Biology subjects

Mifsud, J. C.

Publications and source records attributed to Mifsud, J. C..

15 recordsLinked to original sources

bioRxiv2026

RNA virus discovery in Australian camelids reveals divergent picornaviruses and the convergent evolution of upstream ORFs

Invasive species can impact viral ecology, evolution and emergence by acquiring and disseminating viruses absent from native hosts. However, the extent to which invasive species harbour previously unrecognized RNA viruses and transmit these to native species is uncertain. We performed metatranscriptomic sequencing of invasive camelids in Australia and identified several previously undescribed vertebrate-associated RNA viruses, including an astrovirus closely related to avian-associated viruses suggesting a recent host jump. We also identified highly divergent picornaviruses that differed sufficiently from recognized taxa in genome organization and polyprotein phylogeny to establish a new genus. Notably, one virus encoded a putative upstream ORF (uORF) in the 5' genomic region. Across the Picornaviridae, putative uORF gain and loss appear to have occurred multiple times independently. In addition, although most of these uORF-encoded proteins exhibited little to no amino acid sequence homology, a subset showed overlapping ranges of secondary structure composition and intrinsic disorder, and when heterologously expressed, these proteins were translated and triggered reproducible transcriptional responses in a cell line. While no single pathway was uniformly affected across all uORFs, distinct uORFs from divergent lineages consistently perturbed overlapping sets of cellular pathways, supporting broadly analogous functional effects despite a lack of sequence homology. These findings demonstrate that uORFs represent a recurrent and selectable functional module within RNA virus genomes, suggest that the upstream genomic position itself constitutes a "hotspot" for the repeated acquisition of a functional module, and provide experimental evidence that their functional properties have converged across evolutionarily independent lineages. Author SummaryRNA viruses evolve under strong genomic constraints, forcing them to repeatedly adopt similar solutions to common challenges posed by their host environment. Invasive species can impact viral ecology, evolution and emergence by acquiring and disseminating non-native viruses. By characterizing RNA viruses infecting invasive camelids in Australia, we discovered previously unrecognized RNA viruses and recurrent patterns of genome evolution. Notably, we identified a small additional gene located at the 5' genomic region, known as an upstream open reading frame (uORF). uORFs were independently acquired and lost across multiple viral lineages, revealing that the same genomic region can be repeatedly exploited to acquire new functions. Although uORF-encoded proteins share little or no amino acid sequence homology, some of the encoded proteins showed comparable secondary structure composition and induce overlapping host cellular responses when expressed in cells. Hence, RNA viruses that have followed different evolutionary paths can converge on similar functional strategies.

microbiology↗

bioRxiv2025

Diverse Viral Pathogens in Australian Canines: Limited Geographic Structure and the First Detection of an RNA Virus in Dingoes

Viruses impose a substantial disease burden on dogs and the close relationship between dogs and humans may facilitate zoonotic disease emergence. Australias geographic isolation, strict biosecurity measures and native dingo populations present a unique model for understanding the spread and evolution of canine viruses. However, aside from a few well-characterised pathogens, genomic data are scarce for many common dog viruses, limiting our understanding of their evolution and disease ecology. Using a metatranscriptomic approach we identified the viruses in dogs and dingoes from various geographical locations across mainland Australia and sample types, revealing 86 vertebrate-associated viruses belonging to 16 distinct species, including a new vesivirus-like species. Many of the viruses identified here have not previously been sequenced in Australia. We identified important dog pathogens associated with canine infectious respiratory disease syndrome--such as canine pneumovirus, canine herpesvirus, and canine respiratory coronavirus--and gastroenteritis, including canine parvovirus, canine coronavirus, and rotavirus A. The sequences of Australian canine viruses often occupied multiple distinct clades phylogenetically and had little geographic structure, suggesting multiple virus introductions and subsequent spread across the country. Notably, we identified the first RNA virus - rotavirus A - in a dingo. This virus was phylogenetically distinct from dog-associated rotavirus A sequences and more closely related to viruses found in humans and bats, indicative of the past cross-species transmission of a reassortant virus into dingoes, and shows dingoes and domestic dogs may have distinct viromes. Our findings expand the knowledge of viral diversity in Australian canines, improving our understanding of viral movement into and within Australia, as well as the potential zoonotic risks associated with dogs and dingoes.

microbiology↗

bioRxiv2025

Integrated analysis of protein sequence and structureredefines viral diversity and the taxonomy of the Flaviviridae

