Publications by authors named "Alberto López-Bueno"

21 Publications

  • Page 1 of 1

Methods for Enrichment and Sequencing of Oral Viral Assemblages: Saliva, Oral Mucosa, and Dental Plaque Viromes.

Methods Mol Biol 2018 ;1838:143-161

Centro de Biología Molecular Severo Ochoa (Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas), Madrid, Spain.

The oral cavity is a major portal of entry for human pathogens including viruses. However, metagenomics has revealed that highly personalized and time-persistent bacteriophage assemblages dominate this habitat. Most oral bacteriophages follow lysogenic life cycles, deploying complex strategies to manage bacterial homeostasis. Although bacterial dysbiosis underlies common oral pathologies such as caries and periodontitis, the cause of these bacteria replacements remains obscure, and it is theorized that bacteriophages play an important role. The enormous sensitivity of metagenomics coupled with next-generation sequencing has made technically feasible to address the putative role of bacteriophages in oral dysbiosis and represents a valuable tool to discover new human viruses.This chapter proposes a workflow that consists of a simple viral enrichment protocol, two alternative random amplification methods, and next-generation sequencing to access virome composition in three oral environments: supragingival plaque, saliva, and mucosa. These protocols circumvent some well-known sources of bias, providing genomic information about DNA and RNA viral communities with minimal contamination from human and bacterial sources.
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http://dx.doi.org/10.1007/978-1-4939-8682-8_11DOI Listing
April 2019

Evaluation of bias induced by viral enrichment and random amplification protocols in metagenomic surveys of saliva DNA viruses.

Microbiome 2018 06 28;6(1):119. Epub 2018 Jun 28.

Centro de Biología Molecular Severo Ochoa (Universidad Autónoma de Madrid/Consejo Superior de Investigaciones Científicas), Madrid, Spain.

Background: Viruses are key players regulating microbial ecosystems. Exploration of viral assemblages is now possible thanks to the development of metagenomics, the most powerful tool available for studying viral ecology and discovering new viruses. Unfortunately, several sources of bias lead to the misrepresentation of certain viruses within metagenomics workflows, hindering the shift from merely descriptive studies towards quantitative comparisons of communities. Therefore, benchmark studies on virus enrichment and random amplification protocols are required to better understand the sources of bias.

Results: We assessed the bias introduced by viral enrichment on mock assemblages composed of seven DNA viruses, and the bias from random amplification methods on human saliva DNA viromes, using qPCR and deep sequencing, respectively. While iodixanol cushions and 0.45 μm filtration preserved the original composition of nuclease-protected viral genomes, low-force centrifugation and 0.22 μm filtration removed large viruses. Comparison of unamplified and randomly amplified saliva viromes revealed that multiple displacement amplification (MDA) induced stochastic bias from picograms of DNA template. However, the type of bias shifted to systematic using 1 ng, with only a marginal influence by amplification time. Systematic bias consisted of over-amplification of small circular genomes, and under-amplification of those with extreme GC content, a negative bias that was shared with the PCR-based sequence-independent, single-primer amplification (SISPA) method. MDA based on random priming provided by a DNA primase activity slightly outperformed those based on random hexamers and SISPA, which may reflect differences in ability to handle sequences with extreme GC content. SISPA viromes showed uneven coverage profiles, with high coverage peaks in regions with low linguistic sequence complexity. Despite misrepresentation of certain viruses after random amplification, ordination plots based on dissimilarities among contig profiles showed perfect overlapping of related amplified and unamplified saliva viromes and strong separation from unrelated saliva viromes. This result suggests that random amplification bias has a minor impact on beta diversity studies.

Conclusions: Benchmark analyses of mock and natural communities of viruses improve understanding and mitigate bias in metagenomics surveys. Bias induced by random amplification methods has only a minor impact on beta diversity studies of human saliva viromes.
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http://dx.doi.org/10.1186/s40168-018-0507-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022446PMC
June 2018

Recombination events and variability among full-length genomes of co-circulating molluscum contagiosum virus subtypes 1 and 2.

