A pathogenitásért és az állati sejtek manipulálásáért felelős, új adenovírus fehérjék keresése = Search for novel adenovirus proteins responsible for pathogenicity and the manipulation of animal cells

Az adenovírusok sejtreceptorokhoz történő kapcsolódásában részt vevő kapszidfehérjék (a pentonbázis és fiberfej) határozzák meg a vírus szöveti és sejt-specificitását. A sejtek manipulálásáért és az adenovírusok kórokozó képességéért azonban alapvetően a korai kifejeződésű gének által kódolt fehérjék felelősek. Eddig ismeretlen adenovírusokat találva főleg hüllőkben, vadmadarakban, rágcsálókban, denevérekben és majmokban, valamint vírusgenom szekvenálást és elemzést végezve (pl. teljes hal-, gyík-, liba-, egér-, denevér-, majom- és humán adenovírus genomok) számos feltehetően korai, új gén létezését sikerült kimutatni. Vizsgáltuk az ezek által kódolt fehérjék evolúcióját, sokféleségét, változékonyságát, természetes mutáció okozta rövidülését, delécióját vagy duplikációját, a sejtreceptor-kötő helyek meglétét vagy hiányát. A hal-adenovírus feltételezett szulfotranszferáz homológjának génjét és több állati adenovírusból a fiberfejet kódoló génszakaszt bakteriális kifejező rendszerbe klónoztuk és teszteltük kifejeződésüket. Következtetéseket vontunk le a vizsgált gének funkciójára, esetleges nélkülözhetőségükre, valamint az egyes vírusok között megfigyelhető pathogenitási és más biológiai különbségek okára vonatkozóan. | The capsid proteins (penton base and the fiber knob), which take part in the attachment to the cellular receptors, play crucial role in the organ and tissue tropism of adenoviruses. Nonetheless, the proteins coded by the early genes are primarily responsible for the manipulation of cells and for the pathogenicity of these viruses. By finding novel adenoviruses mainly in reptiles, wild birds, rodents, bats and monkeys, and by sequencing and analysing adenovirus genomes (e.g., the full genome of the sturgeon, lizard, goose, mouse, bat, monkey and human adenoviruses), the existence of numerous such supposedly early genes was revealed. We studied the evolution, diversity, variability, truncation caused by natural mutation, deletion, duplication of these proteins as well as the presence or lack of cellular receptor binding sites. The gene of a sulfotransferase homologue of sturgeon adenovirus and gene fragments coding the fiber knobs of several animal adenoviruses were cloned and expressed in bacterial expression systems. We drew several conclusions concerning the function, possible non-essential status of these genes, and the reason for the observed differences in the pathogenicity and other biological properties of the adenoviruses examined

Random sampling of the Central European bat fauna reveals the existence of numerous hitherto unknown adenoviruses⁺

From over 1250 extant species of the order Chiroptera, 25 and 28 are known to occur in Germany and Hungary, respectively. Close to 350 samples originating from 28 bat species (17 from Germany, 27 from Hungary) were screened for the presence of adenoviruses (AdVs) using a nested PCR that targets the DNA polymerase gene of AdVs. An additional PCR was designed and applied to amplify a fragment from the gene encoding the IVa2 protein of mastadenoviruses. All German samples originated from organs of bats found moribund or dead. The Hungarian samples were excrements collected from colonies of known bat species, throat or rectal swab samples, taken from live individuals that had been captured for faunistic surveys and migration studies, as well as internal organs of dead specimens. Overall, 51 samples (14.73%) were found positive. We detected 28 seemingly novel and six previously described bat AdVs by sequencing the PCR products. The positivity rate was the highest among the guano samples of bat colonies. In phylogeny reconstructions, the AdVs detected in bats clustered roughly, but not perfectly, according to the hosts’ families (Vespertilionidae, Rhinolophidae, Hipposideridae, Phyllostomidae and Pteropodidae). In a few cases, identical sequences were derived from animals of closely related species. On the other hand, some bat species proved to harbour more than one type of AdV. The high prevalence of infection and the large number of chiropteran species worldwide make us hypothesise that hundreds of different yet unknown AdV types might circulate in bats

Identification of two novel adenoviruses in smooth-billed ani and tropical screech owl