The Flaviviridae are a family of non-segmented positive-sense enveloped RNA viruses containing significant pathogens including hepatitis C virus and yellow fever virus. Recent large-scale metagenomic surveys have identified many diverse RNA viruses related to classical orthoflaviviruses and pestiviruses but quite different genome lengths and configurations, and with a hugely expanded host range that spans multiple animal phyla, including molluscs, cnidarians and stramenopiles,, and plants. Grouping of RNA-directed RNA polymerase (RdRP) hallmark gene sequences of flavivirus and flavi-like viruses into four divergent clades and multiple lineages within them was congruent with helicase gene phylogeny, PPHMM profile comparisons, and comparison of RdRP protein structure predicted by AlphFold2. These results support their classification into the established order, Amarillovirales, in three families (Flaviviridae, Pestiviridae, and Hepaciviridae), and 14 genera. This taxonomic framework informed by RdRP hallmark gene evolutionary relationships provides a stable reference from which major genome re-organisational events can be understood.

microbiology↗

bioRxiv2024

Tunicate metatranscriptomes reveal ancient virus-host co-divergence and inter-order recombination in the evolutionary history of disease-causing viruses

Tunicates are a key transitional taxon in animal evolution as the closest extant invertebrate relatives of the vertebrates. Their viruses may also reflect this transitional state. Yet, it is not known whether tunicate viruses are more closely related to vertebrate- or invertebrate-infecting viral lineages. We analysed primary and publicly available RNA libraries to extend the known diversity of tunicate-associated viruses and determine their relationship to viruses of other animals. We present evidence that influenza viruses, alphaviruses, and some mononegaviruses emerged prior to the evolution of vertebrates. We also show that the recombination of glycoproteins between different orders of RNA viruses, including between positive- and negative-sense viruses, may have shaped the evolution of multiple lineages. Our study reveals that some disease-causing RNA virus lineages were present in early chordates and highlights that the evolution of structural genes may be incongruent with that of the highly conserved RNA-dependent RNA polymerase.

microbiology↗

bioRxiv2024

Detection of exotic biosecurity threat ribgrass mosaic virus and novel tobamoviruses through metatranscriptomic sequencing of animal gut content

Ribgrass mosaic virus (RMV) and related viruses of the genus Tobamovirus (Virgaviridae) are cruciferous plant pathogens that represent a threat to global horticultural systems. In Australia, they are considered exotic biosecurity threats, and an incursion of these viruses would require rapid and strict control efforts. However, current surveillance methods for these viruses are limited. We examined whether RMV and related tobamoviruses could be detected by deep sequencing of gut metatranscriptomes of vertebrate animals and ticks. Using this method, we discovered that RMV, as well as a novel relative of RMV, and two highly diverse novel tobamoviruses are present in Australia. RMV was detected in multiple sites in both the Australian Capital Territory (ACT) and Tasmania, two regions separated by approximately 700km of land and 200km of water. The novel relative of RMV was detected in the ACT and New South Wales (NSW), while the highly divergent novel tobamoviruses were each detected in a single state, NSW and Queensland (QLD). In addition, Tobacco mild green mosaic virus, which is already known to be present in Australia, was detected in QLD using this method. This work highlights the potential utility of metatranscriptomic sequencing of wild animal gut for the surveillance of biosecurity threats to native and agricultural plant species. ImportancePlant viruses can have devastating impacts on global horticulture. Tobamoviruses (family Virgaviridae, genus Tobamovirus) are among the most damaging seed-borne viruses in horticultural crops, and Australia is free of many of the tobamoviruses that cause major crop losses in other countries. These viruses are extremely difficult to eradicate. Consequently, early detection of incursions is key to the control of these viruses in Australia, alongside rapid deployment of eradication and management plans. Current biosecurity surveillance methods in Australia rely on visual inspection, immunological assays, and molecular methods such as screening of imported seed lots. This study introduces a complementary approach that utilises unbiased metatranscriptomic sequencing of animal gut material to detect cryptic plant viruses circulating in nature. Using this approach, we detected five different tobamovirus circulating in Australia, including a virus thought to be exotic and three novel viruses. This unique approach highlights alternative options for surveillance/detection of exotic crop viruses.

microbiology↗

bioRxiv2024

Limited transmission of microbial species among coral reef fishes from the Great Barrier Reef, Australia

