Biology subjects

Parry, R. H.

Publications and source records attributed to Parry, R. H..

10 recordsLinked to original sources

bioRxiv2026

Expanding Reverse Genetics of Positive-Strand RNA Viruses: Optimised Rescue Platforms and Construction of a Novel Fluorescent Reporter Nidovirus

Reverse genetics systems are crucial for facilitating the precise manipulation of viruses across a wide spectrum of translational and fundamental research pipelines. Here, we compared Circular polymerase extension reaction (CPER), Gibson assembly, and infectious subgenomic amplicons (ISA) for bacteria-free recovery of a positive sense RNA virus. Through optimisation of CPER, we demonstrated accelerated virus recovery and enhanced viral yields. We further investigated strategies to improve rescue efficiency across diverse positive-sense RNA virus families through incorporation of alternative promoters and non-coding elements. To evaluate the performance of the Aedes aegypti polyubiquitin promoter (AePUb) in tandem with a hammerhead ribozyme (HH Rbz) and a polymerase pause site for virus recovery in insect cells, we constructed a new fluorescent reporter genome using a 20 kb insect-specific mesonivirus. In vitro recovery by CPER of the mesonivirus was achievable in 1 day when using AePUb with HH Rbz, in comparison to a four-day recovery when using the minimal OpIE2-CA promoter. These elements were additionally assessed for rescue of the orthoflaviviruses, Binjari virus (BinJV) and dengue virus 2 (DENV-2), in insect cells (using AePUb); or in mammalian cells (using the CMV promoter) and for launch of DENV2 and SARS-CoV-2. Both BinJV and DENV-2 demonstrated improved rescue with the AePUb promoter and HH Rbz. However, the addition of the HH Rbz and the polymerase pause site to the CMV linker fragment showed no significant differences to the standard CMV promoter systems for both DENV-2 and SARS-CoV-2, highlighting the context-specific benefits of their implementation. In summary, we demonstrated that a potent constitutive promoter system and a hammerhead ribozyme significantly enhance the efficiency of positive-sense RNA virus rescue using CPER.

molecular biology↗

bioRxiv2026

Mapping the global distribution and spread of the Plasmodium vivax-associated virus MaRNAV-1

Matryoshka RNA virus 1 is a bi-segmented and single-stranded RNA virus associated with Plasmodium vivax, a cause of human malaria. Little has been uncovered about the epidemiology and ecology of this virus since its discovery in 2019. To address this, we used a combination of primary and publicly available metatranscriptomic data to map the geographic distribution and host associations of MaRNAV-1. We detected this virus throughout Southeast Asia, in parts of South America, and, for the first time, in Oceania. Despite its broad distribution, MaRNAV-1 was found exclusively in metatranscriptomes containing P. vivax, suggesting that there is a specific virus-host relationship that has shaped the evolutionary history of this virus. We were unable to estimate the emergence date of the MaRNAV-1 lineage; however, phylogeographic mapping analysis suggested that MaRNAV-1 may have radiated from Southeast Asia. Our findings have both evolutionary and public health implications and can serve as the basis for future investigations in these fields.

evolutionary biology↗

bioRxiv2025

Newly identified genomic sequences establish benchmarks for proposed taxonomic classification of jingmenviruses

Jingmenviruses are a group of viruses related to orthoflaviviruses characterized by a segmented genome and multipartite organisation that have been detected worldwide in a wide range of hosts. As next-generation sequencing has become more affordable, increasing numbers of large-scale metagenomics studies have been published, alongside raw sequencing data. With the growing number of new jingmenvirus sequences identified in metagenomics data, it can be difficult to assess whether a new sequence is associated to a new virus species or to a strain of an existing species. In order to propose clear classification criteria for this group, we assembled a large jingmenvirus sequence database, from published and newly assembled sequences. Indeed, we screened data from studies that did not search for or report jingmenvirus sequences, looking for new strains of known jingmenvirus species. We then performed multiple sequence alignments and used the inferred percentage identity values to determine demarcation criteria based on the distribution of evolutionary distances upon pairwise comparisons. We report the identification of almost 60 libraries containing jingmenvirus sequences, in a wide range of sample types and geographical locations. Using these data and published jingmenvirus sequences, we have determined that to classify jingmenvirus sequences into virus species, at least four segments are required, on which eight cut-off values in percentage identity (nucleotide and amino acid) are used for demarcation. The ratification of this proposal would enhance consistency in virus taxonomy and provide a standardized framework for comparative genomics studies of jingmenviruses, a group that is yet under-characterised. ImportanceThere are currently no guidelines to classify jingmenvirus sequences which means sequences are associated to species with no ratified rationale, which can result in missed opportunities at better describing jingmenvirus genomics. In this study we propose criteria to provide a parsimonious framework for classifying jingmenvirus species based on current knowledge, and allow us to propose re-classification of some sequences and to identify outlier sequences which we recommend should be further characterised in vitro.

