Juan Cristina

Analysis of genetic variability in human respiratory syncytial virus by the RNase a mismatch cleavage method: Subtype divergence and heterogeneity

We have applied the RNase A mismatch cleavage method to the analysis of genetic variability among human Respiratory Syncytial (RS) viruses. Antisense RNA probes of the Long strain were hybridized to total RNA extracted from cells infected with other strains. The RNA:RNA heteroduplexes were digested with RNase A and the resistant products analyzed by gel electrophoresis. Each virus generated characteristic band patterns with the different probes. Comparative analyses of the cleavage patterns indicate that antigenic subtypes correlate with genetically distinct viral groups. Viruses within each subtype, however, show substantial genetic heterogeneity and progressive accumulation of genetic changes with time. This heterogeneity is also observed among viruses of the same epidemic outbreak which cannot be distinguished with a panel of monoclonal antibodies. Different genes and gene regions also differ in their rates of change. These results are discussed in terms of RS virus evolution.

Evidence of recombination in natural populations of hepatitis A virus

Genetic analysis of selected genome regions of hepatitis A virus (HAV) suggested that distinct genotypes of HAV could be found in different geographical regions. At least seven HAV genotypes have been identified all over the world, including four human genotypes (I, II, III, and VII) and three simian strains (IV, V, and VI). Phylogenetic analysis using full-length VP1 sequences revealed that human strain 9F94 has a close genetic relation with strain SLF-88 (sub-genotype VII). Nevertheless, the same analysis using full-length VP2 or VP3 sequences revealed that strain 9F94 has a close genetic relation with strain MBB (sub-genotype IB). To test the possibility of genetic recombination, phylogenetic studies were carried out, revealing that a crossing over had taken place in the VP1 capsid protein. These findings indicate that capsid-recombination can play a significant role in shaping the genetic diversity of HAV and, as such, can have important implications for its evolution, biology, and control.

Evidence of structural genomic region recombination in Hepatitis C virus

Background/AimHepatitis C virus (HCV) has been the subject of intense research and clinical investigation as its major role in human disease has emerged. Although homologous recombination has been demonstrated in many members of the family Flaviviridae, to which HCV belongs, there have been few studies reporting recombination on natural populations of HCV. Recombination break-points have been identified in non structural proteins of the HCV genome. Given the implications that recombination has for RNA virus evolution, it is clearly important to determine the extent to which recombination plays a role in HCV evolution. In order to gain insight into these matters, we have performed a phylogenetic analysis of 89 full-length HCV strains from all types and sub-types, isolated all over the world, in order to detect possible recombination events.MethodPutative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning, using putative recombinant sequence as a query.ResultsTwo crossing over events were identified in the E1/E2 structural region of an intra-typic (1a/1c) recombinant strain.ConclusionOnly one of 89 full-length strains studied resulted to be a recombinant HCV strain, revealing that homologous recombination does not play an extensive roll in HCV evolution. Nevertheless, this mechanism can not be denied as a source for generating genetic diversity in natural populations of HCV, since a new intra-typic recombinant strain was found. Moreover, the recombination break-points were found in the structural region of the HCV genome.

Evidence of recombination in quasispecies populations of a Hepatitis C Virus patient undergoing anti-viral therapy

Background/AimHepatitis C virus (HCV) has been the subject of intense research and clinical investigation as its major role in human disease has emerged. HCV circulates in vivo as a complex population of different but closely related viral variants, commonly referred to as a quasispecies. The extent to which recombination plays a role in the evolution of HCV quasispecies when patients are undergoing anti-viral therapy is currently unknown. In order to gain insight into these matters, we have performed a phylogenetic analysis of HCV quasispecies populations from six patients undergoing anti-viral therapy.MethodsPutative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning, using putative recombinant sequence as a query. Statistical support for the presence of a recombination event was done by the use of LARD program.ResultsA crossing-over event in the NS5A gene in a HCV strain recovered after four weeks of treatment was identified in quasispecies from a patient with sustained response. Putative parental-like strains were identified as strains circulating in previous weeks on the same patient.ConclusionOnly one recombinant strain was detected in all patient quasispecies populations studied. The recombination break-point is situated on the PKR-binding region of NS5A. Although recombination may not appeared to be extensive in NS5A genes of HCV quasispecies populations of patients undergoing antiviral therapy, this possibility should be taken into account as a mechanism of genetic variation for HCV.

Hepatitis C virus genetic variability and evolution

Hepatitis C virus (HCV) has infected over 170 million people worldwide and creates a huge disease burden due to chronic, progressive liver disease. HCV is a single-stranded, positive sense, RNA virus, member of the Flaviviridae family. The high error rate of RNA-dependent RNA polymerase and the pressure exerted by the host immune system, has driven the evolution of HCV into 7 different genotypes and more than 67 subtypes. HCV evolves by means of different mechanisms of genetic variation. On the one hand, its high mutation rates generate the production of a large number of different but closely related viral variants during infection, usually referred to as a quasispecies. The great quasispecies variability of HCV has also therapeutic implications since the continuous generation and selection of resistant or fitter variants within the quasispecies spectrum might allow viruses to escape control by antiviral drugs. On the other hand HCV exploits recombination to ensure its survival. This enormous viral diversity together with some host factors has made it difficult to control viral dispersal. Current treatment options involve pegylated interferon-α and ribavirin as dual therapy or in combination with a direct-acting antiviral drug, depending on the country. Despite all the efforts put into antiviral therapy studies, eradication of the virus or the development of a preventive vaccine has been unsuccessful so far. This review focuses on current available data reported to date on the genetic mechanisms driving the molecular evolution of HCV populations and its relation with the antiviral therapies designed to control HCV infection.