Reef fishes account for one-third of all extant marine fishes and exhibit enormous biodiversity within a highly interactive ecosystem. Yet relatively little is known about the diversity and evolution of microbial species (bacteria, viruses, and eukaryotes) associated with reef fish, even though this may provide valuable insights into the factors that shape microbial communities within vertebrate hosts as well as the extent and pattern of cross-species transmission. Through metatranscriptomic sequencing we characterised the viruses, bacteria, and single-celled eukaryotes from 128 reef fish species inhabiting Lizard Island and Orpheus Island on the Great Barrier Reef, Australia. We assessed whether microbial communities differed between islands that are separated by approximately 450 kilometres, and to what extent viruses were able to emerge in new hosts. Notably, despite strong ecological interactions in the reef environment, and the presence of the same families and subfamilies of viruses and bacteria on both islands, there was minimal evidence for the cross-species transmission of individual microorganisms among fish species. An exception was the high prevalence of the bacterial pathogen Photobacterium damselae among apparently healthy cardinalfishes from both islands, indicating that these fish species are natural reservoirs within the reef system. Overall, these data suggest that reef fishes have microbial-host associations that arose prior to the formation of the Great Barrier Reef, leading to strong host barriers to cross-species microbial transmission even within a highly interactive and species-rich environment.

microbiology↗

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

Australian terrestrial environments harbour extensive RNA virus diversity

Australia is home to a diverse range of unique native fauna and flora. To address whether Australian ecosystems also harbour unique viruses, we performed meta-transcriptomic sequencing of 16 farmland and sediment samples taken from the east and west coasts of Australia. We identified 2,562 putatively novel viruses across 15 orders, the vast majority of which belonged to the microbe-associated phylum Lenarviricota. In many orders, the novel viruses identified here comprised entirely new clades, such as the Nodamuvirales and Ghabrivirales. Novel viruses also fell between established genera or families, such as in the Cystoviridae and Picornavirales, while highly divergent lineages were identified in the Martellivirales and Ghabrivirales. Viral abundance and alpha diversity were influenced by sampling site, soil type and land use, but not by depth from the surface. In sum, Australian soils and sediments are home to remarkable viral diversity, reflecting the biodiversity of local fauna and flora.

microbiology↗

bioRxiv2023

Host adaptive radiation is associated with rapid virus diversification and cross-species transmission in African cichlid fishes

Adaptive radiations are generated through a complex interplay of biotic and abiotic factors. Although adaptive radiations have been widely studied in the context of animal and plant evolution, little is known about how they impact the evolution of the viruses that infect these hosts, which in turn may provide insights into the drivers of disease emergence. We examined how the rapid adaptive radiation of the African cichlid fishes of Lake Tanganyika over the last 10 million years has shaped the diversity and evolution of the viruses they carry. Through metatranscriptomic analysis we identified 121 vertebrate-associated viruses among various tissue types that fell into 13 RNA and 4 DNA virus groups. Host-switching was commonplace, particularly within the Astroviridae, Metahepadnavirus, Nackednavirus, Picornaviridae, and Hepacivirus groups, occurring more frequently than in other fish communities. A time-calibrated phylogeny revealed that hepacivirus evolution was not constant throughout the cichlid radiation, but accelerated 2-3 million years ago, coinciding with a period of rapid cichlid diversification and niche packing in Lake Tanganyika, thereby providing more closely related hosts for viral infection. These data show that African cichlids contain a complex interacting pool of virus diversity, likely reflecting their close genetic relationships that lowers the barriers to cross-species virus transmission.

evolutionary biology↗

bioRxiv2023

Divergent hepaciviruses, chuvirus and deltaviruses in Australian marsupial carnivores (Dasyurids) identified through transcriptome mining

