Investigating virus diversity in Australian domestic cats and bats

The field of virus discovery has made dramatic advances since the development of metagenomic next-generation sequencing. Although this technique has been used to identify important novel viruses, to date there have been few studies using metagenomics to reveal the diversity and evolution of those viruses carried by wildlife and companion animal species in Australia. Similarly, the prevalence of enteric viruses in domestic cats is understudied in Australia compared to other countries. To reduce these major knowledge gaps, I used metagenomic next-generation sequencing to characterise the viruses present in five bat species (grey-headed, black and little red flying fox, large footed myotis and eastern-bent wing bat) and in faecal and tissue samples from healthy and diseased domestic cats. This led to the identification of 13 known viruses from the faeces of domestic cats and two from the tissue of bats. Additionally, sequence comparisons and phylogenetic analysis revealed eleven novel mammalian viruses from the families Astroviridae, Caliciviridae, Coronaviridae, Picornaviridae, Papillomaviridae and Retroviridae in domestic cat and bat faecal and tissue samples. By performing two large-scale domestic cat and grey-headed flying fox faecal virome studies, I also determined that a large diversity of viruses are shed via the faeces, including likely host, dietary, bacterial, fungal and invertebrate viruses. In addition, the studies presented in this thesis provide the first evidence of the circulation of feline kobuvirus, feline picornavirus, feline chaphamaparvovirus, bat sapovirus, bat astroviruses, bat kunsagivirus and possible exogenous bat betaretroviruses in Australia. In sum, this thesis presents an overview of the virus diversity in domestic cats and urban/suburban bat populations, highlighting the power of metagenomic sequencing to detect novel virus species in diseased and health mammals

Limited cross-species virus transmission in a spatially restricted coral reef fish community

The Great Barrier Reef (GBR) - the largest coral reef ecosystem in the world - supports over 1,200 fish species with some of the highest population densities and diversities observed in vertebrates, offering a high potential for virus transmission among species. As such, the GBR represents an exceptional natural ecosystem to determine the impact of host community diversity on virus evolution and emergence. In recent decades, the GBR has also experienced significant threats of extinction, making it one of the most vulnerable ecosystems on the planet. Despite the global importance of the GBR, our understanding of virus diversity and connectivity in tropical reef fishes remains poor. Here, we employed metatranscriptomic sequencing to reveal the viromes of sixty-one reef fish species. This identified transcripts representing 132 putative viral sequences, 38 of which exhibited strong phylogenetic relationships with known vertebrate-associated viral genera, including a novel Santee-Cooper ranavirus (Iridoviridae). We found little evidence for virus transmission between fish species living within a very restricted geographical space - a 100-m2 coral reef ecosystem - suggesting that there might be important host barriers to successful cross-species transmission despite regular exposure. We also identified differences in virome composition among reef fish families, such that cryptobenthic reef fishes - characterized by small body sizes and short life spans - exhibited greater virome richness compared to large reef fishes. This study suggests that there are important barriers to cross-species virus transmission and that successful emergence in a reef fish community likely requires active host adaptation, even among closely related host species

Faecal virome of the Australian grey-headed flying fox from urban/ suburban environments contains novel coronaviruses, retroviruses and sapoviruses

Bats are important reservoirs for viruses of public health and veterinary concern. Virus studies in Australian bats usually target the families Paramyxoviridae, Coronaviridae and Rhabdoviridae, with little known about their overall virome composition. We used metatranscriptomic sequencing to characterise the faecal virome of greyheaded flying foxes from three colonies in urban/suburban locations from two Australian states. We identified viruses from three mammalian-infecting (Coronaviridae, Caliciviridae, Retroviridae) and one possible mammalianinfecting (Birnaviridae) family. Of particular interest were a novel bat betacoronavirus (subgenus Nobecovirus) and a novel bat sapovirus (Caliciviridae), the first identified in Australian bats, as well as a potentially exogenous retrovirus. The novel betacoronavirus was detected in two sampling locations 1375 km apart and falls in a viral lineage likely with a long association with bats. This study highlights the utility of unbiased sequencing of faecal samples for identifying novel viruses and revealing broad-scale patterns of virus ecology and evolution