genomics↗

bioRxiv2025

Global transcriptional reprogramming by cytomegalovirus infection suppresses antigen presentation while enhancing migration machinery in murine dendritic cells.

Dendritic cells (DCs) serve dual roles in cytomegalovirus infection: orchestrating antiviral immunity and acting as vehicles for viral dissemination. DC-dependent systemic spread of mouse cytomegalovirus (MCMV) is dependent on expression of a viral G protein-coupled receptor (GPCR) homolog, encoded by M33. We performed global transcriptional profiling of murine DCs infected with either wild-type mouse MCMV or a M33 mutant harbouring a single point mutation (R131Q; denoted M33NQY), which ablates constitutive G protein-dependent signalling. RNA-seq analysis revealed that MCMV induces substantial transcriptional reprogramming, with over 2,400 significantly altered genes affecting key immune and migration pathways. Wild-type MCMV infection resulted in 1,883 upregulated and 658 downregulated genes, while M33NQY infection showed comparable effects with 1,905 upregulated and 668 downregulated genes. Both viruses systematically downregulated the MHC class II antigen presentation machinery, with substantial suppression of H2 molecules, peptide-loading components (H2-DMa/H2-DMb1), and the class II transactivator Ciita (log2FC > -6.4). Pathway analysis revealed coordinated disruption of B cell receptor signalling, leukocyte transendothelial migration, and antigen processing and presentation. A comparison between wild-type and M33NQY-infected DCs demonstrated that while both viruses similarly impair antigen presentation, M33 signalling specifically enhances the expression of genes involved in transendothelial migration, including Spp1 (osteopontin, log2FC = 0.65), cytoskeletal components (Actg1, Actb), adhesion molecules (Icam1), and matrix interaction factors (Tnc, Plau). Network analysis identified critical hub genes, including B2m, Itgb1, Itgam, and Icam1, as key regulatory nodes connecting antigen presentation and migration pathways. This provides molecular evidence for a sophisticated viral strategy that shields MCMV from immune detection while hijacking DC migratory machinery to facilitate dissemination, with M33 serving as a specific enhancer of migration-related pathways. Impact StatementThis study provides the first comprehensive transcriptional profile of mouse cytomegalovirus (MCMV) infection in dendritic cells, revealing a sophisticated viral strategy that simultaneously suppresses antigen presentation while enhancing migration machinery. We identified that MCMV systematically downregulates the entire MHC class II pathway by targeting the master regulator Ciita, while the viral G protein-coupled receptor M33 specifically enhances genes involved in transendothelial migration, including Spp1 (osteopontin). Our network analysis identified critical hub genes connecting immune evasion and migration pathways, providing molecular evidence for how MCMV exploits dendritic cells as vehicles for dissemination while evading immune detection. These findings advance our understanding of betaherpesvirus pathogenesis and highlight potential therapeutic targets to limit viral spread.