J Gen Virol 2017 May 30;98(5):1073-1079. Epub 2017 May 30.

Centro de Investigación en Sanidad Animal; Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Valdeolmos, Madrid 28130, Spain.

Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and causes a highly prevalent human disease of the skin characterized by the formation of a variable number of lesions that can persist for prolonged periods of time. Two major genotypes, subtype 1 and subtype 2, are recognized, although currently only a single complete genomic sequence corresponding to MCV subtype 1 is available. Using next-generation sequencing techniques, we report the complete genomic sequence of four new MCV isolates, including the first one derived from a subtype 2. Comparisons suggest a relatively distant evolutionary split between both MCV subtypes. Further, our data illustrate concurrent circulation of distinct viruses within a population and reveal the existence of recombination events among them. These results help identify a set of MCV genes with potentially relevant roles in molluscum contagiosum epidemiology and pathogenesis.
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http://dx.doi.org/10.1099/jgv.0.000759DOI Listing
May 2017

Concurrence of Iridovirus, Polyomavirus, and a Unique Member of a New Group of Fish Papillomaviruses in Lymphocystis Disease-Affected Gilthead Sea Bream.

J Virol 2016 10 12;90(19):8768-79. Epub 2016 Sep 12.

Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Valdeolmos, Madrid, Spain

Unlabelled: Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought.

Importance: Lymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papillomalike lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the family Iridoviridae In the Mediterranean area, the gilthead sea bream (Sparus aurata), an important farmed fish, is frequently affected. Using next-generation sequencing, we have identified within S. aurata lymphocystis lesions the concurrent presence of an additional LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyomavirus and papillomavirus families, and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses have not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.
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http://dx.doi.org/10.1128/JVI.01369-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021401PMC
October 2016

Metagenomic analysis of lacustrine viral diversity along a latitudinal transect of the Antarctic Peninsula.

FEMS Microbiol Ecol 2016 Jun 7;92(6):fiw074. Epub 2016 Apr 7.

Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid, 28049 Madrid, Spain

Environmental viruses constitute the most abundant biological entities on earth, and harbor an enormous genetic diversity. While their strong influence on the ecosystem is widely acknowledged, current knowledge about their diversity and distribution remains limited. Here we present the metagenomic study of viral communities from freshwater bodies located along a transect of the Antarctic Peninsula. These ecosystems were chosen on the basis of environmental and biogeographical variation. The results obtained indicate that the virus assemblages were diverse, and that the larger fraction represented viruses with no close relatives in the databases. Comparisons to existing metaviromes showed that the communities studied were dissimilar to other freshwater viromes including those from the Arctic. Finally, we observed no indication of there being a reduction in either viral richness or diversity estimates with increasing latitude along the studied transect, further adding to the controversy regarding the possible existence of latitudinal gradients of diversity in the microbial world.
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http://dx.doi.org/10.1093/femsec/fiw074DOI Listing
June 2016

Ecosystem function decays by fungal outbreaks in Antarctic microbial mats.

Sci Rep 2016 Mar 14;6:22954. Epub 2016 Mar 14.

Departamento de Biología, Universidad Autónoma de Madrid, Madrid, Spain.

Antarctica harbours a remarkably diverse range of freshwater bodies and terrestrial ecosystems, where microbial mats are considered the most important systems in terms of biomass and metabolic capabilities. We describe the presence of lysis plaque-like macroscopic blighted patches within the predominant microbial mats on Livingston Island (Antarctic Peninsula). Those blighting circles are associated with decay in physiological traits as well as nitrogen depletion and changes in the spatial microstructure; these alterations were likely related to disruption of the biogeochemical gradients within the microbial ecosystem caused by an unusually high fungal abundance and consequent physical alterations. This phenomenon has been evidenced at a time of unprecedented rates of local warming in the Antarctic Peninsula area, and decay of these ecosystems is potentially stimulated by warmer temperatures.
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http://dx.doi.org/10.1038/srep22954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4789741PMC
March 2016

Biodiversity and distribution of polar freshwater DNA viruses.

