An optimised protocol for the expression and purification of adenovirus core protein VII.

Adenovirus protein VII (pVII) is a highly basic core protein, bearing resemblance to mammalian histones. Despite its diverse functions, a comprehensive understanding of its structural intricacies and the mechanisms underlying its functions remain elusive, primarily due to the complexity of producing a good amount of soluble pVII. This study aimed to optimise the expression and purification of recombinant pVII from four different adenoviruses with a simple vector construct. This study successfully determined the optimal conditions for efficiently purifying pVII across four adenovirus species, revealing the differential preference for bacterial expression systems. The One Shot BL21 Star (DE3) proved favourable over Rosetta 2 (DE3) pLysS with consistent levels of expression between IPTG-induced and auto-induction. We demonstrated that combining chemical and mechanical cell lysis is possible and highly effective. Other noteworthy benefits were observed in using RNase during sample processing. The addition of RNase has significantly improved the quality and quantity of the purified protein as confirmed by chromatographic and western blot analyses. These findings established a solid groundwork for pVII purification methodologies and carry the significant potential to assist in unveiling the core structure of pVII, its arrangement within the core, DNA condensation intricacies, and potential pathways for nuclear transport.

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways.

Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120-128 (120PGGFKRRRL128). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMPα2 and IMPα3 but not IMPß. Structural analysis of the pVII-NLS/IMPα2 complex confirmed a classical interaction, with the major binding site of IMPα occupied by K124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMPα/ß-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMPα/ß nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.

Characterisation of a novel aviadenovirus associated with disease in tawny frogmouths (Podargus strigoides).

Aviadenoviruses are widespread in wild birds but rarely cause disease in nature. However, when naïve species are exposed to poultry or aviaries, aviadenoviruses can lead to disease outbreaks. This study characterised a novel aviadenovirus infection in a native Australian bird, the tawny frogmouth (Podargus strigoides) during an outbreak investigation. The identified complete genome of aviadenovirus, named tawny frogmouth aviadenovirus A (TwAviAdV-A) was 41,175 bp in length containing 52 putative genes. TwAviAdV-A exhibits the common aviadenovirus genomic organisation but with a notable monophyletic subclade in the phylogeny. The TwAviAdV-A virus was hepatotrophic and the six frogmouths presented to the wildlife hospitals in South Eastern Queensland most commonly exhibited regurgitation (in four frogmouths). Three were died or euthanized, two recovered, and one showed no signs. The detection of TwAviAdV-A in frogmouths coming into care re-emphasizes the need for strict biosecurity protocols in wildlife hospitals and care facilities.

Detection of a Novel Alphaherpesvirus and Avihepadnavirus in a Plantar Papilloma from a Rainbow Lorikeet (Trichoglosis moluccanus).

Cutaneous plantar papillomas are a relatively common lesion of wild psittacine birds in Australia. Next-generation sequencing technology was used to investigate the potential aetiologic agent(s) for a plantar cutaneous papilloma in a wild rainbow lorikeet (Trichoglosis moluccanus). In the DNA from this lesion, two novel viral sequences were detected. The first was the partial sequence of a herpesvirus with the proposed name, psittacid alphaherpesvirus 6, from the Mardivirus genus of the family alphaherpesviruses. This represents the first mardivirus to be detected in a psittacine bird, the first mardivirus to be detected in a wild bird in Australia, and the second mardivirus to be found in a biopsy of an avian cutaneous papilloma. The second virus sequence was a complete sequence of a hepadnavirus, proposed as parrot hepatitis B genotype H (PHBV-H). PHBV-H is the first hepadnavirus to be detected in a wild psittacine bird in Australia. Whether other similar viruses are circulating in wild birds in Australia and whether either of these viruses play a role in the development of the plantar papilloma will require testing of biopsies from similar lesions and normal skin from other wild psittacine birds.

Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal.

Adeno-associated viruses (AAV) are important vectors for gene therapy, and accordingly, many aspects of their cell transduction pathway have been well characterized. However, the specific mechanisms that AAV virions use to enter the host nucleus remain largely unresolved. We therefore aimed to reveal the interactions between the AAV Cap protein and the nuclear transport protein importin alpha (IMPα) at an atomic resolution. Herein we expanded upon our earlier research into the Cap nuclear localization signal (NLS) of a porcine AAV isolate, by examining the influence of upstream basic regions (BRs) towards IMPα binding. Using a high-resolution crystal structure, we identified that the IMPα binding determinants of the porcine AAV Cap comprise a bipartite NLS with an N-terminal BR binding at the minor site of IMPα, and the previously identified NLS motif binding at the major site. Quantitative assays showed a vast difference in binding affinity between the previously determined monopartite NLS, and bipartite NLS described in this study. Our results provide a detailed molecular view of the interaction between AAV capsids and the nuclear import receptor, and support the findings that AAV capsids enter the nucleus by binding the nuclear import adapter IMPα using the classical nuclear localization pathway.

