Establishment Of Chronic Infection Research Articles

Identification of a Novel Antimicrobial Peptide from Human Hepatitis B Virus Core Protein Arginine-Rich Domain (ARD)

The rise of multidrug-resistant (MDR) pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV). In this study, we demonstrated that the peptide containing the full-length ARD I–IV (HBc147-183) has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E)-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II–IV (HBc153-176) and ARD I–III (HBc147-167) were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS) in several in vitro binding assays. Peptide ARD I–IV (HBc147-183) had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p.) inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that HBc ARD could be a new promising antimicrobial peptide.

Predicted binding of certain antifilarial compounds with glutathione-S-transferase of human Filariids.

Glutathione-S-transferase is a major phase-II detoxification enzyme in parasitic helminthes. Previous research highlights the importance of GSTs in the establishment of chronic infections in cytotoxic microenvironments. Filarial nematodes depend on these detoxification enzymes for their survival in the host. GST plays an important role in filariasis and other diseases. GST from W.bancrofti and B.malayi are very much different from human GST. This structural difference makes GST potential chemotherapeutic targets for antifilarial treatment. In this study we have checked the efficacy of some well known antifilarial compounds against GST from B.malayi and W.bancrofti. The structure of BmGST was modeled using modeller9v10 and was submitted to PMDB. Molecular docking study reveals arbindazole to be the most potent compounds against GST from both the filarial parasites. Role of some residues playing important role in the binding of compounds within the active site of GST has also been revealed in the present study. The BmGST and WbGST structural information and docking studies could aid in screening new antifilarials or selective inhibitors for chemotherapy against filariasis.AbbreviationsGST - Glutathione-S-transferase, Bm - Brugia malayi, Wb - Wuchereria bancrofti.

The YfiBNR Signal Transduction Mechanism Reveals Novel Targets for the Evolution of Persistent Pseudomonas aeruginosa in Cystic Fibrosis Airways

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

Evolution of hepatitis C virus hypervariable region 1 in chronically infected children

Hepatitis C virus (HCV) quasispecies diversification plays an essential role in the establishment of chronic infections. Our earlier analysis of HCV population structure in children subjected to interferon–ribavirin treatment demonstrated that viral quasispecies is homogenous in patients who failed to respond to the therapy and heterogeneous in sustained responders. We also showed that certain variants of HCV hypervariable region 1 (HVR1) are conserved in non-responders. To better elucidate the pathways of HCV evolution, here we examined the changes of HVR1 in viral populations isolated from sera of eight treatment-naive pediatric patients. We found that HCV evolution in untreated chronically infected children occurs according to two pathways and results in the formation of either genetically homogenous or variable quasispecies. Variable populations are prone to quasispecies shifts. In contrast, homogenous populations are composed of closely related variants that undergo only minor changes. In addition, we observed that a phenomenon of inter-quasispecies conservation of HVR1 is associated with some of the homogenous HCV populations. The collected data suggest that there exist HVR1 variants with superior fitness, capable of persisting in different hosts.

Impact of hydrophobic lipophilic molecules on worm burden in balb/c mice infected with heligmosomoides polygyrus

It was recently discovered that parasitic nematodes of plants, animals and humans secrete a structurally novel Fatty Acid and Retinol (FAR) Binding Protein into the tissues they occupy. The lipid‐binding characteristics of FAR proteins across multiple families of parasitic nematodes, and their presence at the host‐parasite interface, lends support to the idea that this nematode‐restricted family of proteins may play a crucial role in the life cycle of the roundworms. To investigate the possible role of these highly conserved nematode‐specific FAR proteins, our lab designed experiments to address the following hypotheses: 1) the FAR proteins are involved in lipid sequestration and delivery from the host to the nematode of a broad range of essential fatty acids, retinoids and other nutrients the parasites cannot synthesize themselves, 2) FAR proteins interfere with intercellular lipid signaling to manipulate the defense reactions of the host, and lastly, as these hypotheses are not mutually exclusive, 3) both mechanisms (hypotheses 1 and 2) are working together to allow for parasite survival and the establishment of chronic infection. To test these hypotheses, mice were dosed with acute, high levels of retinol and/or oleic acid prior to hookworm infection, to determine the impact on worm burden levels and circulating IL‐4. Our preliminary results demonstrate both a statistically significant increase in worm burdens and a significant decrease in circulating IL‐4, after treatments containing ether retinol or oleic acid, results which support hypothesis #3.

