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Muto ergo sum. Hepatitis B and the forces of evolution.

The hepatitis B virus (HBV) polymerase has no editing function and thus frequently transcribes its template incorrectly to result in viral variants that go under the name of "mutants." These mutants may be viewed as part of a continuing Darwinian evolution of HBV with some survival benefit. They also result in unusual extrahepatic manifestations of HBV infection, which will doubtless continue to divert the hepatologist.

Biological Evolution↗

Evolution of the receptor binding phenotype of influenza A (H5) viruses.

Receptor specificity of influenza A/H5 viruses including human 2003-04 isolates was studied. All but two isolates preserved high affinity to Sia2-3Gal (avian-like) receptors. However, two isolates (February, 2003, Hong Kong) demonstrated decreased affinity to Sia2-3Gal and moderate affinity to a Sia2-6Gal (human-like) receptors. These two viruses had a unique Ser227-Asn change in the hemagglutinin molecule. Thus, a single amino acid substitution can significantly alter receptor specificity of avian H5N1 viruses, providing them with an ability to bind to receptors optimal for human influenza viruses. Asian 2003-04 H5 isolates from chickens and humans demonstrated highest affinity to the sulfated trisaccharide Neu5Acalpha2-3Galbeta1-4(6-HSO3)GlcNAcbeta (Su-3'SLN) receptor but, in contrast to 1997 isolates, had increased affinity to fucosylated Su-3'SLN. American poultry H5 viruses also had increased affinity to Su-3'SLN. These data demonstrate that the genetic evolution of avian influenza A(H5N1) viruses is accompanied during adaptation to poultry by the evolution of their receptor specificity.

Amino Acid Sequence↗

The appearance of escape variants in vivo does not account for the failure of recombinant envelope vaccines to protect against simian immunodeficiency virus.

The presence or evolution of immune escape variants has been proposed to account for the failure of recombinant envelope vaccines to protect macaques against challenge with simian immunodeficiency virus (SIVmac). To address this issue, two groups of three cynomolgus macaques were immunized with recombinant SIV Env vaccines using two different vaccine schedules. One group of macaques received four injections of recombinant SIV gp120 in SAF-1 containing threonyl muramyl dipeptide as adjuvant. A second group were primed twice with recombinant vaccinia virus expressing SIV gp160 and then boosted twice with recombinant SIV gp120. Both vaccine schedules elicited neutralizing antibodies to Env. However, on the day of challenge, titres of anti-Env antibodies measured by ELISA were higher in macaques primed with recombinant vaccinia virus. Following intravenous challenge with 10 monkey infectious doses of the SIVmac J5M challenge stock, five of the six immunized macaques and all four naive controls became infected. The virus burdens in PBMC of macaques that were primed with recombinant vaccinia virus were lower than those of naive controls, as determined by virus titration and quantitative DNA PCR. Sequence analysis was performed on SIV env amplified from the blood of immunized and naive infected macaques. No variation of SIV env sequence was observed, even in macaques with a reduced virus load, suggesting that the appearance of immune escape variants does not account for the incomplete protection observed. In addition, this study indicates that the measurement of serum neutralizing antibodies may not provide a useful correlate for protection elicited by recombinant envelope vaccines.

Amino Acid Sequence↗

Factors influencing host-virus interactions. I. A comparison of viral multiplication and histopathology in infant, adult, and cortisone-treated adult mice infected with the Conn. -5 strain of Coxsackie virus.

A study was made of the pathogenesis of infection due to the Conn.-5 strain of Coxsackie virus in 4 to 5 day old infant mice, untreated adult mice, and adult mice treated with cortisone. The quantitative distribution of virus and the evolution of lesions in different tissues were followed for the first 7 days of the infection. Virus dissemination was prompt and widespread via the blood in all groups. In 4 to 5 day old infant mice viral multiplication and cellular injury occurred in many organs and tissues, while in untreated adult mice these processes were largely limited to the pancreas, even though infecting virus appeared to be equally available to other tissues from the blood. Treatment of adult mice with a single injection of 2.5 mg. cortisone resulted in viral multiplication and tissue damage in several sites in addition to the pancreas, the most marked occurring in the liver and heart. In a consideration of possible mechanisms involved, it was thought unlikely that the differences in the course of the disease in the three groups could be attributed solely to differences in the specific immune response. It is suggested that developmental changes in cells and tissues, perhaps related to cellular metabolism and alterable by cortisone administration, are the major factors determining the location and extent of viral multiplication and tissue injury in this infection in mice.

