Maluquer de Motes C, Clemente-Casares P, Hundesa A, Martin M, Girones R (2004) Detection of bovine and porcine adenoviruses for tracing the source of fecal contamination, Applied Environmental Microbiology 70 (3) pp. 1448-1454
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.
Smith GL, Benfield CTO, Maluquer de Motes C, Mazzon M, Ember SWJ, Ferguson BJ, Sumner RP (2013) Vaccinia virus immune evasion: Mechanisms, virulence and immunogenicity, Journal of General Virology 94 (PART 11) pp. 2367-2392
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. © 2013 SGM.
Sumner RP, Maluquer de Motes C, Veyer DL, Smith GL (2013) Vaccinia virus inhibits NF-ºB-dependent gene expression downstream of p65 translocation, J Virol
The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ºB) transcription factor plays a critical role in host defence against viral infection by inducing the production of pro-inflammatory mediators and type I interferon. Consequently, viruses have evolved many mechanisms to block its activation. The poxvirus vaccinia virus (VACV) encodes numerous inhibitors of NF-ºB activation that target multiple points in the signaling pathway. A derivative of VACV strain Copenhagen, called vv811, lacking 55 open reading frames in the left and right terminal regions of the genome, was reported to still inhibit NF-ºB activation downstream of tumour necrosis factor (TNF)± and interleukin (IL)-1², suggesting the presence of one or more additional inhibitors. In this study we constructed a recombinant vv811 lacking the recently described NF-ºB inhibitor A49 (vv811A49), yielding a virus that lacked all currently described inhibitors downstream of TNF±/IL-1². Unlike vv811, vv811A49 no longer inhibited degradation of phosphorylated IºB± and p65 translocated into the nucleus. However, despite this translocation, vv811A49 still inhibited TNF±- and IL1²-induced NF-ºB-dependent reporter gene expression and the transcription and production of cytokines induced by these agonists. This inhibition did not require late viral gene expression. These findings indicate the presence of another inhibitor of NF-ºB that is expressed early during infection and acts by a novel mechanism downstream of p65 translocation into the nucleus.
Maluquer de Motes C, Cooray S, Ren H, Almeida GMF, McGourty K, Bahar MW, Stuart DI, Grimes JM, Graham SC, Smith GL (2011) Modulation of apoptosis and pro-inflammatory signalling by vaccinia virus N1., Cytokine (56)
Maluquer de Motes C, Hundesa A, Almeida FC, Bofill-Mas S, Girones R (2011) Isolation of a novel monkey adenovirus reveals a new phylogenetic clade in the evolutionary history of simian adenoviruses, Virol J 8
Adenoviruses of primates include human (HAdV) and simian (SAdV) isolates classified into 8 species (Human Adenovirus A to G, and Simian Adenovirus A). In this study, a novel adenovirus was isolated from a colony of cynomolgus macaques (Macaca fascicularis) and subcultured in VERO cells. Its complete genome was purified and a region encompassing the hexon gene, the protease gene, the DNA binding protein (DBP) and the 100 kDa protein was amplified by PCR and sequenced by primer walking. Sequence analysis of these four genes showed that the new isolate had 80% identity to other primate adenoviruses and lacked recombination events. The study of the evolutionary relationships of this new monkey AdV based on the combined sequences of the four genes supported a close relationship to SAdV-3 and SAdV-6, lineages isolated from Rhesus monkeys. The clade formed by these three types is separated from the remaining clades and establishes a novel branch that is related to species HAdV-A, F and G. However, the genetic distance corresponding to the newly isolated monkey AdV considerably differs from these as to belong to a new, not yet established species. Results presented here widen our knowledge on SAdV and represents an important contribution to the understanding of the evolutionary history of primate adenoviruses.
