Virome analysis of New Zealand's bats reveals cross-species viral transmission among the Coronaviridae.

The lesser short-tailed bat (Mystacina tuberculata) and the long-tailed bat (Chalinolobus tuberculatus) are Aotearoa New Zealand's only native extant terrestrial mammals and are believed to have migrated from Australia. Long-tailed bats arrived in New Zealand an estimated two million years ago and are closely related to other Australian bat species. Lesser short-tailed bats, in contrast, are the only extant species within the Mystacinidae and are estimated to have been living in isolation in New Zealand for the past 16-18 million years. Throughout this period of isolation, lesser short-tailed bats have become one of the most terrestrial bats in the world. Through a metatranscriptomic analysis of guano samples from eight locations across New Zealand, we aimed to characterise the viromes of New Zealand's bats and determine whether viruses have jumped between these species over the past two million years. High viral richness was observed among long-tailed bats with viruses spanning seven different viral families. In contrast, no bat-specific viruses were identified in lesser short-tailed bats. Both bat species harboured an abundance of likely dietary- and environment-associated viruses. We also identified alphacoronaviruses in long-tailed bat guano that had previously been identified in lesser short-tailed bats, suggesting that these viruses had jumped the species barrier after long-tailed bats migrated to New Zealand. Of note, an alphacoronavirus species discovered here possessed a complete genome of only 22,416 nucleotides with entire deletions or truncations of several non-structural proteins, thereby representing what may be the shortest genome within the Coronaviridae identified to date. Overall, this study has revealed a diverse range of novel viruses harboured by New Zealand's only native terrestrial mammals, in turn expanding our understanding of bat viral dynamics and evolution globally.

Total infectome investigation of diphtheritic stomatitis in yellow-eyed penguins (Megadyptes antipodes) reveals a novel and abundant megrivirus.

First identified in 2002, diphtheritic stomatitis (DS) is a devastating disease affecting yellow-eyed penguins (Megadyptes antipodes, or hoiho in te reo Maori). The disease is associated with oral lesions in chicks and has caused significant morbidity and mortality. DS is widespread among yellow-eyed penguin chicks on mainland New Zealand yet appears to be absent from the subantarctic population. Corynebacterium spp. have previously been suspected as causative agents yet, due to inconsistent cultures and inconclusive pathogenicity, their role in DS is unclear. Herein, we used a metatranscriptomic approach to identify potential causative agents of DS by revealing the presence and abundance of all viruses, bacteria, fungi and protozoa - together, the infectome. Oral and cloacal swab samples were collected from presymptomatic, symptomatic and recovered chicks along with a control group of healthy adults. Two novel viruses from the Picornaviridae were identified, one of which - yellow-eyed penguin megrivirus - was highly abundant in chicks irrespective of health status but not detected in healthy adults. Tissue from biopsied oral lesions also tested positive for the novel megrivirus upon PCR. We found no overall clustering among bacteria, protozoa and fungi communities at the genus level across samples, although Paraclostridium bifermentans was significantly more abundant in oral microbiota of symptomatic chicks compared to other groups. The detection of a novel and highly abundant megrivirus has sparked a new line of inquiry to investigate its potential association with DS.

Host specificity shapes fish viromes across lakes on an isolated remote island.

Fish viromes often provide insights into the origin and evolution of viruses affecting tetrapods, including those associated with imporant human diseases. However, despite fish being the most diverse vertebrate group, their viruses are still understudied. We investigated the viromes of fish on Chatham Island (Rekohu), a geographically isolated island housing 9% of New Zealand's threatened endemic fish species. Using metatranscriptomics, we analyzed samples from seven host species across 16 waterbodies. We identified 19 fish viruses, including 16 potentially novel species, expanding families such as the Coronaviridae, Hantaviridae, Poxviridae, and the recently proposed Tosoviridae. Surprisingly, virome composition was not influenced by the ecological factors measured and smelt (Retropinna retropinna) viromes were consistent across lakes despite differences in host life history, seawater influence, and community richness. Overall, fish viromes across Rekohu were highly diverse and revealed a long history of co-divergence between host and virus despite their unique and geographically isolated ecosystem.