Sci Adv 2015 Jun 19;1(5):e1400127. Epub 2015 Jun 19.

Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid 28049, Spain.

Viruses constitute the most abundant biological entities and a large reservoir of genetic diversity on Earth. Despite the recent surge in their study, our knowledge on their actual biodiversity and distribution remains sparse. We report the first metagenomic analysis of Arctic freshwater viral DNA communities and a comparative analysis with other freshwater environments. Arctic viromes are dominated by unknown and single-stranded DNA viruses with no close relatives in the database. These unique viral DNA communities mostly relate to each other and present some minor genetic overlap with other environments studied, including an Arctic Ocean virome. Despite common environmental conditions in polar ecosystems, the Arctic and Antarctic DNA viromes differ at the fine-grain genetic level while sharing a similar taxonomic composition. The study uncovers some viral lineages with a bipolar distribution, suggesting a global dispersal capacity for viruses, and seemingly indicates that viruses do not follow the latitudinal diversity gradient known for macroorganisms. Our study sheds light into the global biogeography and connectivity of viral communities.
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http://dx.doi.org/10.1126/sciadv.1400127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640604PMC
June 2015

Establishment of a Zebrafish Infection Model for the Study of Wild-Type and Recombinant European Sheatfish Virus.

J Virol 2015 Oct 5;89(20):10702-6. Epub 2015 Aug 5.

Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Valdeolmos, Spain

Amphibian-like ranaviruses include pathogens of fish, amphibians, and reptiles that have recently evolved from a fish-infecting ancestor. The molecular determinants of host range and virulence in this group are largely unknown, and currently fish infection models are lacking. We show that European sheatfish virus (ESV) can productively infect zebrafish, causing a lethal pathology, and describe a method for the generation of recombinant ESV, establishing a useful model for the study of fish ranavirus infections.
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http://dx.doi.org/10.1128/JVI.01580-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580169PMC
October 2015

Phylogeny and differentiation of reptilian and amphibian ranaviruses detected in Europe.

PLoS One 2015 23;10(2):e0118633. Epub 2015 Feb 23.

Fachgebiet für Umwelt- und Tierhygiene, Universität Hohenheim, Stuttgart, Germany; Laboklin GmbH & Co. KG, Laboratory for Clinical Diagnostics, Bad Kissingen, Germany.

Ranaviruses in amphibians and fish are considered emerging pathogens and several isolates have been extensively characterized in different studies. Ranaviruses have also been detected in reptiles with increasing frequency, but the role of reptilian hosts is still unclear and only limited sequence data has been provided. In this study, we characterized a number of ranaviruses detected in wild and captive animals in Europe based on sequence data from six genomic regions (major capsid protein (MCP), DNA polymerase (DNApol), ribonucleoside diphosphate reductase alpha and beta subunit-like proteins (RNR-α and -β), viral homolog of the alpha subunit of eukaryotic initiation factor 2, eIF-2α (vIF-2α) genes and microsatellite region). A total of ten different isolates from reptiles (tortoises, lizards, and a snake) and four ranaviruses from amphibians (anurans, urodeles) were included in the study. Furthermore, the complete genome sequences of three reptilian isolates were determined and a new PCR for rapid classification of the different variants of the genomic arrangement was developed. All ranaviruses showed slight variations on the partial nucleotide sequences from the different genomic regions (92.6-100%). Some very similar isolates could be distinguished by the size of the band from the microsatellite region. Three of the lizard isolates had a truncated vIF-2α gene; the other ranaviruses had full-length genes. In the phylogenetic analyses of concatenated sequences from different genes (3223 nt/10287 aa), the reptilian ranaviruses were often more closely related to amphibian ranaviruses than to each other, and most clustered together with previously detected ranaviruses from the same geographic region of origin. Comparative analyses show that among the closely related amphibian-like ranaviruses (ALRVs) described to date, three recently split and independently evolving distinct genetic groups can be distinguished. These findings underline the wide host range of ranaviruses and the emergence of pathogen pollution via animal trade of ectothermic vertebrates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0118633PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338083PMC
January 2016

Genome sequence of two novel species of torque teno minivirus from the human oral cavity.