Hundesa A, Maluquer de Motes C, Albinana-Gimenez N, Rodriguez-Manzano J, Bofill-Mas S, Suñen E, Rosina Girones R (2009) Development of a qPCR assay for the quantification of porcine adenoviruses as an MST tool for swine fecal contamination in the environment, Journal of Virological Methods 158 (1-2) pp. 130-135
The Adenoviridae family comprises a wide diversity of viruses that may be excreted for long periods in feces or urine. Previous studies have suggested that the detection of human and animal adenoviruses as well as human and animal polyomaviruses by PCR could be used as an index of fecal contamination of human and animal origin. In this study, quantitative PCR assays targeting specifically porcine adenoviruses have been developed and applied to fecal and environmental samples, including pig slurries, urban sewage, slaughterhouse sewage and river water samples. To develop real-time quantitative PCR for the detection and quantitation of porcine adenoviruses, primers and a TaqMan probe targeting a 68-bp region of the porcine adenovirus hexon gene were designed to amplify specifically porcine adenovirus, and the conditions of the reaction were optimized. The assay detected 1-10 genome copies per test tube and was specific in showing no positive results when samples containing human or bovine adenoviruses were analyzed. Fecal samples contained mean concentrations of porcine adenoviruses of 105 GC/g while slaughterhouse wastewater samples showed mean values of 103 GC/ml. The assay detected porcine fecal pollution in samples that were highly diluted and had been collected at a considerable distance from the input source, such as river water. In general, the data presented here provide a quantitative tool for the analysis of porcine adenoviruses as indicators of the presence of porcine contamination in the environment, and support the detection of porcine adenoviruses by real-time quantitative PCR as a promising and valuable tool for source-tracking studies. © 2009 Elsevier B.V. All rights reserved.
Hundesa A, Bofill-Mas S, Maluquer de Motes C, Rodriguez-Manzano J, Bach A, Casas M, Girones R (2010) Development of a quantitative PCR assay for the quantitation of bovine polyomavirus as a microbial source-tracking tool, Journal of Virological Methods 163 (2) pp. 385-389
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 104 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. © 2009 Elsevier B.V. All rights reserved.
Neidel S, Maluquer de Motes C, Mansur DS, Strnadova P, Smith GL, Graham SC (2015) Vaccinia virus protein A49 is an unexpected member of the B-cell Lymphoma (Bcl)-2 protein family., J Biol Chem 290 (10) pp. 5991-6002
Vaccinia virus (VACV) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor ºB (NF-ºB). VACV protein A49 prevents translocation of NF-ºB to the nucleus by sequestering cellular ²-TrCP, a protein required for the degradation of the inhibitor of ºB. 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 ²-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.
Maluquer de Motes C, Cooray S, Ren H, Almeida GM, McGourty K, Bahar MW, Stuart DI, Grimes JM, Graham SC, Smith GL (2011) Inhibition of apoptosis and NF-ºB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence, PLoS Pathog 7 (12)
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-ºB (NF-ºB). Unusually, N1 inhibits both apoptosis and NF-ºB 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-ºB 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-ºB 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.
Ren H, Ferguson BJ, Maluquer de Motes C, Sumner RP, Harman LE, Smith GL (2014) Enhancement of CD8(+) T-cell memory by removal of a vaccinia virus nuclear factor-ºB inhibitor., Immunology 145 (1) pp. 34-49
Factors influencing T-cell responses are important for vaccine development but are incompletely understood. Here, vaccinia virus (VACV) protein N1 is shown to impair the development of both effector and memory CD8(+) T cells and this correlates with its inhibition of nuclear factor-ºB (NF-ºB) activation. Infection with VACVs that either have the N1L gene deleted (vN1) or contain a I6E mutation (vN1.I6E) that abrogates its inhibition of NF-ºB resulted in increased central and memory CD8(+) T-cell populations, increased CD8(+) T-cell cytotoxicity and lower virus titres after challenge. Furthermore, CD8(+) memory T-cell function was increased following infection with vN1.I6E, with more interferon-³ production and greater protection against VACV infection following passive transfer to naive mice, compared with CD8(+) T cells from mice infected with wild-type virus (vN1.WT). This demonstrates the importance of NF-ºB activation within infected cells for long-term CD8(+) T-cell memory and vaccine efficacy. Further, it provides a rationale for deleting N1 from VACV vectors to enhance CD8(+) T-cell immunogenicity, while simultaneously reducing virulence to improve vaccine safety.