Identification of Novel Astroviruses in the Gastrointestinal Tract of Domestic Cats

Astroviruses, isolated from numerous avian and mammalian species including humans, are commonly associated with enteritis and encephalitis. Two astroviruses have previously been identified in cats, and while definitive evidence is lacking, an association with enteritis is suggested. Using metagenomic next-generation sequencing of viral nucleic acids from faecal samples, we identified two novel feline astroviruses termed Feline astrovirus 3 and 4. These viruses were isolated from healthy shelter-housed kittens (Feline astrovirus 3; 6448 bp) and from a kitten with diarrhoea that was co-infected with Feline parvovirus (Feline astrovirus 4, 6549 bp). Both novel astroviruses shared a genome arrangement of three open reading frames (ORFs) comparable to that of other astroviruses. Phylogenetic analysis of the concatenated ORFs, ORF1a, ORF1b and capsid protein revealed that both viruses were phylogenetically distinct from other feline astroviruses, although their precise evolutionary history could not be accurately determined due to a lack of resolution at key nodes. Large-scale molecular surveillance studies of healthy and diseased cats are needed to determine the pathogenicity of feline astroviruses as single virus infections or in co-infections with other enteric viruses

A novel papillomavirus in a New Zealand fur seal (Arctocephalus forsteri) with oral lesions

Abstract 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 A. forsteri papillomavirus 1 (AforPV1) in an animal with clinically and histologically identified oral papilloma-like lesions. RT-PCR confirmed the presence of AforPV1 only in oral papilloma samples from the affected individual. Phylogenetic analysis of the complete 7926 bp genome of AforPV1 revealed that it grouped with taupapillomaviruses found in related Carnivora species. These findings highlight the need for further research into the disease associations and impact of undiagnosed and novel viruses on New Zealand fur seals

The enteric virome of cats with feline panleukopenia differs in abundance and diversity from healthy cats

Feline panleukopenia (FPL) is a severe, often fatal disease caused by feline parvovirus (FPV). How infection with FPV might impact the composition of the entire eukaryotic enteric virome in cats has not been characterized. We used metatranscriptomic and viral particle enrichment metagenomic approaches to characterize the enteric viromes of 23 cats naturally infected with FPV (FPV-cases) and 36 age-matched healthy shelter cats (healthy controls). Sequencing reads were detected from 11 mammalian infecting viral families mostly belonging to Coronaviridae, Parvoviridae and Astroviridae . Among the healthy control cats the most abundant viruses were Feline coronavirus, Mamastrovirus 2 and Carnivore bocaparvovirus 3 (Feline bocavirus 1) with frequent co-infections of all three. Feline chaphamaparvovirus was only detected in healthy controls (6/36, 16.7%). Among the FPV-cases, in addition to FPV, the most abundant viruses were Mamastrovirus 2 , Feline coronavirus and Carnivore bocaparvovirus 4 (Feline bocaparvovirus 2). The latter and Feline bocaparvovirus 3 were detected significantly more frequently in FPV-cases than in healthy controls. Feline calicivirus was present in a high proportion of FPV-cases (11/23, 47.8%) compared to healthy controls (5/36, 13.9%, p=0.0067). Feline kobuvirus infections were also common among FPV-cases (9/23, 39.1%) and were not detected in any healthy control cats (p<0.0001). While abundant in both groups, astroviruses were more frequently present in FPV-cases (19/23, 82.6%) than in healthy controls (18/36, p=0.0142). The differences in eukaryotic virome composition found in this study indicate that further investigations to determine associations between enteric viral co-infections on clinical disease severity in cats with FPL are warranted

Limited cross-species virus transmission in a spatially restricted coral reef fish community