Genome Announc 2014 Sep 25;2(5). Epub 2014 Sep 25.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Madrid, Spain

Anelloviridae is a family of circular, single-stranded DNA viruses highly prevalent among humans. We report the genome sequence of two torque teno miniviruses found in human oral mucosa samples. Genome organization, phylogenetic analysis, and pairwise comparisons reveal that they belong to novel species within the Betatorquevirus genus.
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http://dx.doi.org/10.1128/genomeA.00868-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175194PMC
September 2014

The genome sequence of ectromelia virus Naval and Cornell isolates from outbreaks in North America.

Virology 2014 Aug 5;462-463:218-26. Epub 2014 Jul 5.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Nicolas Cabrera 1, Campus de Cantoblanco, Madrid, Spain; Department of Medicine, University of Cambridge, United Kingdom. Electronic address:

Ectromelia virus (ECTV) is the causative agent of mousepox, a disease of laboratory mouse colonies and an excellent model for human smallpox. We report the genome sequence of two isolates from outbreaks in laboratory mouse colonies in the USA in 1995 and 1999: ECTV-Naval and ECTV-Cornell, respectively. The genome of ECTV-Naval and ECTV-Cornell was sequenced by the 454-Roche technology. The ECTV-Naval genome was also sequenced by the Sanger and Illumina technologies in order to evaluate these technologies for poxvirus genome sequencing. Genomic comparisons revealed that ECTV-Naval and ECTV-Cornell correspond to the same virus isolated from independent outbreaks. Both ECTV-Naval and ECTV-Cornell are extremely virulent in susceptible BALB/c mice, similar to ECTV-Moscow. This is consistent with the ECTV-Naval genome sharing 98.2% DNA sequence identity with that of ECTV-Moscow, and indicates that the genetic differences with ECTV-Moscow do not affect the virulence of ECTV-Naval in the mousepox model of footpad infection.
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http://dx.doi.org/10.1016/j.virol.2014.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139192PMC
August 2014

Genome Sequence of WAU86/88-1, a New Variant of Vaccinia Virus Lister Strain from Poland.

Genome Announc 2014 Jan 9;2(1). Epub 2014 Jan 9.

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Campus de Cantoblanco, Madrid, Spain.

The poxviruses Warsaw Agricultural University 86 (WAU86) and 88-1 (WAU88-1) were isolated in 1986 to 1988 from separate outbreaks in laboratory mice in Poland and described as ectromelia virus isolates. The genome sequences of these poxviruses reveal that they are almost identical and represent a novel variant of the vaccinia virus Lister strain.
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http://dx.doi.org/10.1128/genomeA.01086-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3886943PMC
January 2014

Complete genome sequence of the European sheatfish virus.

J Virol 2012 Jun;86(11):6365-6

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Madrid, Spain.

Viral diseases are an increasing threat to the thriving aquaculture industry worldwide. An emerging group of fish pathogens is formed by several ranaviruses, which have been isolated at different locations from freshwater and seawater fish species since 1985. We report the complete genome sequence of European sheatfish ranavirus (ESV), the first ranavirus isolated in Europe, which causes high mortality rates in infected sheatfish (Silurus glanis) and in other species. Analysis of the genome sequence shows that ESV belongs to the amphibian-like ranaviruses and is closely related to the epizootic hematopoietic necrosis virus (EHNV), a disease agent geographically confined to the Australian continent and notifiable to the World Organization for Animal Health.
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http://dx.doi.org/10.1128/JVI.00618-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372208PMC
June 2012

The genome sequence of the emerging common midwife toad virus identifies an evolutionary intermediate within ranaviruses.

J Virol 2012 Apr 1;86(7):3617-25. Epub 2012 Feb 1.

Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain.