Animals↗

SARS/avian coronaviruses.

In the hunt for the aetiology of the SARS outbreak in 2003, a newly developed virus DNA micro-array was successfully used to hybridise PCR products obtained by random amplification of nucleic acids extracted from a cell culture infected with material from a SARS patient. The SARS agent was found to hybridise with micro-array probes from both coronaviruses and astroviruses, but one of the coronavirus probes and the four astrovirus probes contained redundant sequences, spanning a highly conserved motif, named s2m, found at the 3' end of the genomes of almost all astroviruses, one picornavirus, and the poultry coronaviruses. The three other coronavirus probes, that hybridised with the SARS agent, were located in the replicase gene, and it could be concluded that the SARS agent was a novel coronavirus, harbouring s2m. The presence of this motif in different virus families is probably the result of recombinations between unrelated viruses, but its presence in both poultry and SARS coronaviruses could suggest a bird involvement in the history of the SARS coronavirus. A recent screening of wild birds for the presence of coronaviruses, using a pan-coronavirus RT-PCR, led to the identification of novel coronaviruses in the three species studied. Phylogenetic analyses performed on both replicase gene and nucleocapsid protein could not add support to a close relationship between avian and SARS coronaviruses, but all the novel avian coronaviruses were found to harbour s2m. The motif is inserted at a homologous place in avian and SARS coronavirus genomes, but in a somewhat different context for the SARS coronavirus. If the presence of s2m in these viruses is a result of two separate recombination events, this suggests that its particular position in these genomes is the only one that would not be deleterious for coronaviral replication, or that it is the result of a copy-choice recombination between coronaviruses, following an ancestral introduction in the coronavirus family by an unrelated virus. In conclusion, the relative high frequencies of recombination observed both experimentally and in the natural evolution of RNA viruses, indicate that horizontal gene transfer does occur, even between unrelated viruses. This might represent a challenge in the rapid identification of novel pathogens with DNA micro-array techniques.

Animals↗

Animal influenza virus surveillance.

Influenza surveillance studies in humans and animals performed for many years have provided a wealth of data on the ecology and evolution of influenza viruses and their public health related and economical impact. Here we provide a brief overview of some of the data from the past and give our view on how the continued and intensified animal influenza virus surveillance may aid in the early recognition and management of outbreaks in the future.

Animals↗

Human immunodeficiency virus (HIV) infection in parenteral drug users: evolution of the epidemic over 10 years. Valencian Epidemiology and Prevention of HIV Disease Study Group.

BACKGROUND: Evaluation of acquired immunodeficiency syndrome (AIDS) prevention strategies requires an on-going follow up of the frequency of human immunodeficiency virus (HIV-1) infection. The aim of this study was to examine the trends in prevalence and incidence of HIV-1 infection among injecting drug users (IDU) during the period 1987-1996. METHODS: Transversal and cohort studies were designed which included a consecutive sample of 7132 IDU who attended three AIDS Prevention and Information Centres in the Region of Valencia (Spain) and voluntarily asked to be tested for HIV antibodies. The prevalence was estimated for each year based on the serological status of HIV-1 when the patient first visited the centre. The annual incidence rates were calculated based on the seronegative patients in which a new determination of HIV-1 was done. In order to control the possible effects on the estimations of age, sex and duration of addiction of the people studied, Poisson and logistic regression models were adjusted. RESULTS: Prevalence and incidence rates of HIV-1 infection showed parallel trends over time. The overall prevalence found was 43.6% (95% confidence intervals [CI]: 42.4-44.7%). Of the 4023 seronegative individuals, 1746 were followed up over the whole of the study period. The incidence rate observed was 6.85 x 100 persons/year (95% CI : 6.04-7.66). The prevalence figures show a decrease, which is most marked from 1990 onwards and then they tend to stabilize over the past few years. The incidence rates increase slightly up to 1991 (9.8 x 100 persons/year), and then begin to decrease. CONCLUSION: Trends of prevalence of HIV-1 infection approximate trends of subjacent incidence rate. Despite decrease in HIV-1 infection frequency observed over 10 years, both the prevalence and incidence figures continue to be high in absolute terms. It is necessary to intensify and adapt preventive measures to each subgroup at risk of infection and in the case of heterosexual transmission ensure that the failure observed in the case of IDU is not repeated.