The Great Barrier Reef (GBR) - the largest coral reef ecosystem in the world - supports over 1,200 fish species with some of the highest population densities and diversities observed in vertebrates, offering a high potential for virus transmission among species. As such, the GBR represents an exceptional natural ecosystem to determine the impact of host community diversity on virus evolution and emergence. In recent decades, the GBR has also experienced significant threats of extinction, making it one of the most vulnerable ecosystems on the planet. Despite the global importance of the GBR, our understanding of virus diversity and connectivity in tropical reef fishes remains poor. Here, we employed metatranscriptomic sequencing to reveal the viromes of sixty-one reef fish species. This identified transcripts representing 132 putative viral sequences, 38 of which exhibited strong phylogenetic relationships with known vertebrate-associated viral genera, including a novel Santee-Cooper ranavirus (Iridoviridae). We found little evidence for virus transmission between fish species living within a very restricted geographical space - a 100-m2 coral reef ecosystem - suggesting that there might be important host barriers to successful cross-species transmission despite regular exposure. We also identified differences in virome composition among reef fish families, such that cryptobenthic reef fishes - characterized by small body sizes and short life spans - exhibited greater virome richness compared to large reef fishes. This study suggests that there are important barriers to cross-species virus transmission and that successful emergence in a reef fish community likely requires active host adaptation, even among closely related host species

Low Intrahost and Interhost Genetic Diversity of Carnivore Protoparvovirus 1 in Domestic Cats during a Feline Panleukopenia Outbreak

Feline panleukopenia (FPL), a highly contagious and frequently fatal disease of cats, is caused by Feline parvovirus (FPV) and Canine parvovirus (CPV). We characterised the diversity of these Carnivore protoparvovirus 1 variants in 18 faecal samples collected from domestic cats with FPL during an outbreak, using targeted parvoviral DNA metagenomics to a mean depth of &gt;10,000 &times; coverage per site. All samples comprised FPV alone. Compared with the reference FPV genome, isolated in 1967, 44 mutations were detected. Ten of these were nonsynonymous, including 9 in nonstructural genes and one in VP1/VP2 (Val232Ile), which was the only one to exhibit interhost diversity, being present in five sequences. There were five other polymorphic nucleotide positions, all with synonymous mutations. Intrahost diversity at all polymorphic positions was low, with subconsensus variant frequencies (SVF) of &lt;1% except for two positions (2108 and 3208) in two samples with SVF of 1.1&ndash;1.3%. Intrahost nucleotide diversity was measured across the whole genome (0.7&ndash;1.5%) and for each gene and was highest in the NS2 gene of four samples (1.2&ndash;1.9%). Overall, intrahost viral genetic diversity was limited and most mutations observed were synonymous, indicative of a low background mutation rate and strong selective constraints

Feline Calicivirus Virulent Systemic Disease: Clinical Epidemiology, Analysis of Viral Isolates and In Vitro Efficacy of Novel Antivirals in Australian Outbreaks

Feline calicivirus (FCV) causes upper respiratory tract disease (URTD) and sporadic outbreaks of virulent systemic disease (FCV-VSD). The basis for the increased pathogenicity of FCV-VSD viruses is incompletely understood, and antivirals for FCV-VSD have yet to be developed. We investigated the clinicoepidemiology and viral features of three FCV-VSD outbreaks in Australia and evaluated the in vitro efficacy of nitazoxanide (NTZ), 2′-C-methylcytidine (2CMC) and NITD-008 against FCV-VSD viruses. Overall mortality among 23 cases of FCV-VSD was 39%. Metagenomic sequencing identified five genetically distinct FCV lineages within the three outbreaks, all seemingly evolving in situ in Australia. Notably, no mutations that clearly distinguished FCV-URTD from FCV-VSD phenotypes were identified. One FCV-URTD strain likely originated from a recombination event. Analysis of seven amino-acid residues from the hypervariable E region of the capsid in the cultured viruses did not support the contention that properties of these residues can reliably differentiate between the two pathotypes. On plaque reduction assays, dose–response inhibition of FCV-VSD was obtained with all antivirals at low micromolar concentrations; NTZ EC50, 0.4–0.6 µM, TI = 21; 2CMC EC50, 2.7–5.3 µM, TI > 18; NITD-008, 0.5 to 0.9 µM, TI > 111. Investigation of these antivirals for the treatment of FCV-VSD is warranted