Although Australian marsupials are characterised by unique biology and geographic isolation, little is known about the viruses present in these iconic wildlife species. The Dasyuromorphia are an order of marsupial carnivores found only in Australia that include both the extinct Tasmanian tiger (Thylacine) and the highly threatened Tasmanian devil. Several other members of the order are similarly under threat of extinction due to habitat loss, hunting, disease, and competition and predation by introduced species such as feral cats. We utilised publicly available RNA-seq data from the NCBI Sequence Read Archive (SRA) database to document the viral diversity within four Dasyuromorphia species. Accordingly, we identified 15 novel virus species from five DNA virus families (Adenoviridae, Anelloviridae, Herpesviridae, Papillomaviridae and Polyomaviridae) and three RNA virus taxa: the order Jingchuvirales, the genus Hepacivirus, and the delta-like virus group. Of particular note was the identification of a marsupial specific clade of delta-like viruses that may indicate an association of deltaviruses and with marsupial species dating back to their origin some 160 million years ago. In addition, we identified a highly divergent hepacivirus in a numbat liver transcriptome that falls outside of the larger mammalian clade, as well as the first detection of the Jingchuvirales in a mammalian host - a chu-like virus in Tasmanian devils - thereby expanding the host range beyond invertebrates and ectothermic vertebrates. As many of these Dasyuromorphia species are currently being used in translocation efforts to reseed populations across Australia, understanding their virome is of key importance to prevent the spread of viruses to naive populations.

microbiology↗

bioRxiv2023

Evidence for an aquatic origin of influenza virus and the order Articulavirales

The emergence of novel disease-causing viruses in mammals is part of the long evolutionary history of viruses. Tracing these evolutionary histories contextualises virus spill over events and may help to elucidate how and why they occur. We used a combination of total RNA sequencing and transcriptome data mining to extend the diversity and evolutionary history of the order Articulavirales, which includes the influenza viruses. From this, we identified the first instance of Articulavirales in the Cnidaria (including corals), constituting a novel and divergent family that we tentatively named the Cnidenomoviridae. This may be the basal group within the Articulavirales. We also extended the known evolutionary history of the influenza virus lineage by identifying a highly divergent, sturgeon-associated influenza virus. This suggests that fish were among the first hosts of influenza viruses. Finally, we substantially expanded the known diversity of quaranjaviruses and proposed that this genus be reclassified as a family (the Quaranjaviridae). We find evidence that vertebrate infecting Quaranjaviridae may have initially evolved in crustaceans before spilling into terrestrial Chelicerata (i.e., ticks). Together, our findings indicate that the Articulavirales has evolved over at least 600 million years, first emerging in aquatic animals. Importantly, the evolution of this order was not shaped by strict virus-host codivergence, but rather by multiple aquatic-terrestrial transitions and substantial host jumps, some of which are still observable today.

evolutionary biology↗

bioRxiv2022

Transcriptome mining extends the host range of the Flaviviridae to non-bilaterians

The Flaviviridae are a family of positive-sense RNA viruses that include well-documented agents of human disease. Despite their importance and ubiquity, the time-scale of flaviviral evolution is uncertain. An ancient origin, spanning time-scales of millions of years, is supported by their presence in both vertebrates and invertebrates and the identification of a flavivirus-derived endogenous viral element in the peach blossom jellyfish genome (Craspedacusta Sowerby, phylum Cnidaria), implying that the flaviviruses arose early in the evolution of the Metazoa. To date, however, no exogenous flavivirus sequences have been identified in these hosts. To help resolve the antiquity of the Flavivirdae we mined publicly available transcriptome data across the Metazoa. From this, we expanded the diversity within the family through the identification of 32 novel viral sequences, and extended the host range of the pestiviruses to include amphibians, reptiles, and ray-finned fish. Through cophylogenetic analysis we found cross-species transmission to be the predominate macroevolutionary event across the non-vectored flaviviral genera (median, 68%), including a cross-species transmission event between bats and rodents, although long-term virus-host co-divergence was still a regular occurrence (median, 23%). Notably, we discovered flavivirus-like sequences in basal metazoan species, including the first associated with Cnidaria. This sequence formed a basal lineage to the genus Flavivirus and was closer to arthropod and crustacean flaviviruses than those in the tamanavirus group that include a variety of invertebrate and vertebrate viruses. Combined, these data attest an ancient origin of the flaviviruses, close to the emergence of the metazoans 750-800 million years ago.

