Carlos Maluquer de Motes

Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity.

Virus infection of mammalian cells is sensed by pattern recognition receptors and leads to an innate immune response that restricts virus replication and induces adaptive immunity. In response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts, despite the host innate immune response. Poxviruses, such as vaccinia virus (VACV), have large DNA genomes and encode many proteins that are dedicated to host immune evasion. Some of these proteins are secreted from the infected cell, where they bind and neutralize complement factors, interferons, cytokines and chemokines. Other VACV proteins function inside cells to inhibit apoptosis or signalling pathways that lead to the production of interferons and pro-inflammatory cytokines and chemokines. In this review, these VACV immunomodulatory proteins are described and the potential to create more immunogenic VACV strains by manipulation of the gene encoding these proteins is discussed.

Identification of Human and Animal Adenoviruses and Polyomaviruses for Determination of Sources of Fecal Contamination in the Environment

ABSTRACT The Adenoviridae and Polyomaviridae families comprise a wide diversity of viruses which may be excreted for long periods in feces or urine. In this study, a preliminary analysis of the prevalence in the environment and the potential usefulness as source-tracking tools of human and animal adenoviruses and polyomaviruses has been developed. Molecular assays based on PCR specifically targeting human adenoviruses (HAdV), porcine adenoviruses (PAdV), bovine adenoviruses (BAdV), and bovine polyomaviruses (BPyV) were applied to environmental samples including urban sewage, slaughterhouse, and river water samples. PAdV and BPyV were detected in a very high percentage of samples potentially affected by either porcine or bovine fecal contamination, respectively. However, BAdV were detected in only one sample, showing a lower prevalence than BPyV in the wastewater samples analyzed. The 22 slaughterhouse samples with fecal contamination of animal origin showed negative results for the presence of HAdV. The river water samples analyzed were positive for the presence of both human and animal adenoviruses and polyomaviruses, indicating the existence of diverse sources of contamination. The identities of the viruses detected were confirmed by analyses of the amplified sequences. All BPyV isolates showed a 97% similarity in nucleotide sequences. This is the first time that PAdV5, BAdV6, and BPyV have been reported to occur in environmental samples. Human and porcine adenoviruses and human and bovine polyomaviruses are proposed as tools for evaluating the presence of viral contamination and for tracking the origin of fecal/urine contamination in environmental samples.

Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination

ABSTRACT In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.

Poxvirus Targeting of E3 Ligase β-TrCP by Molecular Mimicry: A Mechanism to Inhibit NF-κB Activation and Promote Immune Evasion and Virulence

The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKβ causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase β-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished β-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

Development of a quantitative PCR assay for the quantitation of bovine polyomavirus as a microbial source-tracking tool

Adenoviruses and polyomaviruses are two distinct DNA viral families that are excreted in high concentrations and distributed in human and animal populations. Targeting specific virus included in these families has proved to be a promising and useful tool for tracing specifically sources of environmental contamination. In this study, a quantitative PCR assay that is specific for bovine polyomaviruses was developed and used to determine the excretion level and concentration of bovine polyomaviruses in urine and environmental samples, including urban sewage, slaughterhouse sewage, and river water. A set of primers and a TaqMan probe were designed to target a 77-bp region of the bovine polyomavirus VP1 gene, and the conditions of the reaction were optimized. A detection limit was established at 1-10 genome copies per test tube. The assay was specific and produced negative results when samples containing human or porcine fecal contamination were analyzed. This is, to our knowledge, the first description of bovine polyomaviruses excreted in bovine urine samples (mean values of 10(4) GC/l). Bovine polyomaviruses were also detected and quantified in slaughterhouse wastewater and river waters, which shows the spread of these viruses in many environmental samples containing contamination of bovine origin. The procedure described in this paper provides a quantitative source-tracking tool for the analysis of bovine polyomaviruses as indicators of the presence of bovine contamination in environmental samples.

Inhibition of Apoptosis and NF-κB Activation by Vaccinia Protein N1 Occur via Distinct Binding Surfaces and Make Different Contributions to Virulence

Vaccinia virus (VACV) protein N1 is an intracellular virulence factor and belongs to a family of VACV B-cell lymphoma (Bcl)-2-like proteins whose members inhibit apoptosis or activation of pro-inflammatory transcription factors, such as interferon (IFN) regulatory factor-3 (IRF-3) and nuclear factor-κB (NF-κB). Unusually, N1 inhibits both apoptosis and NF-κB activation. To understand how N1 exerts these different functions, we have mutated residues in the Bcl-2-like surface groove and at the interface used to form N1 homodimers. Mutagenesis of the surface groove abolished only the N1 anti-apoptotic activity and protein crystallography showed these mutants differed from wild-type N1 only at the site of mutation. Conversely, mutagenesis of the dimer interface converted N1 to a monomer and affected only inhibition of NF-κB activation. Collectively, these data show that N1 inhibits pro-inflammatory and pro-apoptotic signalling using independent surfaces of the protein. To determine the relative contribution of each activity to virus virulence, mutant N1 alleles were introduced into a VACV strain lacking N1 and the virulence of these viruses was analysed after intradermal and intranasal inoculation in mice. In both models, VACV containing a mutant N1 unable to inhibit apoptosis had similar virulence to wild-type virus, whereas VACV containing a mutant N1 impaired for NF-κB inhibition induced an attenuated infection similar to that of the N1-deleted virus. This indicates that anti-apoptotic activity of N1 does not drive virulence in these in vivo models, and highlights the importance of pro-inflammatory signalling in the immune response against viral infections.