microbiology↗

bioRxiv2025

The fluid genomic organisation of jingmenviruses

Jingmenviruses are a distinct group of flavi-like viruses characterized by a genome consisting of four to five segments. Here, we report the discovery of three novel putative jingmenviruses, identified by mining publicly available metagenomics data from mosquito and arachnid samples. Strikingly, these novel jingmenvirus sequences contain up to six genomic segments, with pairs of homologous segments coding for putative structural proteins. Following this discovery, we found an additional homologous segment for two other jingmenvirus genomes, which had gone unnoticed in the initial publications. The presence of a single version of the segments coding for non-structural proteins suggests we have indeed identified jingmenviruses with infectious units that contain up to six segments. We compared these novel jingmenvirus sequences to published sequences, in particular the segments with multiple open reading frames, and we propose that the putative translation initiation mechanisms involved for these segments are ribosomal frameshift resulting in the fusion of open reading frames and leaky scanning for overlapping ORFs. These putative mechanisms, conserved for all jingmenvirus sequences analysed, including in homologous segments, require biological confirmation. We also generated structural models of two putative structural proteins in the duplicated segments, and the corresponding alignments enabled us to confirm or identify the homologous relationship between sequences that shared limited nucleotide or amino acid identity. Altogether, these results highlight the fluid nature of jingmenviruses, which is a hallmark of multipartite viruses. Different combinations of segments packaged in different virus particles could facilitate the acquisition or loss of genomic segments and a segment duplication following genomic drift. Our data therefore contribute to the evidence of the multipartite nature of jingmenviruses and the evolutionary role this organisation may play.

bioinformatics↗

bioRxiv2024

Vaccine-induced and hybrid immunity against SARS-CoV-2 variants of concern in two cohorts in Queensland, Australia (2021-2022).

The spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for vaccine development, as antibodies generated against the spike protein are the most immunodominant and neutralizing against the virus. However, variants of concern (VOC), often containing multiple mutations within neutralizing epitopes, confer immune evasion of the response generated by current SARS-CoV-2 vaccines. To assess the immunogenicity and virus-neutralisation ability of antibodies induced by individual or combination of Vaxzevria (AstraZeneca: AZ) and Comirnaty (Pfizer: PZ) vaccines and of a hybrid immunity induced in people who were also infected with SARS-CoV-2, longitudinal sera samples of participants recruited through the David Serisier Research biobank (Mater Research Hospital) or at the University of Queensland were collected. ELISA with a panel of purified Spike proteins from ancestral, alpha, delta and omicron BA.1 and BA.2 VOCs showed significantly (by [~]2 to 6-fold) reduced IgG antibody titres against Spike proteins from Omicron BA.1 and BA.2 compared to ancestral strain regardless of the number of vaccinations or presence of infection. Neutralisation assays showed reduced activity against delta and omicron BA.1, BA.5 and BA.5 VOCs. However, the differences were in general less pronounced than in the ELISA assay and some were not statistically significant, particularly after four (two AZ and two PZ) vaccinations. We also generated by circular polymerase extension reaction an attenuated SARS-CoV-2 strain with deletion of all accessory genes, ORF 3, 6, 7 and 8, based on the ancestral (QLD02) virus backbone (QLD02{Delta}3678) and validated it in virus-neutralization assays with our panel of sera samples. We showed the attenuation of the QLD02{Delta}3678 virus in Vero E6 and human Caco2 cells. We demonstrated that neutralization assays with the wild-type QLD02 virus and QLD02{Delta}3678 virus were concordant, providing a safe platform for neutralisation assays in BSL2/PC2 settings.

microbiology↗

bioRxiv2024

The PAZ domain of Aedes aegypti Dicer 2 is critical for accurate and high-fidelity size determination of virus-derived small interfering RNAs.

The exogenous siRNA (exo-siRNA) pathway is a critical RNA interference response involved in controlling arbovirus replication in mosquito cells. It is initiated by the detection of viral long double-stranded RNA (dsRNA) by the RNase III enzyme Dicer 2 (Dcr2), which is processed into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs, or vsiRNAs that are taken up by the Argonaute 2 (Ago2) protein to target viral single-stranded RNAs. The detailed understanding of Dicer structure, function and domains owes much to studies outside the context of viral infection, and how Dcr2 domains contribute to detecting viral dsRNA to mount antiviral responses in infected mosquito cells remains much less understood. Here, we used a Dcr2 reconstitution system in Aedes aegypti derived Dcr2 KO cells to assess the contribution of the PAZ domain to induction of the exo-siRNA pathway following infection with Semliki Forest virus (SFV; Togaviridae, Alphavirus). Amino acids critical for PAZ activity were identified, and loss of PAZ function affected the production of 21 nt vsiRNAs -though not the overall ability of Dcr2 to process viral dsRNA- and silencing activity. This study establishes the importance of correct vsiRNA size in mosquito exo-siRNA antiviral responses, as well as the PAZ domains functional contribution to Dcr2 processing of viral dsRNA to 21 nt vsiRNAs.

