Evolutionary aspects of recombination in RNA viruses.
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Limited information is available concerning the molecular drift of the influenza neuraminidase (NA) genes. We report on the genetic variability of the NA gene from 31 influenza A:H3N2 viruses isolated in the Province of Québec (Canada) during the last three flu seasons (1997-2000). Amino acid substitutions within the NA protein were observed at rates of 1.01% and 0.45% between strains of the 1997-1998 and 1998-1999 seasons and between those of the 1998-1999 and 1999-2000 seasons, respectively. In most strains (28/31), amino acid changes occurred within at least one of four codons (197, 339, 370, and 401) previously implicated as antigenic sites. The 8 functional and 10 framework residues that compose the catalytic site of the NA enzyme were completely conserved over the study period. All isolates contained the seven conserved asparagine-linked glycosylation sites found in the NA of the progenitor A/Hong Kong/8/68 strain. In addition, most strains (30/31) had an eighth potential glycosylation site at position 329, whereas a ninth one was found at position 93 in 16 strains. The NA of all strains in this study was related to that of the vaccine strain A/Beijing/353/89, whereas the HA genes of these strains were related to the A/Beijing/32/92 vaccine strain. Thus, it appears that recent influenza strains continue to evolve from a reassortant produced during the cocirculation of the two above variants. Interestingly, some strains whose HA genes were closely related showed significant differences in their NA genes and conversely. This study confirms the importance of analyzing both the NA and the HA genes to assess the evolution of recent influenza epidemic strains.
We studied spatial and temporal variation in 20-23 Aedes aegypti samples collected in Phnom Penh and its suburbs to estimate the population genetic structure using allozymes and the susceptibility to a dengue-2 virus. Based on seven allozyme systems, we detected low levels of genetic exchanges (i.e. high, significant F(ST) values) between populations collected in the city centre, and different patterns of genetic structure for samples collected in the suburbs, depending on the type of environment and the date of collection. In the southern suburbs and the Chroy Chang Var Peninsula, differentiation became highly significant at the end of the dry season, whereas the opposite situation was observed for collections from the northern suburbs. Vector competence assessed by oral infections with a dengue-2 virus was lower for samples collected in the city centre than in the suburbs. A significant decrease of dengue susceptibility was observed in populations during the dry season. This study allows a model of Ae. aegypti population functioning in Phnom Penh to be suggested. Dynamics of dengue virus diffusion depend on the population genetic structure of the vector and its evolution over space and time.
We have carried out a phylogenetic study of the evolution of the VP1 gene sequence from different serological types and subtypes of foot-and-mouth disease virus (FMDV). The maximum-likelihood method developed by Hasegawa and co-workers (Hasegawa et al. 1985) for the estimation of evolutionary parameters and branching dates has been used to decide between alternative models of evolution: constant versus variable rates. The results obtained indicate that a constant rate model, i.e., a molecular clock, seems to be the most plausible one. However, additional information suggests the possibility that the appearance of serotype CS has been accompanied by an episode of rapid evolution (Villaverde et al. 1991). We discuss the possibility that this evolution of RNA viruses was due to episodic positive Darwinian selection, which would have helped the new variant to escape the immunogenic pressure from the hosts.
HCoV-HKU1 diversity and evolution were scarcely studied. We performed next-generation sequencing (NGS) and analysis of HCoV-HKU1 genomes over 5 years. NGS used Illumina technology on NovaSeq 6000 following whole genome PCR amplification by an in-house set of primers designed using Gemi and PrimalScheme. Genome assembly and analyses used CLC Genomics, Mafft, BioEdit, Nextstrain, Nextclade, MEGA, and iTol bioinformatic tools. Spike molecular modeling and dynamics simulations used Molegro Molecular Viewer and Hyperchem programs. Twenty-eight PCR systems allowed obtaining 158 HCoV-HKU1 genomes including 69 and 89 of genotypes A and B, respectively. Both genotypes co-circulated during the study period but one predominated each year. A total of 1683 amino acid substitutions including 80 in ≥ 10 genomes were detected in genotype A relatively to a 2004 reference. H512R in spike, first detected in 2009 and reported as involved in antibody neutralization, was found in all genotype A, almost always with V387I and K478N, and was predicted here to significantly improve cellular TMPRSS2 protein binding. Also, 1802 amino acid substitutions including 64 in ≥ 10 genomes were detected in genotype B relatively to a 2005 reference. This study substantially expands the global set of HCoV-HKU1 genomes. Genomics with protein structural analyses contributed to our understanding of HCoV-HKU1 evolution.