Acquired Immunodeficiency Syndrome↗

Refining a giant virus lineage: a novel order unifying Mamonoviridae and "Manesviridae," unveiled by the discovery of furtivovirus.

UNLABELLED: The evolutionary origins and taxonomic framework of giant viruses related to the family Mamonoviridae and its relative group, including clandestinovirus, remain unclassified due to gaps in genome size and host range between these two groups. This study aimed to address this gap by integrating our newly isolated virus with publicly available metagenome-assembled genomes (MAGs) to construct a more robust phylogenetic framework. Here, we report the isolation and characterization of a new giant virus, furtivovirus, using the unicellular amoeba Vermamoeba vermiformis as a host. Furtivovirus has a genome of approximately 560 kbp and shares key features with its closest relative, clandestinovirus. Ultrastructural analysis revealed a unique host-nucleus-dependent replication strategy characterized by the breakdown of the nuclear membrane and the packaging of nascent virions directly within the nucleoplasm, distinguishing it from canonical cytoplasmic virion factories. Comprehensive phylogenetic and comparative genomic analyses of shared orthologous groups and nucleocytovirus marker proteins revealed that furtivovirus, clandestinovirus, ushikuvirus, and usurpativirus form a distinct monophyletic clade, for which we propose a new family, "Manesviridae." Further analysis using amino acid-based similarity metrics of Nucleocytoviricota viral genomes, including established MAGs, demonstrated that this new family is robustly placed as a sister group to the family Mamonoviridae. This study elucidated the evolutionary relationships between viruses with large and small genomes that possess similar virion sizes within this lineage. Based on this cumulative evidence, we propose the establishment of a new order to unify these two families, thereby expanding their diversity and clarifying the evolutionary history of this branch within Nucleocytoviricota. IMPORTANCE: Giant viruses challenge our traditional understanding of viral evolution, raising the question of how a single related group can diverge to infect different hosts while evolving into vastly different genome sizes and replication strategies. The family Mamonoviridae and its relatives epitomize this evolutionary divergence: one group possesses massive genomes, whereas the other has genomes that are less than half their size. The discovery of furtivovirus and its unique nucleoplasm-dependent replication cycle provides a critical biological context for this genomic disparity. Through deep comparative genomic analysis, we demonstrated that these seemingly disparate lineages share a cohesive evolutionary origin that is distinct from other established orders. This finding highlights the complexity of genome evolution, demonstrating that giant viruses can expand their overall genome size to adapt to uncertain environments while reducing their core essential genes, thereby providing new insights into the evolutionary pressures that shape the diversity of the virosphere.

Giant Viruses↗

Simian immunodeficiency virus-specific CD8+ lymphocyte response in acutely infected rhesus monkeys.