Critical role for phosphoinositide 3-kinase gamma in parasite invasion and disease progression of cutaneous leishmaniasis

Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana. Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana. These findings reveal a unique role for PI3Kγ in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania.

Antibodies against PfEMP1, RIFIN, MSP3 and GLURP Are Acquired during Controlled Plasmodium falciparum Malaria Infections in Naïve Volunteers

Antibodies to polymorphic antigens expressed during the parasites erythrocytic stages are important mediators of protective immunity against P. falciparum malaria. Therefore, polymorphic blood stage antigens like MSP3, EBA-175 and GLURP and variant surface antigens PfEMP1 and RIFIN are considered vaccine candidates. However, to what extent these antibodies to blood stage antigens are acquired during naive individuals' first infections has not been studied in depth. Using plasma samples collected from controlled experimental P. falciparum infections we show that antibodies against variant surface antigens, PfEMP1 and RIFIN as well as MSP3 and GLURP, are acquired during a single short low density P. falciparum infection in non-immune individuals including strain transcendent PfEMP1 immune responses. These data indicate that the immunogenicity of the variant surface antigens is similar to the less diverse merozoite antigens. The acquisition of a broad and strain transcendent repertoire of PfEMP1 antibodies may reflect a parasite strategy of expressing most or all PfEMP1 variants at liver release optimizing the likelihood of survival and establishment of chronic infections in the new host.

A Quorum Sensing Regulated Small Volatile Molecule Reduces Acute Virulence and Promotes Chronic Infection Phenotypes

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.

A Review: Competence, Compromise, and Concomitance—Reaction of the Host Cell To Toxoplasma gondii Infection and Development

Toxoplasma gondii is an important zoonotic parasite with a worldwide distribution. It infects about one-third of the world's population, causing serious illness in immunosuppressed individuals, fetuses, and infants. Toxoplasma gondii biology within the host cell includes several important phases: (1) active invasion and establishment of a nonfusogenic parasitophorous vacuole in the host cell, (2) extensive modification of the parasitophorous vacuolar membrane for nutrient acquisition, (3) intracellular proliferation by endodyogeny, (4) egress and invasion of new host cells, and (5) stage conversion from tachyzoite to bradyzoite and establishment of chronic infection. During these processes, T. gondii regulates the host cell by modulating morphological, physiological, immunological, genetic, and cellular biological aspects of the host cell. Overall, the infection/development predispositions of T. gondii -host cell interactions overtakes the infection resistance aspects. Upon invasion and development, host cells are modulated to keep a delicate balance between facilitating and eliminating the infection.

Pseudomonas aeruginosa Evasion of Phagocytosis Is Mediated by Loss of Swimming Motility and Is Independent of Flagellum Expression

Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that causes severe opportunistic infections in immunocompromised individuals; in particular, severity of infection with P. aeruginosa positively correlates with poor prognosis in cystic fibrosis (CF) patients. Establishment of chronic infection by this pathogen is associated with downregulation of flagellar expression and of other genes that regulate P. aeruginosa motility. The current paradigm is that loss of flagellar expression enables immune evasion by the bacteria due to loss of engagement by phagocytic receptors that recognize flagellar components and loss of immune activation through flagellin-mediated Toll-like receptor (TLR) signaling. In this work, we employ bacterial and mammalian genetic approaches to demonstrate that loss of motility, not the loss of the flagellum per se, is the critical factor in the development of resistance to phagocytosis by P. aeruginosa. We demonstrate that isogenic P. aeruginosa mutants deficient in flagellar function, but retaining an intact flagellum, are highly resistant to phagocytosis by both murine and human phagocytic cells at levels comparable to those of flagellum-deficient mutants. Furthermore, we show that loss of MyD88 signaling in murine phagocytes does not recapitulate the phagocytic deficit observed for either flagellum-deficient or motility-deficient P. aeruginosa mutants. Our data demonstrate that loss of bacterial motility confers a dramatic resistance to phagocytosis that is independent of both flagellar expression and TLR signaling. These findings provide an explanation for the well-documented observation of nonmotility in clinical P. aeruginosa isolates and for how this phenotype confers upon the bacteria an advantage in the context of immune evasion.