Worldwide amphibian population declines have been ascribed to global warming, increasing pollution levels, and other factors directly related to human activities. These factors may additionally be favoring the emergence of novel pathogens. In this report, we have determined the complete genome sequence of the emerging common midwife toad ranavirus (CMTV), which has caused fatal disease in several amphibian species across Europe. Phylogenetic and gene content analyses of the first complete genomic sequence from a ranavirus isolated in Europe show that CMTV is an amphibian-like ranavirus (ALRV). However, the CMTV genome structure is novel and represents an intermediate evolutionary stage between the two previously described ALRV groups. We find that CMTV clusters with several other ranaviruses isolated from different hosts and locations which might also be included in this novel ranavirus group. This work sheds light on the phylogenetic relationships within this complex group of emerging, disease-causing viruses.
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http://dx.doi.org/10.1128/JVI.07108-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302492PMC
April 2012

High diversity of the viral community from an Antarctic lake.

Science 2009 Nov;326(5954):858-61

Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid, Madrid, Spain.

Viruses are the most abundant biological entities and can control microbial communities, but their identity in terrestrial and freshwater Antarctic ecosystems is unknown. The genetic structure of an Antarctic lake viral community revealed unexpected genetic richness distributed across the highest number of viral families that have been found to date in aquatic viral metagenomes. In contrast to other known aquatic viromes, which are dominated by bacteriophage sequences, this Antarctic virus assemblage had a large proportion of sequences related to eukaryotic viruses, including phycodnaviruses and single-stranded DNA (ssDNA) viruses not previously identified in aquatic environments. We also observed that the transition from an ice-covered lake in spring to an open-water lake in summer led to a change from a ssDNA- to a double-stranded DNA-virus-dominated assemblage, possibly reflecting a seasonal shift in host organisms.
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http://dx.doi.org/10.1126/science.1179287DOI Listing
November 2009

Evolution to pathogenicity of the parvovirus minute virus of mice in immunodeficient mice involves genetic heterogeneity at the capsid domain that determines tropism.

J Virol 2008 Feb 28;82(3):1195-203. Epub 2007 Nov 28.

Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), 28049 Cantoblanco, Madrid, Spain.

Very little is known about the role that evolutionary dynamics plays in diseases caused by mammalian DNA viruses. To address this issue in a natural host model, we compared the pathogenesis and genetics of the attenuated fibrotropic and the virulent lymphohematotropic strains of the parvovirus minute virus of mice (MVM), and of two invasive fibrotropic MVM (MVMp) variants carrying the I362S or K368R change in the VP2 major capsid protein, in the infection of severe combined immunodeficient (SCID) mice. By 14 to 18 weeks after oronasal inoculation, the I362S and K368R viruses caused lethal leukopenia characterized by tissue damage and inclusion bodies in hemopoietic organs, a pattern of disease found by 7 weeks postinfection with the lymphohematotropic MVM (MVMi) strain. The MVMp populations emerging in leukopenic mice showed consensus sequence changes in the MVMi genotype at residues G321E and A551V of VP2 in the I362S virus infections or A551V and V575A changes in the K368R virus infections, as well as a high level of genetic heterogeneity within a capsid domain at the twofold depression where these residues lay. Amino acids forming this capsid domain are important MVM tropism determinants, as exemplified by the switch in MVMi host range toward mouse fibroblasts conferred by coordinated changes of some of these residues and by the essential character of glutamate at residue 321 for maintaining MVMi tropism toward primary hemopoietic precursors. The few viruses within the spectrum of mutants from mice that maintained the respective parental 321G and 575V residues were infectious in a plaque assay, whereas the viruses with the main consensus sequences exhibited low levels of fitness in culture. Consistent with this finding, a recombinant MVMp virus carrying the consensus sequence mutations arising in the K368R virus background in mice failed to initiate infection in cell lines of different tissue origins, even though it caused rapid-course lethal leukopenia in SCID mice. The parental consensus genotype prevailed during leukopenia development, but plaque-forming viruses with the reversion of the 575A residue to valine emerged in affected organs. The disease caused by the DNA virus in mice, therefore, involves the generation of heterogeneous viral populations that may cooperatively interact for the hemopoietic syndrome. The evolutionary changes delineate a sector of the surface of the capsid that determines tropism and that surrounds the sialic acid receptor binding domain.
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http://dx.doi.org/10.1128/JVI.01692-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2224436PMC
February 2008

Minute virus of mice, a parvovirus, in complex with the Fab fragment of a neutralizing monoclonal antibody.

