Abstract
BackgroundAdenoviruses are common pathogens infecting animals and humans. They are classified based on serology, or genome sequence information. These methods have limitations due to lengthy procedures or lack of infectivity data. Adenoviruses are easy to produce and amenable to genetic and biochemical modifications, which makes them a powerful tool for biological studies, and clinical gene-delivery and vaccine applications. Antibodies directed against adenoviral proteins are important diagnostic tools for virus identification in vivo and in vitro, and are used to elucidate infection mechanisms, often in combination with genomic sequencing and type specific information from hyper-variable regions of structural proteins.ResultsHere we describe a novel and readily useable method for cloning, expressing and purifying small fragments of hyper-variable regions 1-6 of the adenoviral hexon protein. We used these polypeptides as antigens for generating polyclonal rabbit antibodies against human adenovirus 3 (HAdV-B3), mouse adenovirus 1 (MAdV-1) and MAdV-2 hexon. In Western immunoblots with lysates from cells infected from thirteen human and three mouse viruses, these antibodies bound to homologous full-length hexon protein and revealed variable levels of cross-reactivity to heterologous hexons. Results from immuno-fluorescence and electron microscopy studies indicated that HAdV-B3 and MAdV-2 hexon antibodies recognized native forms of hexon.ConclusionsThe procedure described here can in principle be applied to any adenovirus for which genome sequence information is available. It provides a basis for generating novel type-specific tools in diagnostics and research, and extends beyond the commonly used anti-viral antibodies raised against purified viruses or subviral components.
Highlights
Adenoviruses are common pathogens infecting animals and humans
Human adenoviruses (HAdV) are comprised of ~60 types classified into seven species, A to G based on serology and DNA genome sequence [2, 3]
Since in our hands, purified Mouse adenoviruses (MAdV) yields were lower compared to HAdVs and purification of MAdV hexon protein from lysates of infected cells was not efficient, we decided to produce recombinant hexon fragments containing Hyper-variable regions (HVRs) to induce specific antibodies
Summary
Adenoviruses are common pathogens infecting animals and humans. They are classified based on serology, or genome sequence information. Adenoviruses (AdV) are ubiquitous pathogens, and affect vertebrates, including humans, livestock and wild animals. They undergo genetic recombination and periodically emerge in the human population, often in geographically distinct patterns [1]. Human adenoviruses (HAdV) are comprised of ~60 types classified into seven species, A to G based on serology and DNA genome sequence [2, 3]. The genetic information of adenoviruses is encoded in a double-stranded linear DNA molecule. The high seroprevalence and immunogenicity of adenoviruses represents a considerable drawback when using these viruses as vectors for gene therapy, as pre-existing immunity leads to virus immune-complexes which can lead to inflammatory reactions [13] and reduced treatment efficacy
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