Human and viral Golgi anti-apoptotic proteins (GAAPs) oligomerize via different mechanisms and monomeric GAAP inhibits apoptosis and modulates calcium

Background: GAAPs are Golgi anti-apoptotic proteins that modulate Ca2+ signaling. Results: Unlike human GAAP, viral GAAP oligomerization is cysteine-dependent. Monomeric vGAAP is functional. Conclusion: Viral and human GAAPs show pH-dependent oligomerization via different mechanisms. Monomeric vGAAP is anti-apoptotic and modulates intracellular Ca2+. Significance: This is the first mechanistic analysis of TMBIM protein oligomerization, and shows that monomers are functional. Golgi anti-apoptotic proteins (GAAPs) are hydrophobic proteins resident in membranes of the Golgi complex. They protect cells from a range of apoptotic stimuli, reduce the Ca2+ content of intracellular stores, and regulate Ca2+ fluxes. GAAP was discovered in camelpox virus, but it is highly conserved throughout evolution and encoded by all eukaryote genomes examined. GAAPs are part of the transmembrane Bax inhibitor-containing motif (TMBIM) family that also includes other anti-apoptotic and Ca2+-modulating membrane proteins. Most TMBIM members show multiple bands when analyzed by SDS-PAGE, suggesting that they may be oligomeric. However, the molecular mechanisms of oligomerization, the native state of GAAPs in living cells and the functional significance of oligomerization have not been addressed. TMBIM members are thought to have evolved from an ancestral GAAP. Two different GAAPs, human (h) and viral (v)GAAP were therefore selected as models to examine oligomerization of TMBIM family members. We show that both hGAAP and vGAAP in their native states form oligomers and that oligomerization is pH-dependent. Surprisingly, hGAAP and vGAAP do not share the same oligomerization mechanism. Oligomerization of hGAAP is independent of cysteines, but oligomerization of vGAAP depends on cysteines 9 and 60. A mutant vGAAP that is unable to oligomerize revealed that monomeric vGAAP retains both its anti-apoptotic function and its effect on intracellular Ca2+ stores. In conclusion, GAAP can oligomerize in a pH-regulated manner, and monomeric GAAP is functional.

Vaccinia Virus Protein A49 is an Unexpected Member of the B-cell Lymphoma (Bcl)-2 Protein Family

Background: Poxviruses contain several virulence factors with no identifiable cellular homologues. Results: Vaccinia virus immunomodulatory protein A49 adopts the same fold as cellular and viral Bcl-2 family proteins but does not share their function. Conclusion: Vaccinia virus Bcl-2-like proteins resemble each other structurally but have divergent functions. Significance: Viruses hijack protein scaffolds and evolve them to subvert host-cell immunity. Vaccinia virus (VACV) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor κB (NF-κB). VACV protein A49 prevents translocation of NF-κB to the nucleus by sequestering cellular β-TrCP, a protein required for the degradation of the inhibitor of κB. A49 does not share overall sequence similarity with any protein of known structure or function. We solved the crystal structure of A49 from VACV Western Reserve to 1.8 Å resolution and showed, surprisingly, that A49 has the same three-dimensional fold as Bcl-2 family proteins despite lacking identifiable sequence similarity. Whereas Bcl-2 family members characteristically modulate cellular apoptosis, A49 lacks a surface groove suitable for binding BH3 peptides and does not bind proapoptotic Bcl-2 family proteins Bax or Bak. The N-terminal 17 residues of A49 do not adopt a single well ordered conformation, consistent with their proposed role in binding β-TrCP. Whereas pairs of A49 molecules interact symmetrically via a large hydrophobic surface in crystallo, A49 does not dimerize in solution or in cells, and we propose that this hydrophobic interaction surface may mediate binding to a yet undefined cellular partner. A49 represents the eleventh VACV Bcl-2 family protein and, despite these proteins sharing very low sequence identity, structure-based phylogenetic analysis shows that all poxvirus Bcl-2 proteins are structurally more similar to each other than they are to any cellular or herpesvirus Bcl-2 proteins. This is consistent with duplication and diversification of a single BCL2 family gene acquired by an ancestral poxvirus.

Persistence of the bovine spongiform encephalopathy infectious agent in sewage

Horizontal transmission of prion diseases through the environment represents a considerable concern. Prions are extremely resistant to inactivation and are thought to enter the environment after burial of animal mortalities or through biosolids from wastewater treatment plants. In addition, deposition of prions in the environment through biological fluids and/or faeces has been proved in the last years. Little is known about the behaviour of prion infectivity in the environment. In this study, the persistence of BSE infectious agent in sewage has been assessed by both PrP(Res) immunoblotting and mouse bioassay in a long-term incubation study. Results indicated that no PrP(Res) was detected after 150 day of incubation and consistent with this, a statistical regression model estimated 2-logs decay in 151 day. In contrast, no reduction in infectivity was observed during this period. Similarly, BSE infectivity remained unaltered after incubation in PBS for 265 day, whereas PrP(Res) levels dropped progressively over the length of the study. These results indicate that in sewage and PBS, prion infectivity persists longer and with different dynamics than its commonly used marker PrP(Res). Thus, mathematical models computed on the basis of PrP(Res) detection were unable to predict inactivation of prion infectivity. It is also reasonable to assume that conventional wastewater treatments with low retention times could have a very limited impact on prion infectivity. This data is essential for the development of accurate risk assessment analysis for BSE and other prion diseases in the environment.

Cost-effective method for microbial source tracking using specific human and animal viruses.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown. Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples. In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method. The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.