Biology subjects

Hobson-Peters, J.

Publications and source records attributed to Hobson-Peters, J..

6 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

Sequence Homology and Tissue Tropism Determine Superinfection Exclusion of Zika virus in Aedes aegypti Mediated by an insect-specific Binjari-Zika Virus Chimera

Arboviruses such as dengue, Zika, and chikungunya viruses cause widespread disease and continue to expand their geographical range due to climate change and vector spread. Insect-specific flaviviruses (ISFs) are promising biocontrol candidates of arboviruses, due to recent studies showing that prior infection with an ISF can reduce arbovirus replication in mosquitoes through superinfection exclusion (SIE). However, the route of infection, tissue tropism, pathogenesis and the mechanisms underlying SIE of ISFs in mosquitoes remain unclear. RNA interference (RNAi) is a potent antiviral response in insects, therefore it is expected that sequence homology between the ISF and the arbovirus will strengthen SIE. Here, we used ISF Binjari virus and a chimera containing the Zika virus structural proteins prME (BinJ-ZIKV) as a model system. Intrathoracic injection of BinJ-ZIKV in Aedes aegypti led to rapid systemic infection that excluded the midgut, subsequently blocking ZIKV dissemination from the midgut. SIE was strongest in tissues where primary-virus replication was highest. This spatial component of SIE was stronger when there was sequence homology between the ISF and arbovirus and displayed a strong 21nt siRNA response, suggesting RNAi contributed to the observed SIE. Upon oral inoculation, BinJ-ZIKV replicated efficiently in mosquitoes, was detected across multiple tissues, and saliva. BinJ-ZIKV also had higher infection establishment than BinJV at lower oral titres. SIE was observed for BinJ-ZIKV infection after oral exposure interfered with subsequent ZIKV midgut infection. Together, these findings support engineered ISF-chimeras as valuable experimental tools to dissect viral determinants of SIE and to optimize mosquito-based arbovirus interference strategies. ImportanceAnnually, over 400 million people are infected with mosquito-transmitted viruses. Insect-specific flaviviruses (ISFs) can interfere with the transmission of clinically important viruses through a phenomenon termed superinfection exclusion (SIE). However, the mechanisms of SIE remain poorly understood. Using a Binjari virus chimera expressing Zika virus (ZIKV) structural proteins, we show that SIE is highly tissue-specific, with exclusion of ZIKV only occurring at sites where the chimera actively replicates and induces the mosquito antiviral RNA interference pathway. We further demonstrate that incorporation of Zika virus prM and E proteins into the ISF backbone enhances infection of the mosquito midgut following oral exposure, which enables direct inhibition of ZIKV infection after a subsequent infectious blood meal. Together, these findings define a replication-dependent, tissue-specific mechanism of ISF-mediated protection and provide a framework for reducing mosquito-borne virus transmission through SIE.

microbiology↗

bioRxiv2025

Insect-specific Yada Yada virus chimeric vaccines protect against chikungunya and Ross River virus-induced arthritis

Arthritogenic alphaviruses such as chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-borne viruses that can cause debilitating polyarthritis/polyarthralgia in humans. Although two CHIKV vaccines have been licensed, there are no licensed vaccines for RRV. Herein we generate a host-restricted, insect-specific alphavirus, Yada Yada virus (YYV), chimeric vaccine for CHIKV (YYV-CHIKVMauritius) and for RRV (YYV-RRVTT). YYV-CHIKVMauritius and YYV-RRVTT was able to replicate in C6/36 mosquito cells to similar titres as wild-type CHIKV and RRV. YYV-CHIKVMauritius was also neutralised by CHIKV monoclonal antibodies to the same titres as wild-type CHIKV, indicating its potential as a diagnostic antigen to detect neutralising CHIKV antibodies in human or animal sera. YYV-CHIKVMauritius further demonstrated protection against CHIKV infection and disease in a wild-type mouse model. Two doses of YYV-CHIKVMauritius showed anti-CHIKV ELISA and neutralising antibody responses, with protection against foot swelling, viraemia and viral feet tissue titres. Protection against CHIKV histopathology including myositis, tendonitis, arthritis, subcutaneous oedema and haemorrhage was also observed. YYV-RRVTT also demonstrated protection against RRV infection and disease in a wild-type mouse model, with two vaccine doses inducing anti-RRV ELISA and neutralising antibody responses. Protection against foot swelling, viraemia and viral feet tissue titres and RRV histopathology including myositis, tendonitis, arthritis and subcutaneous oedema was also observed. Cross-protection was also evaluated between YYV-CHIKVMauritius and RRV. Although cross-reactive total IgG were observed for YYV-CHIKVMauritius vaccinated mice, this offered no cross-neutralising antibodies and no protection against RRV infection and disease. Overall, our findings show that YYV-CHIKVMauritius and YYV-RRVTT are safe and efficacious vaccines against CHIKV and RRV, respectively, but do not offer cross-protection.

microbiology↗

bioRxiv2025

Characterisation and phylogenetic analysis of Murray Valley encephalitis virus (MVEV) from the Ralph Doherty collection.