J Virol 2007 Sep 11;81(18):9851-8. Epub 2007 Jul 11.

Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054, USA.

The structure of virus-like particles of the lymphotropic, immunosuppressive strain of minute virus of mice (MVMi) in complex with the neutralizing Fab fragment of the mouse monoclonal antibody (MAb) B7 was determined by cryo-electron microscopy to 7-A resolution. The Fab molecule recognizes a conformational epitope at the vertex of a three-fold protrusion on the viral surface, thereby simultaneously engaging three symmetry-related viral proteins in binding. The location of the epitope close to the three-fold axis is consistent with the previous analysis of MVMi mutants able to escape from the B7 antibody. The binding site close to the symmetry axes sterically forbids the binding of more than one Fab molecule per spike. MAb as well as the Fab molecules inhibits the binding of the minute virus of mice (MVM) to permissive cells but can also neutralize MVM postattachment. This finding suggests that the interaction of B7 with three symmetry-related viral subunits at each spike hinders structural transitions in the viral capsid essential during viral entry.
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http://dx.doi.org/10.1128/JVI.00775-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045413PMC
September 2007

Host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence.

J Virol 2006 Feb;80(3):1563-73

Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, 28049 Cantoblanco, Madrid, Spain.

The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 A away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts.
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http://dx.doi.org/10.1128/JVI.80.3.1563-1573.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1346950PMC
February 2006

Virulent variants emerging in mice infected with the apathogenic prototype strain of the parvovirus minute virus of mice exhibit a capsid with low avidity for a primary receptor.

J Virol 2005 Sep;79(17):11280-90

Centro de Biología Molecular Severo Ochoa (UAM-CSIC), Universidad Autónoma de Madrid, Cantoblanco, Spain.

The mechanisms involved in the emergence of virulent mammalian viruses were investigated in the adult immunodeficient SCID mouse infected by the attenuated prototype strain of the parvovirus Minute Virus of Mice (MVMp). Cloned MVMp intravenously inoculated in mice consistently evolved during weeks of subclinical infection to variants showing altered plaque phenotypes. All the isolated large-plaque variants spread systemically from the oronasal cavity and replicated in major organs (brain, kidney, liver), in sharp contrast to the absolute inability of the MVMp and small-plaque variants to productively invade SCID organs by this natural route of infection. The virulent variants retained the MVMp capacity to infect mouse fibroblasts, consistent with the lack of genetic changes across the 220-to-335 amino acid sequence of VP2, a capsid domain containing main determinants of MVM tropism. However, the capsid of the virulent variants shared a lower affinity than the wild type for a primary receptor used in the cytotoxic infection. The capsid gene of a virulent variant engineered in the MVMp background endowed the recombinant virus with a large-plaque phenotype, lower affinity for the receptor, and productive invasiveness by the oronasal route in SCID mice, eventually leading to 100% mortality. In the analysis of virulence in mice, both MVMp and the recombinant virus similarly gained the bloodstream 1 to 2 days postoronasal inoculation and remained infectious when adsorbed to blood cells in vitro. However, the wild-type MVMp was cleared from circulation a few days afterwards, in contrast to the viremia of the recombinant virus, which was sustained for life. Significantly, attachment to an abundant receptor of primary mouse kidney epithelial cells by both viruses could be quantitatively competed by wild-type MVMp capsids, indicating that virulence is not due to an extended receptor usage in target tissues. We conclude that the selection of capsid-receptor interactions of low affinity, which favors systemic infection, is a major evolutionary process in the adaptation of parvoviruses to new hosts and in the cause of disease.
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http://dx.doi.org/10.1128/JVI.79.17.11280-11290.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1193584PMC
September 2005

Enhanced cytoplasmic sequestration of the nuclear export receptor CRM1 by NS2 mutations developed in the host regulates parvovirus fitness.