Hepatitis C virus with a naturally occurring single amino-acid substitution in the E2 envelope protein escapes neutralization by naturally-induced and vaccine-induced antibodies

Mutations arising in neutralizing epitopes of hepatitis C virus may play a role in the ability of the virus to escape control by neutralizing antibodies and in the establishment of chronic infections. An amino-acid substitution, Q412H, within a major conserved neutralization epitope EP I (aa 412–426) in the E2 glycoprotein is observed in chronic HCV carriers. We found that naturally acquired polyclonal EP I-specific antibodies have an equivalent binding capacity toward either the wild type or the Q412H mutant peptide encompassing the EP I epitope. While EP I-specific antibodies neutralized J6/JFH1 virus in vitro, they did not neutralize J6/JFH1 virus containing the Q412H mutation. Furthermore, we found that plasma obtained from a chimpanzee that had anti-E1/E2 antibodies following experimental immunization, neutralized the wild type J6/JFH1 virus but failed to neutralize the mutant virus. Thus, mutation Q412H found in naturally occurring variants could represent an antibody escape mutation. These data may have important implications for vaccine design.

Estimation of the Initial Viral Growth Rate and Basic Reproductive Number during Acute HIV-1 Infection

During primary infection, the number of HIV-1 particles in plasma increases rapidly, reaches a peak, and then declines until it reaches a set point level. Understanding the kinetics of primary infection, and its effect on the establishment of chronic infection, is important in defining the early pathogenesis of HIV. We studied the viral dynamics of very early HIV-1 infection in 47 subjects identified through plasma donation screening. We calculated how fast the viral load increases and how variable this parameter is among individuals. We also estimated the basic reproductive ratio, the number of new infected cells generated by an infectious cell at the start of infection when target cells are not limiting. The initial viral doubling time had a median of 0.65 days with an interquartile range of 0.56 to 0.91 days. The median basic reproductive ratio was 8.0 with an interquartile range of 4.9 to 11. In 15 patients, we also observed the postpeak decay of plasma virus and found that the virus decay occurred at a median rate of 0.60 day(-1), corresponding to a half-life of 1.2 days. The median peak viral load was 5.8 log(10) HIV-1 RNA copies/ml, and it was reached 14 days after the virus was quantifiable with an assay, with a lower limit of detection of 50 copies/ml. These results characterize the early plasma viral dynamics in acute HIV infection better than it has been possible thus far. They also better define the challenge that the immune response (or therapeutic intervention) has to overcome to defeat HIV at this early stage.

Up-regulate HLA Class I Expression Following Hepatitis B Virus Transfection in a Hepatocellular Carcinoma Cell Line BEL7405

Chronic hepatitis B virus infection is associated with a high risk of developing into hepatocellular carcinoma, while tumor recognition is important during the immune surveillance process that prevents cancer development in humans. The mechanisms of immune evasion and the role of the early immune response in chronic infection caused by hepatitis B virus (HBV) are still unclear. In the present study, 1 copy or 1.2 copies of HBV genome was transfected into a hepatocellular carcinoma cell line BEL7405. RT-PCR, Western blot and flow cytometry analysis were used to evaluate the expression of HLA class I molecules and transporter associated with antigen processing 1 (TAP1). Finally, the cytotoxic activity of natural killer (NK) cells against HBV transfected liver cells was detected by MTT colorimetry method. Following transfection of 1 copy or 1.2 copies of HBV genome, HLA class I expression was up-regulated in BEL7405 cell line in a dose-dependent manner. Furthermore, increased the surface HLA class I expression were caused by enhanced expression of TAP1 at mRNA and protein levels in those transfected cells. Consequently, a significantly down-regulated cytotoxic activity of NK cells against HBV transfected liver cells was observed. These results may demonstrate a way by which HBV avoids recognition by NK cells that might be associated with the establishment of chronic infection and tumor formation.

Peroxisome Proliferator-Activated Receptor (PPAR): Balance for Survival in Parasitic Infections

Parasitic infections induce a magnitude of host responses. At the opposite ends of the spectrum are those that ensure the host's needs to eliminate the invaders and to minimize damage to its own tissues. This review analyzes how parasites would manipulate immunity by activating the immunosuppressive nuclear factor, peroxisome proliferator-activated receptors (PPARs) with type 2 cytokines and free fatty acids from arachidonic acid metabolism. PPARs limit the action of type 1 immunity, in which classically activated macrophages act through the production of proinflammatory signals, to spare the parasites. They also favor the development of alternately activated macrophages which control inflammation so the host would not be destroyed. Possibly, the nuclear factors hold a pivotal role in the establishment of chronic infection by delicately balancing the pro- and anti-inflammatory signaling mechanisms and their ligands may be used as combination therapeutics to limit host pathology.