To assess the possible role of cytotoxic T lymphocytes (CTLs) in containing the spread of human immunodeficiency virus in acutely infected individuals, the temporal evolution of the virus-specific CD8+ lymphocyte response was defined in simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys. A brief period of SIVmac plasma antigenemia was seen 9 to 16 days following intravenous infection with SIVmac, ending as the absolute number of CD8+ peripheral blood lymphocytes (PBLs) increased. In a prospective assessment of the ability of CD8+ lymphocytes of these monkeys to suppress SIVmac replication in autologous PBLs, inhibitory activity was detected as early as 4 days, with a more pronounced effect 12 to 16 days following infection. SIVmac Gag- and Nef-specific CD8+ effector cell activities were demonstrable in PBLs of animals by 2 weeks following virus inoculation. In fact, SIVmac-specific CTL precursors were documented in the PBLs of rhesus monkeys 4 to 6 days after SIVmac infection. These studies indicate that AIDS virus-specific CD8+ CTLs are present in PBLs within days of infection and may play an important role in containing the early spread of virus.

Acute Disease↗

Sequence and phylogenetic analyses of a new STLV-I from a naturally infected tantalus monkey from Central Africa.

Simian T-cell leukemia virus (STLV-I) is an oncovirus highly related to human T-cell leukemia virus type I (HTLV-I). To further examine the extent of variability, dissemination patterns, phylogeny, and evolution of these viruses, we analyzed a new STLV-I variant from a naturally infected Cercopithecus aethiops var. tantalus from the Central African Republic. Sequence analyses of its LTR, gag, pol, env, and pX (OrfII) genes indicated that this isolate, STLV-I (Tan 90), is 6% divergent from the prototype HTLV-I (ATK) and is the most divergent African STLV-I characterized to date. Our phylogenetic data indicate that southeast Asian and African STLV-I and HTLV-I strains segregated from each other thousands of years ago and that Japanese HTLV-I strains represent a relatively recent introduction of African or New World isolates. The data also indicate that interspecies transmission occurred several times on different continents over prolonged periods of time.

Africa, Central↗

Variant influenza virus hemagglutinin that induces fusion at elevated pH.

The hemagglutinin (HA) glycoprotein of influenza virus performs two critical roles during infection: it binds virus to cell surface sialic acids, and under mildly acidic conditions it induces fusion of the virion with intracellular membranes, liberating the genome into the cytoplasm. The pH dependence of fusion varies for different influenza virus strains. Here we report the isolation and characterization of a naturally occurring variant of the X31 strain that fuses at a pH 0.2 units higher than the parent strain does and that is less sensitive to the effects of ammonium chloride, a compound known to elevate endosomal pH. The bromelain-solubilized ectodomain of the variant HA displayed a corresponding shift in the pH at which it changed conformation and bound to liposomes. Cloning and sequencing of the variant HA gene revealed amino acid substitutions at three positions in the polypeptide. Two substitutions were in antigenic determinants in the globular region of HA1, and the third occurred in HA2 near the base of the molecule. By using chimeric HA molecules expressed in CV-1 cells from simian virus 40-based vectors, we demonstrated that the change in HA2 was solely responsible for the altered fusion phenotype. This substitution, asparagine for aspartic acid at position 132, disrupted a highly conserved interchain salt bridge between adjacent HA2 subunits. The apparent role of this residue in stabilizing the HA trimer is consistent with the idea that the trimer dissociates at low pH. Furthermore, the results demonstrate that influenza virus populations contain fusion variants, raising the possibility that such variants may play a role in the evolution of the virus.

Amino Acid Sequence↗

Selection profiles in RNA viruses reflect the characteristics of viruses more than individual proteins.

Proteins that are exposed on the surface of a virus are frequently subject to strong selection to escape from neutralizing antibodies. To investigate whether surface-exposed (SE) and non-exposed (NE) proteins encoded by RNA viruses exhibit different patterns of evolution under selection, we analyzed 244 protein-coding genes from 28 species of RNA viruses representing 15 taxonomic families. First, we show that gene-wide rates of non-synonymous (dN) and synonymous (dS) substitutions do not differentiate between SE and NE proteins. To incorporate variation in substitution rates among codon sites, we inferred the posterior distribution over a fixed grid of dN and dS rates for each alignment. This 'evolutionary fingerprint' provides a common framework for comparing the selection profiles of non-homologous genes. Next, we computed the Wasserstein distance for every pair of fingerprints, which is analogous to amount of work required to reshape one distribution to another. After compensating for differences in genetic variation among alignments, we found a small but significant difference between the fingerprints of SE and NE proteins (PERMANOVA, P&#x2009;=&#x2009;0.03). However, we observed larger and more significant effects of whether the virus is enveloped (P&#x2009;<&#x2009;10-5) and the interaction between these factors (P=6.9&#xd7;10-4). The latter effects were driven by high levels of purifying selection in capsid proteins of Picornaviruses. Furthermore, greater amounts of variation in fingerprints were explained by significant differences among virus families and modes of transmission (P&#x2009;<&#x2009;10-5). These results imply the pattern of selection on a virus protein is shaped more by characteristics of the virus than the protein itself.