Dengue and dengue hemorrhagic fever re-emerged in Bangladesh in 2000 and 2001 and nearly all viruses isolated were dengue type 3. Phylogenetic analyses of the envelope genes of examples of these viruses indicated that they were most closely related to recently emerged dengue type 3 viruses from neighboring Thailand and Myanmar but distinct from those from India and Sri Lanka. Since this strain of dengue virus type 3 had not been associated with unusual patterns of disease in Thailand or Myanmar, it suggested that the outbreak in Bangladesh was due to local factors after the introduction of viruses from countries to the east rather than to the evolution of an unusually virulent strain of virus in Bangladesh.
Nucleotide sequences of the 5'-untranslated region (5'-UTR), at the 3'-side of the poly C tract, have been compared for 21 isolates of foot-and-mouth disease virus (FMDV) of serotype C from Europe, South America and The Philippines. A deletion of 43 nucleotides is present in the European isolates as compared with most American isolates. A larger deletion of 86 nucleotides is present in some viruses from South America and The Philippines. These deletions include the loss of one or two pseudoknot structures predicted in this region of the 5'-UTR. In addition, multiple point mutations have allowed the derivation of a phylogenetic tree which defines a grouping of isolates very similar to that derived from the capsid gene sequences of the same viruses. The study provides evidence that deletion (or addition) events must be very frequent during evolution of FMDV type C, since viruses which are phylogenetically very closely related (they belong to the same tree branch) may differ in the presence or absence of these deletions. Implications for FMDV evolution are discussed.
BACKGROUND/AIMS: To describe the clinical and virological evolution of a polyarteritis nodosa (PAN) case associated with a hepatitis B virus (HBV) pre-core promoter mutant infection that was successfully treated with plasma exchanges, corticosteroids, and interferon alpha (IFN-alpha). METHODS: Viral markers were used, including HBV DNA quantified by the branched DNA assay and detected by PCR, the HBV genome sequence, pre-S1Ag and anti-HBC IgM which were studied throughout the treatment period and the entire follow-up in the serum, while the presence of virus in extrahepatic sites was detected by immuno-staining. RESULTS: The patient was infected with a typical pre-core promoter mutant harboring four point mutations. Pre-S1Ag was cleared rapidly from serum, most likely via the formation of immune complexes since HBV DNA declined more progressively. Viral infection was then cleared after a second episode of hepatocyte lysis. This was accompanied by a recovery from all clinical manifestations. CONCLUSIONS: The favorable treatment outcome observed in this first case of pre-core promoter HBV mutant associated PAN underlines that combination therapy based on IFN-alpha can clear pre-core promoter HBV infection and cure PAN. It also provides new insight in the pathogenesis of HBV associated PAN.
Recently published evidence for sequence repetition in potyvirus genomes prompted us to analyse the published complete genome sequences and coat protein gene sequences of viruses of this family for evidence of replication slippage. Five of nine complete genomic sequences and 17 of 32 coat protein genes had significant sequence repetitions. Most of these were in coat protein genes, although the 5' region of the turnip mosaic virus genome also showed evidence of slippage. The results suggest that replication slippage may be involved in the evolution of viruses, as well as prokaryotes and eukaryotes, and that slippage can occur in both RNA and DNA when it is being replicated.