Evidence of recombination in Hepatitis C Virus populations infecting a hemophiliac patient

Background/AimHepatitis C virus (HCV) infection is an important cause of morbidity and mortality in patients affected by hereditary bleeding disorders. HCV, as others RNA virus, exploit all possible mechanisms of genetic variation to ensure their survival, such as recombination and mutation. In order to gain insight into the genetic variability of HCV virus strains circulating in hemophiliac patients, we have performed a phylogenetic analysis of HCV strains isolated from 10 patients with this kind of pathology.MethodsPutative recombinant sequence was identified with the use of GARD program. Statistical support for the presence of a recombination event was done by the use of LARD program.ResultsA new intragenotypic recombinant strain (1b/1a) was detected in 1 out of the 10 hemophiliac patient studied. The recombination event was located at position 387 of the HCV genome (relative to strain AF009606, sub-type 1a) corresponding to the core gene region.ConclusionAlthough recombination may not appear to be common among natural populations of HCV it should be considered as a possible mechanism for generating genetic diversity in hemophiliacs patients.

Evolution of the G and P genes of human respiratory syncytial virus (subgroup A) studied by the RNase A mismatch cleavage method

The G and P genes of human respiratory syncytial viruses (subgroup A), isolated between 1961 and 1989, were analyzed by RNase A one-dimensional fingerprinting, using the Long strain as the reference. Total RNA extracted from cells infected with the different isolates was hybridized to radiolabeled antisense G or P RNA probes of the Long virus. The RNA:RNA heteroduplexes were digested with RNase A and the resistant products analyzed by gel electrophoresis. Comparative analysis of the cleavage patterns revealed extensive genetic heterogeneity in both genes among viruses isolated in different epidemics. In contrast, 13 viruses isolated in Montevideo during a 3-month period showed much more restricted heterogeneity; thus, 11 viruses represented the predominant type of this outbreak and only 2 other viruses generated different RNA cleavage patterns distantly related to the major type. Statistical analysis of the results obtained indicated progressive accumulation of genetic changes with time along cocirculating evolutionary lineages within the same antigenic subgroup of RS virus. The results are discussed in terms of a model for RS virus evolution.

Evolution of Dengue Virus Type 3 Genotype III in Venezuela: Diversification, Rates and Population Dynamics

BackgroundDengue virus (DENV) is a member of the genus Flavivirus of the family Flaviviridae. DENV are comprised of four distinct serotypes (DENV-1 through DENV-4) and each serotype can be divided in different genotypes. Currently, there is a dramatic emergence of DENV-3 genotype III in Latin America. Nevertheless, we still have an incomplete understanding of the evolutionary forces underlying the evolution of this genotype in this region of the world. In order to gain insight into the degree of genetic variability, rates and patterns of evolution of this genotype in Venezuela and the South American region, phylogenetic analysis, based on a large number (n = 119) of envelope gene sequences from DENV-3 genotype III strains isolated in Venezuela from 2001 to 2008, were performed.ResultsPhylogenetic analysis revealed an in situ evolution of DENV-3 genotype III following its introduction in the Latin American region, where three different genetic clusters (A to C) can be observed among the DENV-3 genotype III strains circulating in this region. Bayesian coalescent inference analyses revealed an evolutionary rate of 8.48 × 10-4 substitutions/site/year (s/s/y) for strains of cluster A, composed entirely of strains isolated in Venezuela. Amino acid substitution at position 329 of domain III of the E protein (A→V) was found in almost all E proteins from Cluster A strains.ConclusionsA significant evolutionary change between DENV-3 genotype III strains that circulated in the initial years of the introduction in the continent and strains isolated in the Latin American region in recent years was observed. The presence of DENV-3 genotype III strains belonging to different clusters was observed in Venezuela, revealing several introduction events into this country. The evolutionary rate found for Cluster A strains circulating in Venezuela is similar to the others previously established for this genotype in other regions of the world. This suggests a lack of correlation among DENV genotype III substitution rate and ecological pattern of virus spread.

Comparison of hepatitis C viral loads in patients with or without coinfection with different genotypes.

ABSTRACT Hepatitis C virus genotyping was assessed for 257 chronic hepatitis C patients with viral loads above 1,000 IU/ml. Twelve patients were coinfected with more than one genotype. Their median viral loads did not differ significantly from those observed for monoinfected patients, which in turn did not vary significantly among different genotypes.

Genome-wide analysis of codon usage bias in Ebolavirus.

Ebola virus (EBOV) is a member of the family Filoviridae and its genome consists of a 19-kb, single-stranded, negative sense RNA. EBOV is subdivided into five distinct species with different pathogenicities, being Zaire ebolavirus (ZEBOV) the most lethal species. The interplay of codon usage among viruses and their hosts is expected to affect overall viral survival, fitness, evasion from hosts immune system and evolution. In the present study, we performed comprehensive analyses of codon usage and composition of ZEBOV. Effective number of codons (ENC) indicates that the overall codon usage among ZEBOV strains is slightly biased. Different codon preferences in ZEBOV genes in relation to codon usage of human genes were found. Highly preferred codons are all A-ending triplets, which strongly suggests that mutational bias is a main force shaping codon usage in ZEBOV. Dinucleotide composition also plays a role in the overall pattern of ZEBOV codon usage. ZEBOV does not seem to use the most abundant tRNAs present in the human cells for most of their preferred codons.