Avian adenoviruses (AdVs) are a very diverse group of pathogens causing diseases in poultry and wild birds. Wild birds, endangered by habitat loss and habitat fragmentation in the tropical forests, are recognised to play a role in the transmission of various AdVs. In this study, two novel, hitherto unknown AdVs were described from faecal samples of smooth-billed ani and tropical screech owl. The former was classified into genus Aviadenovirus, the latter into genus Atadenovirus, and both viruses most probably represent new AdV species as well. These results show that there is very limited information about the biodiversity of AdVs in tropical wild birds, though viruses might have a major effect on the population of their hosts or endanger even domesticated animals. Surveys like this provide new insights into the diversity, evolution, host variety, and distribution of avian AdVs

Structure and N-acetylglucosamine binding of the distal domain of mouse adenovirus 2 fibre

15 pags, 8 figsMurine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple ß-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutagenesis, we show that the fibre specifically binds to the monosaccharide N-acetylglucosamine (GlcNAc). The crystal structure of the complex reveals that GlcNAc binds between the AB and CD loops at the top of each of the three monomers of the MAdV-2 fibre head. However, infection competition assays show that soluble GlcNAc monosaccharide and natural GlcNAc-containing polymers do not inhibit infection by MAdV-2. Furthermore, site-directed mutation of the GlcNAc-binding residues does not prevent the inhibition of infection by soluble fibre protein. On the other hand, we show that the MAdV-2 fibre protein binds GlcNAc-containing mucin glycans, which suggests that the MAdV-2 fibre protein may play a role in viral mucin penetration in the mouse gut.This research was sponsored by grant BFU2014-53425-P (to M. J. v. R.), coordinated grants CTQ2015-64597-P-C02-01 and CTQ2015-64597-P-C02-02 (to J. J. B. and F. J. C., respectively), grant BFU2015-70052-R (to M. M.) and the Spanish Adenovirus Network (AdenoNet, BIO2015-68990-REDT), all from the Spanish Agencia Estatal de Investigación. Financial support to M. M. from the CIBER of Respiratory Diseases (CIBERES) from the Spanish Institute of Health Carlos III is also acknowledged. These grants are co-financed by the European Regional Development Fund of the European Union. A. K. S. and T. H. N. were recipients of pre-doctoral fellowships from La Caixa and CSIC-VAST, respectively. The expression vectors were designed and created in Hungary, and this was financed by the Hungarian Scientific Research Fund (OTKA K100163). M. K. thanks Enterprise Ireland for a Commercialisation Fund grant (CF/2015/0089), A. K. acknowledges the National University of Ireland for a Cancer Care West Hardiman PhD scholarship and L. J. acknowledges the EU FP7 programme in support of the GlycoHIT consortium (grant no. 260600). This work was supported by R01 AI104920 (to J. G. S.) from the National Institute for Allergy and Infectious Diseases (www.niaid.nih.gov). S. S. W. was also supported by the Helen Riaboff Whiteley Endowment to the University of Washington and by Public Health Service, National Research Service Awards T32 AI083203 from the National Institute for Allergy and Infectious Diseases and T32 GM007270 from the National Institute of General Medical Sciences