The nucleotide sequence of 17 variants of the satellite RNA of cucumber mosaic virus (CMV-satRNA) isolated from field-infected tomato plants in the springs of 1989, 1990, and 1991 was determined. The sequence of each of the 17 satRNAs was unique and was between 334 and 340 nucleotides in length; 57 positions were polymorphic. There was much genetic divergence, ranging from 0.006 to 0.141 nucleotide substitutions per site for pairwise comparisons, and averaging 0.074 for any pair. When the polymorphic positions were analyzed relative to a secondary structure model proposed for CMV-satRNAs, it was found that there were significantly different numbers of changes in base-paired and non-base-paired positions, and that mutations that did not disrupt base pairing were preferred at the putatively paired sites. This supports the concept that the need to maintain a functional structure may limit genetic divergence of CMV-satRNA. Phylogenetic analyses showed that the 17 CMV-satRNA variants clustered into two subgroups, I and II, and evolutionary lines proceeding by the sequential accumulation of mutations were apparent. Three satRNA variants were outliers for these two phylogenetic groups. They were shown to be recombinants of subgroup I and II satRNAs by calculating phylogenies for different molecular regions and by using Sawyer's test for gene conversion. At least two recombination events were required to produce these three recombinant satRNAs. Thus, recombinants were found to be frequent ( approximately 17%) in natural populations of CMV-satRNA, and recombination may make an important contribution to the generation of new variants. To our knowledge this is the first report of data allowing the frequency of recombinant isolates in natural populations of an RNA replicon to be estimated.
In order to evaluate the evolution of transfusional hepatitis C in haemophiliacs, we performed a retrospective study of ALT levels and HCV viraemia with a RNA PCR assay in 57 patients. We found that the vast majority of HCV-infected patients remained viraemic (43/57 = 75%) and higher ALT levels correlated with HCV viraemia. Although indicators of the transfusional viral load (age, severity of haemophilia) and HBV co-infection did not correlate with HCV RNA replication, HIV seropositivity was strongly associated with persistence of HCV viraemia (23/25 = 92% in HIV-positive versus 20/32 = 62% in HIV-negative patients), without any correlation with CD4 counts. Genotyping of HCV in the 43 viraemic patients shows more frequent genotype 1 in the HIV-seropositive group (14/23) than in the seronegative group (6/20). Our data emphasize that besides the role of the immunodeficiency status, the genotypes of HCV might be involved in the differences observed in terms of HCV RNA replication between the HIV-seropositive and seronegative haemophiliacs.
We have studied the evolution of sequences which include the RNA polymerase binding sites at the 5' and 3' termini of vesicular stomatitis virus mutants (Sdi-) resistant to defective-interfering particles. We observed a striking stepwise accumulation of stable base substitutions within the area of replication initiation at the 5'-terminal 54 nucleotides of Sdi- mutants isolated at intervals from persistent infections and undiluted lytic passage series. Fewer mutations accumulated in the region of transcription initiation at the 3' end and in those portions of the N and L protein coding cistrons examined. The termini changes are not strictly required to obtain the Sdi- phenotype. However, it is possible that they represent stepwise compensatory changes to accommodate Sdi- mutations affecting viral replication or encapsidation gene products or both. These results have important implications for RNA virus genome evolution.
Annual influenza epidemics are caused by rapid evolution of the viral genome. Continuous and extensive antigenic variation has been shown for hemagglutinin (HA), the principal immunizing antigen of the virus. Monitoring of the antigenicity of circulating influenza viruses is necessary for selection of the most suitable vaccine strains. In this study, characterization of influenza A/H3N2 and influenza B viruses recently circulating in Germany was performed by molecular and antigenic analysis. Sequencing and phylogenetic analysis of the HA1 gene revealed that two distinct groups of H3N2 viruses co-circulated during 1997/1998. The majority of isolates clustered with the new drift variant A/Sydney/5/97, as was also shown by antigenic characterization. A noteworthy genetic drift of H3N2 viruses was evident during the winter 1998/1999. However, serological characterization using hemagglutinin inhibition tests did not result in detection of viruses belonging to different groups as confirmed by molecular analysis. Influenza B viruses isolated during 1996/1997 were antigenically closely related to the prototype vaccine strains B/Beijing/184/93 or B/Harbin/7/94. Molecular analysis demonstrated that our German 1996/1997 isolates differed by nine amino acids from B/Harbin/7/94 and represented a group of viruses that was completely different from the Harbin strain. Retrospective studies revealed the circulation of B/Yamanshi/166/98-like viruses in Germany already during the 1996/1997 season. Our results suggest that molecular analysis of the HA gene is important to complement the antigenic characterization for a better selection of appropriate vaccine strains.