Maluquer de Motes C, Mansur DS, Unterholzner L, Sumner RP, Ren H, Strnadova P, Ferguson BJ, Bowie AG, Smith GL (2012) The E3 ligase ²-TrCP is a target for the vaccinia virus virulence factor A49 to inhibit NF-ºB and promote immune evasion, Immunology (137 (Suppl. 1))
Mansur DS, Maluquer de Motes C, Unterholzner L, Sumner RP, Ferguson BJ, Ren H, Strnadova P, Bowie AG, Smith GL (2013) Poxvirus Targeting of E3 Ligase ²-TrCP by Molecular Mimicry: A Mechanism to Inhibit NF-ºB Activation and Promote Immune Evasion and Virulence, PLoS Pathogens 9 (2)
The transcription factor NF-ºB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IºB±. Here we describe an inhibitor of NF-ºB 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-ºB activation by molecular mimicry and contains a motif conserved in IºB± which, in IºB±, is phosphorylated by IKK² causing ubiquitination and degradation. Like IºB±, A49 binds the E3 ligase ²-TrCP, thereby preventing ubiquitination and degradation of IºB±. Consequently, A49 stabilised phosphorylated IºB± (p-IºB±) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IºB±-like motif of A49 abolished ²-TrCP binding, stabilisation of p-IºB± and inhibition of NF-ºB activation. Remarkably, despite encoding nine other inhibitors of NF-ºB, a VACV lacking A49 showed reduced virulence in vivo. © 2013 Mansur et al.
Maluquer De Motes C, Simon S, Grassi J, Torres JM, Pumarola M, Girones R (2008) Assessing the presence of BSE and scrapie in slaughterhouse wastewater, Journal of Applied Microbiology 105 (5) pp. 1649-1657
Aims: This paper describes a procedure for evaluating the presence and the stability of the proteinase K-resistant form of the prion protein (PrP res) in slaughterhouse wastewater. Methods and Results: Wastewater samples were spiked with either scrapie or bovine spongiform encephalopathy agents and PrPres was concentrated and detected by western blotting. The detection limit was estimated to be 2-4 ¼g of either scrapie or BSE-infected brain tissue in 15 ml of sewage. Wastewater samples from three abattoirs were analysed, two of which had processed BSE-infected animals. No PrPres was detected. The effect of sewage on the inoculum and the persistence of transmissible spongiform encephalopathy agents in wastewater were also considered. Conclusions: The results of the assay suggest that wastewaters from abattoirs where one positive BSE case has been identified would contain titres lower than 0.6-26 × 10-4 cattle oral ID50 per litre resulting from specified risk material tissue contamination. Moreover, the effect of abattoir wastewaters is to reduce the persistence of PrP res. Significance and Impact of the Study: The assay may be a useful tool for risk assessment studies and for reducing the potential risk of contamination with BSE via sewage sludge fertilizer procedures. © 2008 The Authors.
Maluquer de Motes C, Espinosa JC, Esteban A, Calvo M, Girones R, Torres JM (2012) Persistence of the bovine spongiform encephalopathy infectious agent in sewage, Environmental Research 117 pp. 1-7
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. © 2012 Elsevier Inc.
Bofill-Mas S, Hundesa A, Calgua B, Rusiñol M, Maluquer de Motes C, Girones R (2011) Cost-effective method for microbial source tracking using specific human and animal viruses, J Vis Exp (58)
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 b
Rodriguez-Manzano J, Hundesa A, Calgua B, Carratala A, Maluquer de Motes C, Rusiñol M, Moresco V, Ramos AP, Martínez-Marca F, Calvo M, Monte Barardi CR, Girones R, Bofill-Mas S (2014) Adenovirus and Norovirus Contaminants in Commercially Distributed Shellfish, Food and Environmental Virology 6 (1) pp. 31-41
Shellfish complying with European Regulations based on quantification of fecal bacterial indicators (FIB) are introduced into markets; however, information on viruses, more stable than FIB, is not available in the literature. To assess the presence of noroviruses (NoVs) GI and GII and human adenoviruses (HAdV) in domestic and imported mussels and clams (n = 151) their presence was analyzed during winter seasons (2004-2008) in north-west Spanish markets through a routine surveillance system. All samples tested negative for NoV GI and 13 % were positive for NoV GII. The role of HAdV as viral indicator was evaluated in 20 negative and 10 positive NoV GII samples showing an estimated sensitivity and specificity of HAdV to predict the presence of NoV GII of 100 and 74 % (cut-off 0.5). The levels of HAdV and NoVs and the efficiency of decontamination in shellfish depuration plants (SDP) were evaluated analyzing pre- and post-depurated mussels collected in May-June 2010 from three different SDP. There were no statistically significant differences in the prevalence and quantification of HAdV between pre- and post-depurated shellfish and between seawater entering and leaving the depuration systems. Moreover, infectious HAdV were detected in depurated mussels. These results confirm previous studies showing that current controls and depuration treatments limiting the number of FIB do not guarantee the absence of viruses in shellfish. © 2013 Springer Science+Business Media New York.