J Virol 2004 Oct;78(19):10674-84

Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.

To investigate whether a DNA virus can evade passive immunotherapy with a polyclonal antiserum, we analyzed the protection of a neutralizing capsid antiserum against a lethal infection of the immunosuppressive strain of the parvovirus minute virus of mice (MVMi) in 42 immunodeficient mice over a period of 200 days. A few mice were effectively protected, but most developed a delayed lethal leukopenic syndrome during the treatment or weeks afterwards. Unexpectedly, viruses isolated from treated but also from control leukopenic mice showed no amino acid changes throughout the entire capsid coding region, although the viral populations were genetically heterogeneous, mainly in the second exon of the coding sequence of the NS2 nonstructural protein. The NS2 point amino acid changes (T88A, K96E, L103P, and L153 M) that were consistently selected in several mice clustered within the nuclear exportin CRM1 binding domain, in a reading frame that did not alter the overlapping NS1 coding region. These mutations endowed emerging viruses with an increased fitness that was demonstrable by their relative resistance to the neutralizing capsid antiserum in a postentry plaque-forming assay, the rapid overgrowth of a competing wild-type (wt) population in culture, and a larger yield of infectious particles. Mutant NS2 proteins interacted with a higher affinity and sequestered CRM1 in the perinuclear region of the cytoplasm more efficiently than the wt. Correspondingly this phenomenon, as well as the following timely ordered release of the NS1 nonstructural protein and the empty capsid from the nucleus to the cytoplasm, occurred markedly earlier in the infection cycle of the mutant viruses. We hypothesize that the enhanced cytoplasmic sequestration of CRM1 by the NS2 mutations selected in mice may trigger pleiotropic effects leading to an accelerated MVMi life cycle and thus to increased fitness. These results strengthen our earlier report on the rapid evolutionary capacity of this mammalian-specific DNA virus in vivo and indicate that the NS2-CRM1 interaction is an important determinant of parvovirus virulence that can be modulated in nature, hampering the effectiveness of passive antibody therapies in the long term.
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http://dx.doi.org/10.1128/JVI.78.19.10674-10684.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC516389PMC
October 2004

High mutant frequency in populations of a DNA virus allows evasion from antibody therapy in an immunodeficient host.

J Virol 2003 Feb;77(4):2701-8

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Cantoblanco, 28049 Madrid, Spain.

The degree of genetic heterogeneity of DNA virus populations in nature and its consequences for disease control are virtually unknown. The parvovirus minute virus of mice (MVMi) was used here to investigate (i) the frequency of antibody-escape mutants in populations of a DNA virus and (ii) the ability of a DNA virus to evade in the long-term a passive monoclonal antibody (MAb) therapy in an immunodeficient natural host. Independent clonal populations of MVMi harbored a high proportion of mutants resistant to neutralizing MAb (mutant frequency = [2.8 +/- 0.5] x 10(-5)) that rapidly evolved under antibody pressure in culture to become mixtures dominated by genotypically diverse escape mutants. Immunodeficient mice naturally infected with clonal populations of MVMi and subsequently treated by intravenous injections of MAb were initially protected from the characteristic viral induced lethal leukopenia. However, some treated animals developed a delayed severe leukopenic syndrome associated with the emergence of genetically heterogeneous populations of MAb-resistant mutants in the MVMi main target organs. The 11 plaque-purified viruses analyzed from an antibody-resistant population obtained from one animal corresponded to four different mutant genotypes, although their consensus sequence remained wild type. All cloned escape mutants harbored single radical amino acid changes within a stretch of seven residues in a surface-exposed loop at the threefold axes of the capsid. This antigenic site, which can tolerate radical changes preserving MVMi pathogenic potential, may thereby allow the virus to evade the immune control. These findings indicate a high genetic heterogeneity and rapid adaptation of populations of a mammal DNA virus in vivo and provide a genetic basis for the failure of passive immunotherapy in the natural host.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC141124PMC
http://dx.doi.org/10.1128/jvi.77.4.2701-2708.2003DOI Listing
February 2003