The genotypic-phenotypic correlates of human immunodeficiency virus type 1 (HIV-1) env gene evolution were investigated in samples from eight infected children under antiretroviral therapy (ART) and virological failure. Evolution of the gp120 C2-V5 env sequence was demonstrated in all subjects but one by sequence analysis of the replicating plasma virus collected at baseline and 12-15 months after ART initiation. The analysis of the host's selective pressure showed that in four subjects, the ratio of nonsynonymous to synonymous amino acid substitutions was higher in the V3 sequences than in the C2-V5 region ([K (a)/K (s)](V3)/[K (a)/K (s)](C2V5) >1.0). Interestingly, this feature was observed only in subjects (four of five) showing an increase in T cell receptor rearrangement excision circle (TREC)-bearing cells and in CD4(+) T-lymphocytes despite persistent viremia (discordant therapeutic response) (p = 0.02), thus suggesting that the V3 loop is a target of the immune reconstitution under ART. Using phenotypic analysis of recombinant viruses expressing exogenous V3 sequences, a reverse shift from CXCR4-tropic to CCR5-tropic variants was demonstrated in two of the four subjects, further indicating that the host's selective pressure sharply forces the V3 evolution of replicating variants. The data indicate that a complex HIV-1-host interplay occurs in children receiving antiretroviral treatments and suggest that the recovery of thymic function places a selective constraint on the viral V3 loop.
The XIX Symposium of the International Association for Comparative Research on Leukemia and Related Diseases (IACRLRD, President: Prof. Dr. R. Hehlmann) was held in Mannheim and Heidelberg, Germany, July 13 - 18, 1997. Comparative research in cancer was systematically established in the 1950s. Similarities in morphology, biology and pathology between animal and human leukemias and related diseases and the viral origin of a variety of animal leukemias and related diseases (lymphomas, sarcomas, breast tumors etc.) led to the concept that comparative research should promote the understanding of human leukemias and related diseases. In 1960 the World Health Organization inaugurated the establishment of a World Committee for Comparative Leukemia Research. The first symposium took place in Hannover, Germany, in 1963. After the fifth symposium in Padova, Italy, in 1971 the International Association for Comparative Research on Leukemia and Related Diseases (IACRLRD) was founded to complement the World Committee and to expand the international effort. The history of the symposium shows the evolution from a meeting on animal leukemia viruses into one dealing with viral and genetic aspects of human and animal leukemia and related diseases. The scientific evolution of the Abelson murine leukemia virus with its abl oncogene in the 1970s to what currently appears as the most reliable marker for human chronic myeloid leukemia is merely one example. Comparative research has reached a new dimension with the the recent advances in sequencing of the genomes of a variety of species and of humans. Many genes identified in the human genome and relevant for disease can be found in the genomes of animal species and even in the genomes of bacteria and of yeast. This reminds us that not just human and animal biology but also pathology must be regarded as a continuum of evolution and that much can be learned from comparing the genetic information of different species. Comparative genome research will allow conclusions to be drawn from principles recognized in animal species which are relevant to human diseases. It is likely that the application of comparative research to genome analysis will provide basic new insights in molecular medicine into the function of living beings for both animal species and humans. The current revolution in genomics is the latest phase in a rich history of medical progress related to the comparative approach. Meetings and organizations that have grown out of IACRLRD, include, at least to some extent: the Meeting of the International Human Retrovirology Association, the Gallo Lab Meeting , the Feline Retrovirus Meeting, the Cold Spring Habor Retrovirus Meeting, international and regional AIDS meetings, and many others. The XIX symposium in Mannheim included five memorial lectures, seven plenary sessions, 18 parallel sessions, two round table discussions and a public forum. In addition, six associated satellite symposia were held. The general meeting, attended by participants from 27 countries, integrated thematically contributions of genetic, cellular, and viral factors toward the development of leukemia and lymphoma and sought unifying concepts in leukemogenesis.