RNA Viruses↗

Molecular intermediates of fitness gain of an RNA virus: characterization of a mutant spectrum by biological and molecular cloning.

The mutant spectrum of a virus quasispecies in the process of fitness gain of a debilitated foot-and-mouth disease virus (FMDV) clone has been analysed. The mutant spectrum was characterized by nucleotide sequencing of three virus genomic regions (internal ribosome entry site; region between the two AUG initiation codons; VP1-coding region) from 70 biological clones (virus from individual plaques formed on BHK-21 cell monolayers) and 70 molecular clones (RT--PCR products cloned in E. coli). The biological and molecular clones provided statistically indistinguishable definitions of the mutant spectrum with regard to the distribution of mutations among the three genomic regions analysed and with regard to the types of mutations, mutational hot-spots and mutation frequencies. Therefore, the molecular cloning procedure employed provides a simple protocol for the characterization of mutant spectra of viruses that do not grow in cell culture. The number of mutations found repeated among the clones analysed was higher than expected from the mean mutation frequencies. Some components of the mutant spectrum reflected genomes that were dominant in the prior evolutionary history of the virus (previous passages), confirming the presence of memory genomes in virus quasispecies. Other components of the mutant spectrum were genomes that became dominant at a later stage of evolution, suggesting a predictive value of mutant spectrum analysis with regard to the outcome of virus evolution. The results underline the observation that greater insight into evolutionary processes of viruses may be gained from detailed clonal analyses of the mutant swarms at the sequence level.

Adaptation, Physiological↗

Evolution of H5 subtype avian influenza A viruses in North America.

The phylogenetic relationships of the hemagglutinin (HA) and non-structural (NS) genes from avian influenza (AI) H5 subtype viruses of North American origin are presented. Analysis of the HA genes of several previously uncharacterized isolates from waterfowl and turkeys provided clear evidence of significant sequence variation and existence of multiple virus clades or sub-lineages, maintained in migratory waterfowl. Phylogenetic analysis of NS gene sequences further demonstrated multiple sub-lineages and also demonstrated re-assortment of two NS alleles in wild duck populations. Based on currently available HA1 gene sequences, at least four clades exist with waterfowl isolates included in three of the four groups. The most genetically unstable of these sub-lineages is composed of recent poultry isolates from the outbreak of AI in Central Mexico. This group of viruses, which replicated unabated in chickens for at least 16 months, exhibited an increased rate of mutation in both the HA and NS gene. Comparison of the HA1 sequence data for all available North American H5 subtype viruses demonstrated minimal variation both in and around the amino acids predicted to be involved in the HA receptor binding site. The sequences also revealed that migratory waterfowl, live poultry market chicken, and turkey isolates uniformly lack a glycosylation site at amino acid 236 in the HA protein which is present in commercial chicken isolates.

Animals↗

Contrasting use of CCR5 structural determinants by R5 and R5X4 variants within a human immunodeficiency virus type 1 primary isolate quasispecies.