Maluquer de Motes C, Grassi J, Simon S, Herva ME, Torres JM, Pumarola M, Girones R (2008) Excretion of BSE and scrapie prions in stools from murine models, Vet Microbiol 131 (1-2) pp. 205-211
Faeces from infected animals have been suggested as a potential source of contamination and transmission of prion diseases in the environment. This work describes the development of a procedure for the detection of PrP(res) in stools which is based on a detergent-based extraction and immunoprecipitation (IP). The procedure was evaluated by analyzing TSE-spiked sheep and mice faeces, and proved to be specific for PrP(res) with sensitivities of 5-10 microg of infected brain tissue. In order to analyze the shedding of prions, we studied stools from orally inoculated mice over 4-days post-inoculation and also stools from terminally sick scrapie-infected mice. PrP(res) was only detected in stools shortly after the oral ingestion of TSE agents. The procedure described could be a useful tool for studying the excretion of prions and for evaluating potential environmental contamination by prions.
Veyer DL, Maluquer de Motes C, Sumner RP, Ludwig L, Johnson BF, Smith GL (2014) Analysis of the anti-apoptotic activity of four vaccinia virus proteins demonstrates that B13 is the most potent inhibitor in isolation and during viral infection., J Gen Virol 95 (Pt 12) pp. 2757-2768
Vaccinia virus (VACV) is a large dsDNA virus encoding ~200 proteins, several of which inhibit apoptosis. Here, a comparative study of anti-apoptotic proteins N1, F1, B13 and Golgi anti-apoptotic protein (GAAP) in isolation and during viral infection is presented. VACVs strains engineered to lack each gene separately still blocked apoptosis to some degree because of functional redundancy provided by the other anti-apoptotic proteins. To overcome this redundancy, we inserted each gene separately into a VACV strain (vv811) that lacked all these anti-apoptotic proteins and that induced apoptosis efficiently during infection. Each protein was also expressed in cells using lentivirus vectors. In isolation, each VACV protein showed anti-apoptotic activity in response to specific stimuli, as measured by immunoblotting for cleaved poly(ADP ribose) polymerase-1 and caspase-3 activation. Of the proteins tested, B13 was the most potent inhibitor, blocking both intrinsic and extrinsic stimuli, whilst the activity of the other proteins was largely restricted to inhibition of intrinsic stimuli. In addition, B13 and F1 were effective blockers of apoptosis induced by vv811 infection. Finally, whilst differences in induction of apoptosis were barely detectable during infection with VACV strain Western Reserve compared with derivative viruses lacking individual anti-apoptotic genes, several of these proteins reduced activation of caspase-3 during infection by vv811 strains expressing these proteins. These results illustrated that vv811 was a useful tool to determine the role of VACV proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 was the most potent.