microbiology↗

bioRxiv2022

Transcriptome Mining Reveals a Spectrum of RNA Viruses in Primitive Plants

Current knowledge of plant viruses stems largely from those affecting economically important plants. Yet, plant species in cultivation represent a small and bias subset of the plant kingdom. Here, we describe virus diversity and abundance from a survey of 1079 transcriptomes from species across the breadth of the plant kingdom (Archaeplastida) by analysing open-source data from the One Thousand Plant Transcriptomes Initiative (1KP). We identified 104 potentially novel viruses, of which 40% comprised single-stranded positive-sense RNA viruses across eight orders, including members of the Hepelivirales, Tymovirales, Cryppavirales, Martellivirales and Picornavirales. One-third of the newly described viruses comprised double-stranded RNA viruses from the orders Durnavirales and Ghabrivirales. The remaining were negative-sense RNA viruses from the Rhabdoviridae, Aspiviridae, Yueviridae, Phenuiviridae and the newly proposed Viridisbunyaviridae. Our analysis considerably expands the known host range of 13 virus families to include lower plants (e.g., Benyviridae and Secoviridae) and four virus families to include algae hosts (e.g., Tymoviridae and Chrysoviridae). The discovery of the first 30 kDa movement protein in a non-vascular plant, suggests that the acquisition of plant virus movement proteins occurred prior to the emergence of the plant vascular system. More broadly, however, a co-phylogeny analysis revealed that the evolutionary history of these families is largely driven by cross-species transmission events. Together, these data highlight that numerous RNA virus families are associated with older evolutionary plant lineages than previously thought and that the scarcity of RNA viruses found in lower plants to date likely reflects a lack of investigation rather than their absence. ImportanceOur knowledge of plant viruses is mainly limited to those infecting economically important host species. In particular, we know little about those viruses infecting primitive plant lineages such as the ferns, lycophytes, bryophytes and charophytes. To expand this understanding, we conducted a broad-scale viral survey of species across the breadth of the plant kingdom. We find that primitive plants harbour a wide diversity of RNA viruses including some that are sufficiently divergent to comprise a new virus family. The primitive plant virome we reveal offers key insights into the evolutionary history of core plant virus gene modules and genome segments. More broadly, this work emphasises that the scarcity of viruses found in these species to date likely reflects the absence of research in this area.

microbiology↗

bioRxiv2020

Host evolutionary history and ecology shape virome composition in fishes

Revealing the determinants of virome composition is central to placing disease emergence in a broader evolutionary context. Fish are the most species-rich group of vertebrates and so provide an ideal model system to study the factors that shape virome compositions and their evolution. We characterised the viromes of 19 wild-caught species of marine fish using total RNA sequencing (meta-transcriptomics) combined with analyses of sequence and protein structural homology to identify divergent viruses that often evade characterisation. From this, we identified 25 new vertebrate-associated viruses and a further 22 viruses likely associated with fish diet or their microbiomes. The vertebrate-associated viruses identified here included the first fish virus in the Matonaviridae (single-strand, negative-sense RNA virus). Other viruses fell within the Astroviridae, Picornaviridae, Arenaviridae, Reoviridae, Hepadnaviridae, Paramyxoviridae, Rhabdoviridae, Hantaviridae, Filoviridae and Flaviviridae and were sometimes phylogenetically distinct from known fish viruses. We also show how key metrics of virome composition - viral richness, abundance and diversity - can be analysed along with host ecological and biological factors as a means to understand virus ecology. Accordingly, these data suggest that that the vertebrate-associated viromes of the fish sampled here are predominantly shaped by the phylogenetic history (i.e. taxonomic order) of their hosts, along with several biological factors including water temperature, habitat depth, community diversity and swimming behaviour. No such correlations were found for viruses associated with porifera, molluscs, arthropods, fungi and algae, that are unlikely to replicate in fish hosts. Overall, these data indicate that fish harbour particularly large and complex viromes and the vast majority of fish viromes are undescribed.

microbiology↗

bioRxiv2020

Global shortfalls in extinction risk assessments for endemic flora

The Global Strategy for Plant Conservation (GSPC) ambitiously calls for an assessment of extinction risk for all recognised plant taxa by 20201. It is now clear that this target will not be met in the short-term; only 21-26% of known plant species have been assessed2 - a monumental shortfall in anticipated knowledge. Yet the need for risk assessments has never been more urgent. Plants are rapidly going extinct3,4 and face threats such as climate change5 and permanent deforestation6. Extinction risk assessments continue to provide the critical foundation to inform protection, management and recovery of plant species7,8, the loss of which will have clear consequences for maintaining planetary systems and human well-being9. Here, we rank countries of the world based on progress towards assessing the extinction risk to their endemic flora. Overall, 67% of country-based endemic species do not have an extinction risk assessment completed (143,294 species). We show that some of the worlds wealthiest nations, which also have relatively strong species protections, are failing to protect their unique flora by not systematically assessing risks to their endemic species.

ecology↗