Macrophagetropic R5 human immunodeficiency virus type 1 (HIV-1) isolates often evolve into dualtropic R5X4 variants during disease progression. The structural basis for CCR5 coreceptor function has been studied in a limited number of prototype strains and suggests that R5 and R5X4 Envs interact differently with CCR5. However, differences between unrelated viruses may reflect strain-specific factors and do not necessarily represent changes resulting from R5 to R5X4 evolution of a virus in vivo. Here we addressed CCR5 domains involved in fusion for a large set of closely related yet functionally distinct variants within a primary isolate swarm, employing R5 and R5X4 Envs derived from the HIV-1 89.6(PI) quasispecies. R5 variants of 89.6(PI) could fuse using either N-terminal or extracellular loop CCR5 sequences in the context of CCR5/CXCR2 chimeras, similar to the unrelated R5 strain JRFL, but R5X4 variants of 89.6(PI) were highly dependent on the CCR5 N terminus. Similarly, R5 89.6(PI) variants and isolate JRFL tolerated N-terminal CCR5 deletions, but fusion by most R5X4 variants was markedly impaired. R5 89.6(PI) Envs also tolerated multiple extracellular domain substitutions, while R5X4 variants did not. In contrast to CCR5 use, fusion by R5X4 variants of 89.6(PI) was largely independent of the CXCR4 N-terminal region. Thus, R5 and R5X4 species from a single swarm differ in how they interact with CCR5. These results suggest that R5 Envs possess a highly plastic capacity to interact with multiple CCR5 regions and support the concept that viral evolution in vivo results from the emergence of R5X4 variants with the capacity to use the CXCR4 extracellular loops but demonstrate less-flexible interactions with CCR5 that are strongly dependent on the N-terminal region.

Gene Products, env↗

Structures of endogenous nonecotropic murine leukemia virus (MLV) long terminal repeats in wild mice: implication for evolution of MLVs.

To develop a better understanding of the interaction between retroviruses and their hosts, we have investigated the polymorphism in endogenous murine leukemia proviruses (MLVs). We used genomic libraries of wild mouse DNAs and PCR to analyze genetic variation in the proviruses found in wild mouse species, including Mus musculus (M. m. castaneus, M. m. musculus, M. m. molossinus, and M. m. domesticus), Mus spretus, and Mus spicelegus, as well as some inbred laboratory strains. In this analysis, we detected several unique forms of sequence organization in the U3 regions of the long terminal repeats of these proviruses. The distribution of the proviruses with unique U3 structures demonstrated that xenotropic MLV-related proviruses were present only in M. musculus subspecies, while polytropic MLV-related proviruses were found in both M. musculus and M. spretus. Furthermore, one unique provirus from M. spicelegus was found to be equidistant from ecotropic provirus and nonecotropic provirus by phylogenetic analysis. This provirus, termed HEMV, was thus likely to be related to the common ancestor of these MLVs. Moreover, an ancestral type of polytropic MLV-related provirus was detected in M. spretus species. Despite their "ancestral" phylogenetic position, proviruses of these types are not widespread in mice, implying more-recent spread by infection rather than inheritance. These results imply that recent evolution of these proviruses involved alternating periods of replication as virus and residence in the germ line.

Amino Acid Sequence↗

Evolution of the Drosophila melanogaster-sigma virus system in natural populations from Languedoc (southern France).

An analysis of natural populations of Drosophila melanogaster in a southern French region (Languedoc) was started in 1983, concerning two non Mendelian systems: the P-M system of transposable elements and the sigma virus. This virus is not contagious, but only transmitted through gametes; it is usually present in a minority of individuals in natural populations. The first data collected revealed unexpectedly clear and fast-evolving phenomena; they also gave evidence of some interesting correlations between the two systems. This paper presents all the results gathered from 1983 to 1991 in the Drosophila-sigma system. Striking correlations were observed for three interconnected parameters: frequency of infected flies, frequency of an allele of the fly giving resistance to the virus, and adaptation of the virus to this allele. This adaptation consisted of a qualitative step (change of viral type) followed by quantitative variation (better adaptation to the allele). This analysis also showed, firstly, that the evolution of natural populations differs completely in Languedoc from the rest of France; secondly, that three geographical zones where selective forces worked differently persisted over time in Languedoc.

Adaptation, Physiological↗