Maluquer de Motes C, Cano MJ, Torres JM, Pumarola M, Girones R (2008) Detection and survival of prion agents in aquatic environments, Water Res 42 (10-11) pp. 2465-2472
Environmental contamination is considered a potential mechanism of transmission of prion diseases. Sheep scrapie and cervid chronic wasting diseases (CWD) epizootics are thought to be maintained by natural horizontal transmission through the environment. Here, we describe a method for the detection of prion proteins (PrPres) in aquatic environments. The procedure is based on a glycine buffer-mediated extraction, sonication, and an ultracentrifugation step. The detection limit of the method was estimated to be over 5-10 microg of infected tissue. In order to determine the inactivation of these agents, we spiked infected brain tissue in urban sewage, seawater and a buffered solution (final concentrations of 0.1-0.2% brain in matrix), and studied the decay of BSE- and scrapie-associated PrPres over time (up to 265 days). Densitometric data from Western blots were plotted in logarithmic scale against time. Reduction of PrPres titer in sewage was quantified in one logarithm after 13.5 days for BSE, 27.9 days for mouse-passaged scrapie and 32.6 days for sheep scrapie. In the buffered solution, a logarithm of BSE-associated PrPres also disappeared earlier than that of scrapie (113.9 and 214.3 days, respectively). By means of the covariance analysis, these differences in the inactivation patterns were shown to be statistically significant. According to the data, prions may be stable for extended periods of time in buffered solutions like PBS, but would show limited survival in aquatic environmental matrices.
Hundesa A, Maluquer de Motes C, Bofill-Mas S, Albinana-Gimenez N, Girones R (2006) Identification of human and animal adenoviruses and polyomaviruses for determination of sources of fecal contamination in the environment, Appl Environ Microbiol 72 (12) pp. 7886-7893
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.
Saraiva N, Prole DL, Carrara G, Maluquer de Motes C, Johnson BF, Byrne B, Taylor CW, Smith GL (2013) Human and viral Golgi anti-apoptotic proteins (GAAPs) oligomerize via different mechanisms and monomeric GAAP inhibits apoptosis and modulates calcium, J Biol Chem 288 (18) pp. 13057-13067
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 Ca(2+) content of intracellular stores, and regulate Ca(2+) 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 Ca(2+)-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 Ca(2+) stores. In conclusion, GAAP can oligomerize in a pH-regulated manner, and monomeric GAAP is functional.
Maluquer de Motes C, Schiffner T, Sumner RP, Smith GL (2014) Vaccinia virus virulence factor N1 can be ubiquitylated on multiple lysine residues., J Gen Virol 95 (Pt 9) pp. 2038-2049
Ubiquitylation is a covalent post-translational modification that regulates protein stability and is involved in many biological functions. Proteins may be modified with mono-ubiquitin or ubiquitin chains. Viruses have evolved multiple mechanisms to perturb the cell ubiquitin system and manipulate it to their own benefit. Here, we report ubiquitylation of vaccinia virus (VACV) protein N1. N1 is an inhibitor of the nuclear factor NF-ºB and apoptosis that contributes to virulence, has a Bcl-2-like fold, and is highly conserved amongst orthopoxviruses. The interaction between N1 and ubiquitin occurs at endogenous protein levels during VACV infection and following ectopic expression of N1. Biochemical analysis demonstrated that N1 is covalently ubiquitylated, and heterodimers of ubiquitylated and non-ubiquitylated N1 monomers were identified, suggesting that ubiquitylation does not inhibit N1 dimerization. Studies with other VACV Bcl-2 proteins, such as C6 or B14, revealed that although these proteins also interact with ubiquitin, these interactions are non-covalent. Finally, mutagenesis of N1 showed that ubiquitylation occurs in a conventional lysine-dependent manner at multiple acceptor sites because only an N1 allele devoid of lysine residues remained unmodified. Taken together, we described a previously uncharacterized modification of the VACV protein N1 that provided a new layer of complexity to the biology of this virulence factor, and provided another example of the intricate interplay between poxviruses and the host ubiquitin system.
Regulated cell death is a powerful anti-viral mechanism capable of aborting the virus replicative cycle and alerting neighbouring cells to the threat of infection. The biological importance of regulated cell death is illustrated by the rich repertoire of host signalling cascades causing cell death and by the multiple strategies exhibited by viruses to block death signal transduction and preserve cell viability. Vaccinia virus (VACV), a poxvirus and the vaccine used to eradicate smallpox, encodes multiple proteins that interfere with apoptotic, necroptotic and pyroptotic signalling. Here the current knowledge on cell death pathways and how VACV proteins interact with them is reviewed. Studying the mechanisms evolved by VACV to counteract host programmed cell death has implications for its successful use as a vector for vaccination and as an oncolytic agent against cancer.