Murray Valley encephalitis virus (MVEV) is a zoonotic flavivirus endemic to Australia and Papua New Guinea. A recent outbreak of MVEV has prompted renewed concerns regarding the potential for MVEV to generate disease outbreaks. Currently, nine full length sequences of MVEV are publicly available, divided into four genotypes (G1-G4). Herein, we sequenced MVEV isolates from the Ralph Doherty Virus Collection, a virus bank with Australian field isolates dating between the 1950s-1980s, and determined their phylogenetic relationship with existing isolates to provide insights into virus evolution and genetic diversity. Additionally, we characterised isolates from different genotypes both in vitro using human neuronal cells, and in vivo using C57BL/6J mice, to provide additional insight into MVEV pathogenicity and establish models of MVEV disease that recapitulate MVEV human disease. We found 15 new full length sequences of MVEV, which primarily clustered into the dominant genotype, G1. Additionally, we show MVEV can be lethal and neuroinvasive in C57BL/6J mice, recapitulating histological lesions identified in human infection. Overall, our study contributes significant genomic sequences to the current MVEV database and establishes mouse models of disease and infection which can be used for mechanistic studies and evaluation of new interventions.

microbiology↗

bioRxiv2024

Application of chimeric antigens to paper-based diagnostics for detection of West Nile virus infections of Crocodylus porosus: a novel animal test case

Laboratory-based diagnostics like plaque reduction neutralization tests (PRNT) and ELISA are commonly used to detect seroconversion to flavivirus infections. However, faster, qualitative screening methods are needed for quicker diagnosis and better patient outcomes. Lateral flow assays (LFAs) can provide rapid results (5-15 mins) at the point-of-care, yet few commercial flavivirus antibody detection LFAs are available. We developed an LFA using novel chimeric viral antigens produced by genetically modifying the mosquito restricted Binjari virus (BinJV) to display the outer virion proteins of pathogenic viruses such as West Nile virus (WNV). The BinJV chimeric platform offers various advantages for diagnostic assay development, including rapid construction of new chimeras in response to emerging viral variants, safe, scalable antigen manufacturing, and structural indistinguishability to the wild-type pathogenic virion. As a demonstration of feasibility, we applied chimeric WNV (BinJV/WNV) antigen to LFA as the capture/test line reagent for detection of seroconversion of crocodilians to WNV - a virus affecting crocodilians on multiple continents. We verified the antigenic conservation of the chimera when applied to the LFA detection surface using monoclonal antibodies. Using well-characterised sera (n=60) from WNV seropositive or flavivirus naive Australian saltwater crocodiles (Crocodylus porosus), we illustrated 100% sensitivity and specificity, with results achieved in less than 15 minutes. The LFA further accurately detected seroconversion in animals experimentally infected with WNV. This qualitative screening method can be performed both inside and outside of a laboratory, and the assay design will guide the optimization of similar tests for vector borne virus infection detection in both humans and other animals.

biochemistry↗

bioRxiv2021

Structural analysis of 3'UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution

Insect-specific flaviviruses (ISFs) circulate in nature due to vertical transmission in mosquitoes and do not infect vertebrates. ISFs include two distinct lineages - classical ISFs (cISFs) that evolved independently and dual host associated ISFs (dISFs) that are proposed to diverge from mosquito-borne flaviviruses (MBFs). Compared to pathogenic flaviviruses, ISFs are relatively poorly studied, and their molecular biology remains largely unexplored. In this study we focused on the characterisation of ISF 3UTRs and their ability to produce subgenomic flaviviral RNAs - noncoding viral RNAs that are known as important determinants of transmission and replication of pathogenetic flaviviruses. We demonstrated that cISFs and dISFs produce sfRNAs by employing a highly conserved mechanism of resistance to degradation by the cellular 5-3 exoribonuclease XRN1. We determined the secondary structures of complete 3UTRs and experimentally identified structured RNA elements that resist degradation by XRN1 (xrRNAs) in divergent representatives of cISF and dISF clades. We discovered a novel class of xrRNAs in dISFs and identified structurally divergent xrRNA in Anopheles-associated cISFs. Phylogenetic analyses based on sequences and secondary structures of xrRNAs and complete 3UTRs reveal that xrRNAs of cISFs and MBFs/dISFs evolved from a common xrRNA ancestor similar to the xrRNA of Anopheles-associated cISFs. Additionally, we found that duplications of xrRNAs occurred independently in ISF and MBF clades. Using ISF mutants deficient in the production of sfRNAs, we found that individual sfRNAs of ISFs have redundant functions. We conclude that duplicated xrRNAs were selected in the evolution of flaviviruses to ensure that sfRNA is produced if one of the xrRNAs lose XRN1 resistance due to mutations or misfolding.

microbiology↗