Cloacal virome of an ancient host lineage - The tuatara (Sphenodon punctatus) - Reveals abundant and diverse diet-related viruses.

Tuatara (Sphenodon punctatus) are one of the most phylogenetically isolated vertebrate species and provide a unique host system to study virus evolution. While the tuatara genome, sequenced in 2020, revealed many endogenous viral elements, we know little of the exogenous viruses that infect tuatara. We performed a metatranscriptomics study of tuatara cloaca samples from a wild population on Takapourewa (Stephens Island), Aotearoa New Zealand. From these data we identified 49 potentially novel viral species that spanned 19 RNA viral families and/or orders, the vast majority (48) of which were likely dietary-related. Notably, using a protein structure homology search, we identified a highly divergent novel virus within the Picornaviridae which may directly infect tuatara. Additionally, two endogenous tuatara adintoviruses were characterised that exhibited long-term viral-host co-divergence. Overall, our results indicate that the tuatara cloacal virome is highly diverse, likely due to a large number of dietary-related viruses.

Slippery when wet: cross-species transmission of divergent coronaviruses in bony and jawless fish and the evolutionary history of the Coronaviridae.

The Nidovirales comprise a genetically diverse group of positive-sense single-stranded RNA virus families that infect a range of invertebrate and vertebrate hosts. Recent metagenomic studies have identified nido-like virus sequences, particularly those related to the Coronaviridae, in a range of aquatic hosts including fish, amphibians, and reptiles. We sought to identify additional members of the Coronaviridae in both bony and jawless fish through a combination of total RNA sequencing (meta-transcriptomics) and data mining of published RNA sequencing data and from this reveal more of the long-term patterns and processes of coronavirus evolution. Accordingly, we identified a number of divergent viruses that fell within the Letovirinae subfamily of the Coronaviridae, including those in a jawless fish-the pouched lamprey. By mining fish transcriptome data, we identified additional virus transcripts matching these viruses in bony fish from both marine and freshwater environments. These new viruses retained sequence conservation in the RNA-dependant RNA polymerase across the Coronaviridae but formed a distinct and diverse phylogenetic group. Although there are broad-scale topological similarities between the phylogenies of the major groups of coronaviruses and their vertebrate hosts, the evolutionary relationship of viruses within the Letovirinae does not mirror that of their hosts. For example, the coronavirus found in the pouched lamprey fell within the phylogenetic diversity of bony fish letoviruses, indicative of past host switching events. Hence, despite possessing a phylogenetic history that likely spans the entire history of the vertebrates, coronavirus evolution has been characterised by relatively frequent cross-species transmission, particularly in hosts that reside in aquatic habitats.

A Novel Rubi-Like Virus in the Pacific Electric Ray (Tetronarce californica) Reveals the Complex Evolutionary History of the Matonaviridae.

Rubella virus (RuV) is the causative agent of rubella ("German measles") and remains a global health concern. Until recently, RuV was the only known member of the genus Rubivirus and the only virus species classified within the Matonaviridae family of positive-sense RNA viruses. Recently, two new rubella-like matonaviruses, Rustrela virus and Ruhugu virus, have been identified in several mammalian species, along with more divergent viruses in fish and reptiles. To screen for the presence of additional novel rubella-like viruses, we mined published transcriptome data using genome sequences from Rubella, Rustrela, and Ruhugu viruses as baits. From this, we identified a novel rubella-like virus in a transcriptome of Tetronarce californica-order Torpediniformes (Pacific electric ray)-that is more closely related to mammalian Rustrela virus than to the divergent fish matonavirus and indicative of a complex pattern of cross-species virus transmission. Analysis of host reads confirmed that the sample analysed was indeed from a Pacific electric ray, and two other viruses identified in this animal, from the Arenaviridae and Reoviridae, grouped with other fish viruses. These findings indicate that the evolutionary history of the Matonaviridae is more complex than previously thought and highlights the vast number of viruses that remain undiscovered.