Vaccinia virus (VACV) encodes multiple proteins inhibiting the NF-kB signalling pathway. One of these, A49, targets the E3
ubiquitin ligase b-TrCP, which is responsible for the ubiquitylation and consequential proteosomal degradation of IkBa and
the release of the NF-kB heterodimer. b-TrCP is a pleiotropic enzyme ubiquitylating multiple cellular substrates, including
the transcriptional activator b-catenin. Here we demonstrate that A49 can activate the Wnt signalling pathway, a critical
pathway that is involved in cell cycle and cell differentiation, and is controlled by b-catenin. The data presented show that
the expression of A49 ectopically or during VACV infection causes accumulation of b-catenin, and that A49 triggering of Wnt
signalling is dependent on binding b-TrCP. This is consistent with A49 blocking the ability of b-TrCP to recognize b-catenin
and IkBa, and possibly other cellular targets. Thus, A49 targeting of b-TrCP affects multiple cellular pathways, including the
NF-kB and Wnt signalling cascades.
Cytosolic recognition of DNA has emerged as a critical cellular mechanism of host immune activation upon pathogen invasion. The central cytosolic DNA sensor cGAS activates STING, which is phosphorylated, dimerises and translocates from the ER to a perinuclear region to mediate IRF-3 activation. Poxviruses are dsDNA viruses replicating in the cytosol and hence likely to trigger cytosolic DNA sensing. Here we investigated the activation of innate immune signalling by 4 different strains of the prototypic poxvirus vaccinia virus (VACV) in a cell line proficient in DNA sensing. Infection with the attenuated VACV strain MVA activated IRF-3 via cGAS and STING, and accordingly STING dimerised and was phosphorylated during MVA infection. Conversely, VACV strains Copenhagen and Western Reserve inhibited STING dimerisation and phosphorylation during infection and in response to transfected DNA and cGAMP, thus efficiently suppressing DNA sensing and IRF-3 activation. A VACV deletion mutant lacking protein C16, thought to be the only viral DNA sensing inhibitor acting upstream of STING, retained the ability to block STING activation. Similar inhibition of DNA-induced STING activation was also observed for cowpox and ectromelia viruses. Our data demonstrate that virulent poxviruses possess mechanisms for targeting DNA sensing at the level of the cGAS-STING axis and that these mechanisms do not operate in replication-defective strains such as MVA. These findings shed light on the role of cellular DNA sensing in poxvirus-host interactions and will open new avenues to determine its impact on VACV immunogenicity and virulence.
Bovine tuberculosis (bTB) caused by Mycobacterium bovis is the most serious endemic disease affecting
livestock in the UK. The European badger (Meles meles) is the most important wildlife reservoir of bTB transmission to
cattle, making eradication particularly difficult. In this respect, oral vaccination with the attenuated M. bovis vaccine Bacillus
Calmette-Guerin (BCG) has been suggested as a wide-scale intervention to reduce bTB infection in badgers. However,
experimental studies show variable protection. Among the possibilities for this variation is that the resident gut bacteria
may influence the success of oral vaccination in badgers; either through competitive exclusion and/or inhibition, or via
effects on the host immune system. In order to explore this possibility, we have tested whether typical gut commensals
such as Lactic Acid Bacteria (LAB) have the capacity to impact on the viability and survival rate of BCG and to modulate
the immune response to BCG using an in vitromodel.
Twelve LAB isolated from badger faeces displayed inhibitory activity to BCG that was species-dependent.
Weissella had a bacteriostatic effect, whereas isolates of enterococci, lactobacilli and pediococci had a more bactericidal
activity. Furthermore, BCG-induced activation of the pro-inflammatory transcription factor NF-ºB in human THP-1
macrophages was modulated by LAB in a strain-dependent manner. Most pediococci enhanced NF-ºB activation but
one strain had the opposite effect. Interestingly, isolates of enterococci, lactobacilli and weissella had different effects as
immunomodulators of BCG-induced macrophage responses as some had no significant influence on NF-ºB activation,
but others increased it significantly.