Adenoviruses in Avian Hosts: Recent Discoveries Shed New Light on Adenovirus Diversity and Evolution.

While adenoviruses cause infections in a wide range of vertebrates, members of the genus Atadenovirus, Siadenovirus, and Aviadenovirus predominantly infect avian hosts. Several recent studies on avian adenoviruses have encouraged us to re-visit previously proposed adenovirus evolutionary concepts. Complete genomes and partial DNA polymerase sequences of avian adenoviruses were extracted from NCBI and analysed using various software. Genomic analyses and constructed phylogenetic trees identified the atadenovirus origin from an Australian native passerine bird in contrast to the previously established reptilian origin. In addition, we demonstrated that the theories on higher AT content in atadenoviruses are no longer accurate and cannot be considered as a species demarcation criterion for the genus Atadenovirus. Phylogenetic reconstruction further emphasised the need to reconsider siadenovirus origin, and we recommend extended studies on avian adenoviruses in wild birds to provide finer evolutionary resolution.

Novel Picornavirus Detected in Wild Deer: Identification, Genomic Characterisation, and Prevalence in Australia.

The use of high-throughput sequencing has facilitated virus discovery in wild animals and helped determine their potential threat to humans and other animals. We report the complete genome sequence of a novel picornavirus identified by next-generation sequencing in faeces from Australian fallow deer. Genomic analysis revealed that this virus possesses a typical picornavirus-like genomic organisation of 7554 nt with a single open reading frame (ORF) encoding a polyprotein of 2225 amino acids. Based on the amino acid identity comparison and phylogenetic analysis of the P1, 2C, 3CD, and VP1 regions, this novel picornavirus was closely related to but distinct from known bopiviruses detected to date. This finding suggests that deer/bopivirus could belong to a novel species within the genus Bopivirus, tentatively designated as "Bopivirus C". Epidemiological investigation of 91 deer (71 fallow, 14 sambar and 6 red deer) and 23 cattle faecal samples showed that six fallow deer and one red deer (overall prevalence 7.7%, 95% confidence interval [CI] 3.8-15.0%) tested positive, but deer/bopivirus was undetectable in sambar deer and cattle. In addition, phylogenetic and sequence analyses indicate that the same genotype is circulating in south-eastern Australia. To our knowledge, this study reports for the first time a deer-origin bopivirus and the presence of a member of genus Bopivirus in Australia. Further epidemiological and molecular studies are needed to investigate the geographic distribution and pathogenic potential of this novel Bopivirus species in other domestic and wild animal species.

Metagenomic detection and characterisation of multiple viruses in apparently healthy Australian Neophema birds.

Emerging viral pathogens are a significant concern, with potential consequences for human, animal and environmental health. Over the past several decades, many novel viruses have been found in animals, including birds, and often pose a significant threat to vulnerable species. However, despite enormous interest in virus research, little is known about virus communities (viromes) in Australian Neophema birds. Therefore, this study was designed to characterise the viromes of Neophema birds and track the evolutionary relationships of recently emerging psittacine siadenovirus F (PsSiAdV-F) circulating in the critically endangered, orange-bellied parrot (OBP, Neophema chrysogaster), using a viral metagenomic approach. This study identified 16 viruses belonging to the families Adenoviridae, Circoviridae, Endornaviridae, Picobirnaviridae and Picornaviridae. In addition, this study demonstrated a potential evolutionary relationship of a PsSiAdV-F sequenced previously from the critically endangered OBP. Strikingly, five adenoviral contigs identified in this study show the highest identities with human adenovirus 2 and human mastadenovirus C. This highlights an important and unexpected aspects of the avian virome and warrants further studies dedicated to this subject. Finally, the findings of this study emphasise the importance of testing birds used for trade or in experimental settings for potential pathogens to prevent the spread of infections.

Genomic Characterisation of a Highly Divergent Siadenovirus (Psittacine Siadenovirus F) from the Critically Endangered Orange-Bellied Parrot (Neophema chrysogaster).

Siadenoviruses have been detected in wild and captive birds worldwide. Only nine siadenoviruses have been fully sequenced; however, partial sequences for 30 others, many of these from wild Australian birds, are also described. Some siadenoviruses, e.g., the turkey siadenovirus A, can cause disease; however, most cause subclinical infections. An example of a siadenovirus causing predominately subclinical infections is psittacine siadenovirus 2, proposed name psittacine siadenovirus F (PsSiAdV-F), which is enzootic in the captive breeding population of the critically endangered orange-bellied parrot (OBP, Neophema chrysogaster). Here, we have fully characterised PsSiAdV-F from an OBP. The PsSiAdV-F genome is 25,392 bp in length and contained 25 putative genes. The genome architecture of PsSiAdV-F exhibited characteristics similar to members within the genus Siadenovirus; however, the novel PsSiAdV-F genome was highly divergent, showing highest and lowest sequence similarity to skua siadenovirus A (57.1%) and psittacine siadenovirus D (31.1%), respectively. Subsequent phylogenetic analyses of the novel PsSiAdV-F genome positioned the virus into a phylogenetically distinct sub-clade with all other siadenoviruses and did not show any obvious close evolutionary relationship. Importantly, the resulted tress continually demonstrated that novel PsSiAdV-F evolved prior to all known members except the frog siadenovirus A in the evolution and possibly the ancestor of the avian siadenoviruses. To date, PsSiAdV-F has not been detected in wild parrots, so further studies screening PsSiAdV-F in wild Australian parrots and generating whole genome sequences of siadenoviruses of Australian native passerine species is recommended to fill the siadenovirus evolutionary gaps.

