HIV. Lymphocytic activation varies during the course of the vital cycle of HIV. For each individual, this lymphocytic activation depends on both the HLA repertoire acquired during thymic ontogenesis and the antigenic experience before and after HIV infection. Thus intercurrent infections alter the immune condition of the organism and influence the outcome of HIV. We described a synthetic analysis of the effects of HIV on the surface protein expression and the cellular activation pathways which should provide insights in the evolutionary relationship between HIV and man and should permit to do a more physiological therapeutic approach.
Can evolutionary principles help resolve the acquired immune deficiency syndrome crisis?
The population of retroviruses inside each infected host can quickly adapt its genes in response to survival threats. For this reason, therapeutics attempting to eliminate or inhibit the human immunodeficiency virus have been unsuccessful. However, it may be possible to apply evolutionary principles to the problem of human immunodeficiency virus infection/acquired immune deficiency syndrome. The rapid evolution capabilities of this pathogen might assist researchers in evolving human immunodeficiency virus toward the goal of virus-host coexistence. In this relationship, infecting viruses do not harm their host. Two different examples illustrate the theoretical application of evolutionary principles to this problem. First, by viewing the virus as a new gene of the immune system, it is possible to evolve this gene toward the goal of improving immune function. Second, the retroviral population of each host is maladapted for long-term survival. Retroviral genes could be evolved with respect to continued existence in the host.
Evolutionary strategies of human T-cell lymphotropic virus type II.
Human T-cell lymphotropic virus type II (HTLV-II) primarily infects two different populations in which the virus is transmitted in very diverse ways. In endemically infected populations, the virus is propagated through sexual contact, and by mother to child transmission via breast-feeding, among intravenous drug users (IDUs), spread is mainly due to blood-borne transmission via needle sharing. The phylogeny of HTLV-II strains isolated from American Indian and Pygmy tribes and strains from IDUs, reveal that the virus originated on the African continent as a result of a simian to human transmission at least 400,000 years ago. HTLV-II was very likely introduced into the American continent during one or more migrations of HTLV-II infected Asian populations over the Bering land bridge, some 15,000-35,000 years ago. During the last few decades, HTLV-II has been transmitted from native American Indians to IDUs at least twice, followed by a rapid spread of the virus in the drug users population world-wide due to the practice of needle sharing. Molecular clock analysis showed that HTLV-II has two different evolutionary rates, with the molecular clock for the virus in IDUs ticking 150-350 times faster than the one in endemically infected tribes: 2.7x10(-4) compared to 1.7/7.3x10(-7) nucleotide substitutions per site per year in the LTR region. Although many of the HTLV-II infected drug users are co-infected with HIV, the dramatic acceleration of the evolutionary rate seems to be mainly related to the different modes of transmission in the two populations. These contrasting evolutionary rates correlate with an endemic spread of HTLV-II in infected tribes compared to an epidemic spread in IDUs.
Bacteriophages with tails: chasing their origins and evolution.
Comparative genomic analysis of the tailed bacteriophages shows that they are genetically mosaic with respect to each other, implying that horizontal exchange of sequences is an important component of their evolution. Horizontal exchange occurs intensively among closely related phages but also at reduced frequency across the entire population of tailed phages. It results in exchange of homologous functions, exchange of analogous but non-homologous functions as with the prophage integrases, and introduction of novel functions into the genome as with the morons. Extrapolation of these processes back in evolutionary time leads to a speculative model for the origins and early evolution of phages.
The diversity and evolution of the T4-type bacteriophages.
Recent studies suggest that viruses are the most numerous entities in the biosphere; bacteriophages, the viruses that infect Eubacteria and Archaea, constitute a substantial fraction of this population. In spite of their ubiquity, the vast majority of phages in the environment have never been studied and nothing is known about them. For the last 10 years our research has focused on an extremely widespread group of phages, the T4-type. It has now become evident that phage T4 has a myriad of relatives in nature that differ significantly in their host range. The genomes of all these phages have homology to the T4 genes that determine virion morphology. Although phylogenetically related, these T4-type phages can be subdivided into four groups that are increasingly distant from T4: the T-evens, the pseudo T-evens, the schizo T-evens and the exo T-evens. Genomic comparisons between the various T4-type phages and T4 indicate that these genomes share homology not only for virion structural components but also for most of the essential genes involved in the T4 life cycle. This suggests that horizontal transmission of the genetic information may have played a less general role in the evolution of these phages than has been supposed. Nevertheless, we have identified several regions of the T4-type genome, such as the segment containing the tail fiber genes that exhibit evidence of extensive modular shuffling during evolution. The T4-type genomes appear to be a mosaic containing a large and fixed group of essential genes as well as highly variable set of non-essential genes. These non-essential genes are probably important for the adaptation of these phages to their particular life-style. Furthermore, swapping autonomous domains within the essential proteins may slightly modify their function(s) and contribute to the adaptive ability of the T4-type phage family. Regulatory sequences also display considerable evolutionary plasticity and this too may facilitate the adaptation of phage gene expression to new environments and stresses.
Evolution of influenza A(H1N1) viruses during a period of low antigenic drift in 1986-91: sequence of the HA1 domain of influenza A/Finland/158/91.
This study used the nucleotide sequence coding for the HA1 domain of virus haemagglutinin to show that influenza A/Finland/158/91, which represents the H1N1 subtype viruses prevalent in Finland in 1990/91, was a direct descendant of a virus (A/NN/1605/88) isolated during the 1988/89 epidemic season in Japan. The elevated rate of 7.4 x 10(-3) nucleotide substitutions per site per year is discussed. The new branch of H1N1 subtype viruses is characterized by loss of a glycosylation site, which may affect subsequent antigenic drift.
RNA viruses: genome structure and evolution.
The explosive pace of sequencing of RNA viruses is leading to rapid advances in our understanding of the evolution of these viruses and of the ways in which their genomes are organized and expressed. New insights are coming not only from genomic nucleotide sequence comparisons, but also from direct sequencing of transcribed mRNAs and of RNAs that serve as intermediates in replication.
Endogenous retroviruses: still active after all these years?
Embedded in the genomes of all vertebrates are the proviral remnants of previous retroviral infections. Although the overwhelming majority has suffered inactivating mutations, current research suggests that members of one family of human retroelements may still be capable of movement.
The origins and ongoing evolution of viruses.
Genome analyses of double strand DNA tailed bacteriophages argue that they evolve by recombinational reassortment of genes and by the acquisition of novel genes as simple genetic elements termed morons. These processes suggest a model for early virus evolution, wherein viruses can be regarded less as having derived from cells and more as being partners in their mutual co-evolution.
Human retroviral sequences associated with extracellular particles in autoimmune diseases: epiphenomenon or possible role in aetiopathogenesis?
Publications describing retroviral sequences associated with extracellular particles in Sjögren's syndrome or systemic lupus erythematosus, multiple sclerosis, and type I diabetes present novel arguments and raise complex questions about eventual relationships between retroviruses and autoimmunity. They are presented and discussed in the present review, preceded by an overview of the biology of retroviral elements.