Localization of Phosphatidylinositol (3,4,5)-Trisphosphate to Phagosomes in Entamoeba histolytica Achieved Using Glutathione S -Transferase- and Green Fluorescent Protein-Tagged Lipid Biosensors

Entamoeba histolytica is an intestinal protozoan parasite that causes amoebic dysentery and liver abscess. Phagocytosis by the parasite is a critical virulence process, since it is a prerequisite for tissue invasion and establishment of chronic infection. While the roles of many of the proteins that regulate phagocytosis-related signaling events in E. histolytica have been characterized, the functions of lipids in this cellular process remain largely unknown in this parasite. In other systems, phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), a major product of phosphoinositide 3 kinase (PI3-kinase) activity, is essential for phagocytosis. Pleckstrin homology (PH) domains are protein domains that specifically bind to PIP(3). In this study, we utilized glutathione S-transferase (GST)- and green fluorescent protein (GFP)-labeled PH domains as lipid biosensors to characterize the spatiotemporal aspects of PIP(3) distribution during various endocytic processes in E. histolytica. PIP(3)-specific biosensors accumulated at extending pseudopodia and in phagosomal cups in trophozoites exposed to erythrocytes but did not localize to pinocytic compartments during the uptake of a fluid-phase marker, dextran. Our results suggest that PIP(3) is involved in the early stages of phagosome formation in E. histolytica. In addition, we demonstrated that PIP(3) exists at high steady-state levels in the plasma membrane of E. histolytica and that these levels, unlike those in mammalian cells, are not abolished by serum withdrawal. Finally, expression of a PH domain in trophozoites inhibited erythrophagocytosis and enhanced motility, providing genetic evidence supporting the role of PI3-kinase signaling in these processes in E. histolytica.

Regulatory T Cells Suppress Antiviral Immune Responses and Increase Viral Loads during Acute Infection with a Lymphotropic Retrovirus

Regulatory T Cells Suppress Antiviral Immune Responses and Increase Viral Loads during Acute Infection with a Lymphotropic Retrovirus

Calpain activation by hepatitis C virus proteins inhibits the extrinsic apoptotic signaling pathway #

An unresolved question regarding the physiopathology of hepatitis C virus (HCV) infection is the remarkable efficiency with which host defenses are neutralized to establish chronic infection. Modulation of an apoptotic response is one strategy used by viruses to escape immune surveillance. We previously showed that HCV proteins down-regulate expression of BH3-only Bcl2 interacting domain (Bid) in hepatocytes of HCV transgenic mice. As a consequence, cells acquire resistance to Fas-mediated apoptosis, which in turn leads to increased persistence of experimental viral infections in vivo. This mechanism might participate in the establishment of chronic infections and the resulting pathologies, including hepatocellular carcinoma. We now report that Bid is also down-regulated in patients in the context of noncirrhotic HCV-linked tumorigenesis and in the HCV RNA replicon system. We show that the nonstructural HCV viral protein NS5A is sufficient to activate a calpain cysteine protease, leading to degradation of Bid. Moreover, pharmacological inhibitors of calpains restore both the physiological levels of Bid and the sensitivity of cells toward a death receptor-mediated apoptotic signal. Finally, human HCV-related tumors and hepatocytes from HCV transgenic mice that display low Bid expression contain activated calpains. Calpains activated by HCV proteins degrade Bid and thus dampen apoptotic signaling. These results suggest that inhibiting calpains could lead to an improved efficiency of immune-mediated elimination of HCV-infected cells.