Molecular Characterisation of a Novel and Highly Divergent Passerine Adenovirus 1.

Wild birds harbour a large number of adenoviruses that remain uncharacterised with respect to their genomic organisation, diversity, and evolution within complex ecosystems. Here, we present the first complete genome sequence of an atadenovirus from a passerine bird that is tentatively named Passerine adenovirus 1 (PaAdV-1). The PaAdV-1 genome is 39,664 bp in length, which was the longest atadenovirus to be sequenced, to the best of our knowledge, and contained 42 putative genes. Its genome organisation was characteristic of the members of genus Atadenovirus; however, the novel PaAdV-1 genome was highly divergent and showed the highest sequence similarity with psittacine adenovirus-3 (55.58%). Importantly, PaAdV-1 complete genome was deemed to contain 17 predicted novel genes that were not present in any other adenoviruses sequenced to date, with several of these predicted novel genes encoding proteins that harbour transmembrane helices. Subsequent analysis of the novel PaAdV-1 genome positioned phylogenetically to a distinct sub-clade with all others sequenced atadenoviruses and did not show any obvious close evolutionary relationship. This study concluded that the PaAdV-1 complete genome described here is not closely related to any other adenovirus isolated from avian or other natural host species and that it should be considered a separate species.

Disease surveillance in wild Victorian cacatuids reveals co-infection with multiple agents and detection of novel avian viruses.

Wild birds are known reservoirs of bacterial and viral pathogens, some of which have zoonotic potential. This poses a risk to both avian and human health, since spillover into domestic bird populations may occur. In Victoria, wild-caught cockatoos trapped under licence routinely enter commercial trade. The circovirus Beak and Feather Disease Virus (BFDV), herpesviruses, adenoviruses and Chlamydia psittaci have been identified as significant pathogens of parrots globally, with impacts on both aviculture and the conservation efforts of endangered species. In this study, we describe the results of surveillance for psittacid herpesviruses (PsHVs), psittacine adenovirus (PsAdV), BFDV and C. psittaci in wild cacatuids in Victoria, Australia. Samples were collected from 55 birds of four species, and tested using genus or family-wide polymerase chain reaction methods coupled with sequencing and phylogenetic analyses for detection and identification of known and novel pathogens. There were no clinically observed signs of illness in most of the live birds in this study (96.3%; n = 53). Beak and Feather Disease Virus was detected with a prevalence of 69.6% (95% CI 55.2-80.9). Low prevalences of PsHV (1.81%; 95% CI 0.3-9.6), PsAdV (1.81%; 95% CI 0.3-9.6), and C. psittaci (1.81%; 95% CI 0.3-9.6) was detected. Importantly, a novel avian alphaherpesvirus and a novel avian adenovirus were detected in a little corella (Cacatua sanguinea) co-infected with BFDV and C. psittaci. The presence of multiple potential pathogens detected in a single bird presents an example of the ease with which such infectious agents may enter the pet trade and how novel viruses circulating in wild populations have the potential for transmission into captive birds. Genomic identification of previously undescribed avian viruses is important to further our understanding of their epidemiology, facilitating management of biosecurity aspects of the domestic and international bird trade, and conservation efforts of vulnerable species.

A novel pathogenic aviadenovirus from red-bellied parrots (Poicephalus rufiventris) unveils deep recombination events among avian host lineages.

Competing roles of coevolution, selective pressure and recombination are an emerging interest in virus evolution. We report a novel aviadenovirus from captive red-bellied parrots (Poicephalus rufiventris) that uncovers evidence of deep recombination among aviadenoviruses. The sequence identity of the virus was most closely related to Turkey adenovirus D (42% similarity) and other adenoviruses in chickens, turkeys and pigeons. Sequencing and comparative analysis showed that the genome comprised 40,930 nucleotides containing 42 predicted open reading frames (ORFs) 19 of which had strong similarity with genes from other adenovirus species. The new genome unveiled a lineage that likely participated in deep recombination events across the genus Aviadenovirus accounting for an ancient evolutionary relationship. We hypothesize frequent host switch events and recombination among adenovirus progenitors in Galloanserae hosts caused the radiation of extant aviadenoviruses and the newly assembled Poicephalus adenovirus genome points to a potentially broader host range of these viruses among birds.