Our in vitro results show that LAB isolated from badgers exhibit significant inhibitory activity against BCG
and influence the immune activation mediated by BCG in a human macrophage assay. These findings suggest that gut
commensal bacteria could play a role in influencing the outcome of oral BCG vaccination. Inactivated cells of LAB, or
LAB that are bacteriostatic but have a synergistic immunostimulatory effect with BCG, could be potential adjuvants to
be used for oral vaccination in badgers. Further work is needed to take into account the complex nature of the gut
microbiome, specific immunity of the badger and the in vivo context.
Ankyrin repeat (ANK) domains are one of the most abundant motifs in eukaryotic proteins. ANK proteins are rare
amongst viruses with the exception of poxviruses, which presumably acquired them from the host via horizontal gene
transfer. The architecture of poxvirus ANK proteins is however different from their cellular counterparts and this
precludes a direct acquisition event. Here we combine bioinformatics analysis and quantitative proteomics to discover
a new class of viral ANK proteins with a domain organisation that relates to cellular ANK proteins. These non-canonical
viral ANK proteins, termed ANK/BC, interact with host Cullin-2 via a C-terminal BC box resembling that of cellular
Cullin-2 substrate adaptors such as the von Hippel-Lindau protein. Mutagenesis of the BC box-like sequence
abrogates binding to Cullin-2, whereas fusion of this motif to an ANK-only protein confers Cullin-2 association. We
demonstrate that these viral ANK/BC proteins are potent immunomodulatory proteins suppressing the activation of the
pro-inflammatory transcription factors NF-ºB and IRF-3 and the production of cytokines and chemokines including
interferon, and that association with Cullin-2 is required for optimal inhibitory activity. ANK/BC proteins exist in several
orthopoxviruses and cluster into 2 closely related orthologue groups in a phylogenetic lineage that is separate from
canonical ANK/F-box proteins. Given the existence of cellular proteins with similar architecture, viral ANK/BC proteins
may be closely related to the original ANK gene acquired by an ancestral orthopoxvirus. These findings uncover a
novel viral strategy to antagonise innate immunity and shed light on the origin of the poxviral ANK protein family.
Vaccinia virus (VACV) morphogenesis is controlled by a temporal cascade enacted by intermediate and late promoter sequences, in conjunction with virally encoded transcription factors. VACV morphogenesis culminates with the formation of two structurally and functionally distinct virion morphotypes from a single mature virus (MV) progenitor; the intracellular mature virus (IMV) and extracellular enveloped virus (EEV). IMV are typified by their single membrane and the presence of the A26 protein, which is expressed at 10 hours post infection. EEVs are characterized by their double membrane and suite of virally encoded proteins, the most crucial of which is the F13 protein expressed at 4 hpi.
Previous investigations have implicated the temporal expression of A26 protein as a regulatory switch, negatively regulating the formation of EEV during late times of infection. However, reverse genetic approaches have refuted this claim. Despite this, these studies do not consider the temporal organisation of key IMV and EEV proteins, in addition to their potential to regulate one another.The aim of this study was to investigate the concept of altering the temporal regulation of both A26 and F13 to generate novel phenotypes, exploring the mechanism underpinning IMV vs EEV balance. Recombinant viruses were generated with F13L and A26L alleles expressed under the control of either F13L or A26L promoter sequences.
When A26L was introduced under the intermediate F13L promoter sequence a significant plaque size reduction was observed.In addition, a further deduction was observed when F13 expression was delayed under the late A26 promoter. Temporally advanced A26 expression significantly altered its rate of association with the key MV membrane protein A27, which is required for EEV formation. When A26 was expressed under the intermediate F13L promoter, incorporation of A26 on virions was significantly enhanced when compared to A26 expressed under its native promoter sequence.This was correlated with a small reduction in EEV formation.
The data presented in this thesis reveals the A26 protein as a potential negative regulator of EEV formation.The temporal segregation of A26 from the EEV morphogenesis proteins; F13 and A27 underpins the transition from EEV to IMV production. In addition, this thesis introduces the concept of altering temporal regulation to explore the constraints of the poxviral genome and its ability to acquire variation. These findings will assist in the refinement of sequencing algorithms used to characterise novel pathogen populations.