A Transmembrane Domain-Containing Surface Protein fromToxoplasma gondiiAugments Replication in Activated Immune Cells and Establishment of a Chronic Infection

Toxoplasma gondii mutants identified as defective in the establishment of chronic infection were screened to isolate those specifically impaired in their ability to replicate within activated macrophages. One of the identified mutants contains an insertion in the hypothetical gene TGME49_111670. Genetic complementation restores the ability of the mutant to replicate in immune cells and produce cysts in the brains of mice. While the mutant is more sensitive to nitric oxide than is its parental strain, it is not defective in its ability to suppress nitric oxide. The disrupted protein has no significant homology to proteins with known functions, but is predicted to have one transmembrane domain. Immunofluorescence shows the protein on the parasite surface, even in activated macrophages, colocalizing with a tachyzoite surface antigen, SAG1, and oriented with its C-terminal end external. Western analysis reveals that the protein is downregulated in bradyzoites. Despite the tachyzoite specificity of this protein, mice infected with the mutant succumb to acute infection similarly to those infected with the parent strain. Serum samples from mice with chronic T. gondii infection react to a polypeptide from TGME49_11670, indicating that the protein is seen by the immune system during infection. This study is the first to characterize a T. gondii surface protein that contains a transmembrane domain and show that the protein contributes to parasite replication in activated immune cells and the establishment of chronic infection.

Staphylococcal Alpha-Toxin Is Not Sufficient To Mediate Escape from Phagolysosomes in Upper-Airway Epithelial Cells

Intracellular Staphylococcus aureus has been implicated in the establishment of chronic infections. It is therefore imperative to understand by what means S. aureus is able to survive within cells. Here we use two expression systems with a fluorescent readout to assay alpha-toxin expression and function within phagolysosomes of infected upper-airway epithelial cells: avirulent Staphylococcus carnosus TM300 and phenotypically alpha-toxin-negative S. aureus laboratory strains. Data from CFU recovery assays suggest that the presence of alpha-toxin is not beneficial for the intracellular survival of recombinant Staphylococcus strains. This finding was corroborated by immunofluorescence studies: whereas S. carnosus and S. aureus are able to deliver S. aureus alpha-toxin to lumina of host cell phagolysosomes, the membrane integrity of these organelles was not affected. Alpha-toxin-expressing strains were detected exclusively within lysosome-associated membrane protein 1 (LAMP1)-yellow fluorescent protein (YFP)-positive vesicles. Measurements of intraphagosomal pH illustrated that all infected phagolysosomes acidified regardless of alpha-toxin expression. In contrast, S. aureus expressing Listeria monocytogenes listeriolysin O leads to the breakdown of the phagolysosomal membrane, as indicated by staphylococci that are not associated with LAMP1-YFP-decorated vesicles and that do not reside within an acidic cellular environment. Thus, our results suggest that staphylococcal alpha-toxin is not sufficient to mediate phagolysosomal escape in upper-airway epithelial cells.

The 30th anniversary of quasispecies

The meeting on ‘Quasispecies: past, present and future’ took place between 17 and 18 November 2008, in Barcelona, Spain, and was organized by J. Gomez, C. Lopez‐Galindez, M.A. Martinez & A. Mas. ![][1] A meeting was held in Barcelona, Spain, in November 2008 to celebrate the 30th anniversary of the publication of the article that described the extensive genetic heterogeneity of bacteriophage Qβ (Domingo et al , 1978), which is considered to mark the beginning of experimental studies on viral quasispecies. This meeting was held at the impressive fifteenth century building of the ancient Hospital de la Santa Creu in the old town of Barcelona, which is now the headquarters of the Institut d'Estudis Catalans, and was attended by C. Weissmann (Jupiter, FL, USA), M. Billeter (Zurich, Switzerland) and E. Domingo (Madrid, Spain), who were three early protagonists of the phage Qβ work at the University of Zurich in the 1970s. Several speakers presented their results on the theoretical aspects of the population dynamics of cells and viruses, the clinical implications of quasispecies, and extensions of the quasispecies concept to cellular genes and prions. The meeting was introduced by A. Mas (Albacete, Spain), who reflected on the increasing impact that quasispecies have had in the scientific literature over the past three decades, and quoted some of the key references on viral quasispecies (Martell et al , 1992; Meyerhans et al , 1989; Najera et al , 1995; Vignuzzi et al , 2006; for a historical review of the impact of quasispecies in virology, see Holland, 2006). The scientific presentations were opened by Weissmann and Domingo, who were the last and first authors of the 1978 paper, respectively. Their talks conveyed the scientific atmosphere of the 1970s—when molecular biology was carried out with few recombinant‐DNA techniques—to a young audience. Nucleic‐acid sequencing was in … [1]: /embed/graphic-1.gif