microbiology↗

bioRxiv2024

A 39.8kb flavi-like virus uses a novel strategy for overcoming the RNA virus error threshold

It is commonly held that there is a fundamental relationship between genome size and error rate, manifest as a notional "error threshold" that sets an upper limit on genome sizes. The genome sizes of RNA viruses, which have intrinsically high mutation rates due to a lack of mechanisms for error correction, must therefore be small to avoid accumulating an excessive number of deleterious mutations that will ultimately lead to population extinction. The proposed exceptions to this evolutionary rule are RNA viruses from the order Nidovirales (such as coronaviruses) that encode an error correcting exonuclease, enabling them to reach genome lengths greater than 40kb. The recent discovery of large genome flavi-like viruses (Flaviviridae), which comprise genomes up to 27kb in length yet seemingly do not encode exonuclease domains, has led to the proposal that a proofreading mechanism is required to facilitate the expansion of RNA virus genomes above 30kb. Herein, we describe a 39.8kb flavi-like virus identified in a Haliclona sponge metatranscriptome that does not encode an exonuclease. Structural analysis revealed that this virus may have instead captured bacterial domains associated with nucleic acid metabolism that have not been previously found in RNA viruses. Phylogenetic analysis placed this virus as a divergent pesti-like lineage, such that we have provisionally termed it Maximus pesti-like virus. This virus represents the first instance of a flavi-like virus achieving a genome size comparable to that of the Nidovirales and demonstrates that RNA viruses have evolved multiple solutions to overcome the error threshold.

evolutionary biology↗

bioRxiv2023

Identification of a novel papillomavirus from a New Zealand fur seal (Arctocephalus forsteri) with oral papilloma-like lesions

Despite being the predominant seal species in the Australian-New Zealand region and serving as a key indicator of marine environmental health, little is known about infectious diseases in New Zealand fur seals (Long-nosed fur seal; Arctocephalus forsteri). Several papillomaviruses have been identified in earless seals and sea lions, with the latter linked to cutaneous plaques and invasive squamous cell carcinoma. To date, no papillomaviruses have been reported in fur seals. We used traditional veterinary diagnostic techniques and metatranscriptomic sequencing of tissue samples to investigate the virome of New Zealand fur seals. We identified a novel papillomavirus, provisionally termed Arctocephalus forsteri papillomavirus 1 (AfPV1) in an animal with clinically and histologically identified oral papilloma-like lesions. RT-PCR confirmed the presence of AfPV1 only in oral papilloma samples from the affected individual. Phylogenetic analysis of the complete 7,926 bp genome of AfPV1 revealed that it clustered with taupapillomaviruses found in related Carnivora species. In addition, we identified the partial genome of a novel Gammaherpesvirus, Arctocephalus forsteri gammaherpesvirus 1 (AfGHV1), in a different individual without pathological evidence of viral infection. These findings highlight the need for further research into the disease associations and impact of undiagnosed and novel viruses on New Zealand fur seals.

microbiology↗

bioRxiv2023

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) encodes a divergent class 1b XRN1-resistant RNA element.

Flavivirids are small, enveloped, positive-sense RNA viruses from the Flaviviridae family with genomes between [~]9-13kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. To date, however, no flavivirus-like or flavivirid has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the United Kingdom, utilising high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid [~]11.2kb in length. The virus encodes a single open reading frame of 3456 amino acids and 5 and 3 untranslated regions (UTRs) of 227 and 666nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, 95.64-97.39% pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of putative secondary structures in the 3'-UTR of RaTV indicated a potential exoribonuclease-resistant RNA (xrRNA) structure identified in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro XRN1 digestion assay showing that RaTV likely forms a divergent but functionally homologous XRN1-resistant xrRNA.

microbiology↗