Rágcsálók és denevérek adenovírusainak genetikai elemzése

Comprehensive PCR screenings were made to estimate the diversity of adenoviruses (AdVs) in rodents and bats. Besides the domestic samples, we tested dead bats and squirrels originating from Germany. We detected and partially characterized previously unknown AdVs in the individuals of two local rodent species, the striped field mouse (Apodemus agrarius) and the common vole (Microtus arvalis). A third novel AdV was detected in the sample of a zoo animal, namely a capybara (Hydrochoerus hydrochaeris). In the Hungarian population of the striped field mouse, we demonstrated the presence of murine adenovirus 3 (MAdV-3), which has been described in Slovakia recently. The presence of the squirrel AdV-1 (SqAdV-1), a virus that had been detected only in Great Britain previously, was proven by us in Germany during the examination of dead captive squirrels. The sequence of a 20,602-bp-long genome fragment of the SqAdV-1, encompassing 17 genes, was determined. By screening a collection of more than 150 samples, representing every bat species that is known to occur in Hungary, we revealed the presence of 13 novel AdVs. We also tested a sample collection from Germany with about 200 samples representing 17 bat species. Eighteen novel AdVs were detected in these samples. Additionally, we demonstrated the presence of 3 AdVs, which have already been reported in other countries previously. I participated in the determination and analysis of the full genomic sequence of two AdVs. In a Swiss collaboration, we studied the MAdV-2, isolated from house mouse. The length of its genome (35,203 bp) was found to be surprisingly long, especially compared to the other two MAdVs. The substantial size difference could primarily be attributed to the longer length rather than the greater number of the genes. The phylogeny reconstructions also confirmed that the MAdV-2 is more different from the other two MAdVs than those from each other. Thus the introduction of a new viral species (Murine mastadenovirus B) was justified. The other AdV was the bat adenovirus 2 (BtAdV-2) isolated from a common pipistrelle (Pipistrellus pipistrellus) in Germany. In collaboration with the German colleagues, we sequenced and annotated the entire genome of BtAdV-2. The phylogenetic analyses also supported our conclusion that this virus represents a new species (Bat mastadenovirus B). Our taxonomic proposals were approved by the International Committee on Taxonomy for Viruses (ICTV). I proved that in the MAdV-3, circulating among the Hungarian striped field mice, a full variant of the E1B 19K gene is present. This gene has a length (174 amino acid) almost identical to that of its counterparts in other AdVs, while the Slovakian prototype MAdV-3 strain, deposited to the GenBank contains a truncated version of the E1B 19K. Due to a mutation, a stop codon is present after the 23rd aa. However, the rest of the aa sequence is also homologous with the corresponding part of the 19K proteins of other AdVs

Genome analysis of bat adenovirus 2: Indications of interspecies transmission

The genome of bat adenovirus 2 was sequenced and analyzed. It is similar in size (31,616 bp) to the genomes of bat adenovirus 3 and canine adenoviruses 1 and 2. These four viruses are monophyletic and share an identical genome organization, with one E3 gene and four E4 genes unique to this group among the mastadenoviruses. These findings suggest that canine adenoviruses may have originated by interspecies transfer of a vespertilionid bat adenovirus

Crystal structure of the fibre head domain of bovine adenovirus 4, a ruminant atadenovirus

[Background] In adenoviruses, primary host cell recognition is generally performed by the head domains of their homo-trimeric fibre proteins. This first interaction is reversible. A secondary, irreversible interaction subsequently takes place via other adenovirus capsid proteins and leads to a productive infection. Although many fibre head structures are known for human mastadenoviruses, not many animal adenovirus fibre head structures have been determined, especially not from those belonging to adenovirus genera other than Mastadenovirus.[Methods] We constructed an expression vector for the fibre head domain from a ruminant atadenovirus, bovine adenovirus 4 (BAdV-4), consisting of amino acids 414–535, expressed the protein in Escherichia coli, purified it by metal affinity and cation exchange chromatography and crystallized it. The structure was solved using single isomorphous replacement plus anomalous dispersion of a mercury derivative and refined against native data that extended to 1.2 Å resolution.[Results] Like in other adenoviruses, the BAdV-4 fibre head monomer contains a beta-sandwich consisting of ABCJ and GHID sheets. The topology is identical to the fibre head of the other studied atadenovirus, snake adenovirus 1 (SnAdV-1), including the alpha-helix in the DG-loop, despite of them having a sequence identity of only 15 %. There are also differences which may have implications for ligand binding. Beta-strands G and H are longer and differences in several surface-loops and surface charge are observed.[Conclusions] Chimeric adenovirus fibres have been used to retarget adenovirus-based anti-cancer and gene therapy vectors. Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for such a vector. Here, we present the high-resolution atomic structure of the BAdV-4 fibre head domain, the second atadenovirus fibre head structure known and the first of an atadenovirus that infects a mammalian host. Future research should focus on the receptor-binding properties of these fibre head domains.The research leading to these results was sponsored by grant BFU2011-24843 (to MJvR, THN, MSG and AKS) from the Spanish Ministry of Economy and Competitiveness, a VAST-CSIC PhD fellowship to THN, a FEMS short-term Research Fellowship award to MZB, a RISAM fellowship to MSG, a La Caixa fellowship to AKS, and by grant OTKA NN107632 from the Hungarian Scientific Research Fund (to BH)