Paola Karinna Vaz

Background: Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal haemorrhagic disease (EEHV-HD) in young Asian elephants ( Elephas maximus ), posing a significant threat to the conservation of the species. Understanding the dynamics of EEHV infection within a herd may help to understand risk factors associated with EEHV-HD, however, only few longitudinal EEHV surveillance studies exist. Methods: This study documented EEHV viral dynamics in nine Asian elephants over a two-year period spanning an anticipated major stressor: relocation from Melbourne Zoo to Werribee Open Range Zoo. Antibodies specific to each EEHV (sub)species were measured by enzyme-linked immunosorbent assays (ELISA). Viral DNA was detected in blood and trunk wash samples by quantitative PCR (qPCR). Results: Contrary to our hypothesis, EEHV detection by qPCR was more common prior to relocation than after relocation. Pre-relocation viral detection was largely attributable to a series of systemic EEHV5 infections in five elephants, unrelated to any changes in herd management or dynamics. Prospective serological testing demonstrated an increase in EEHV5A-specific antibodies in three young elephants with low pre-existing antibodies in response to systemic EEHV5 infection. Trunk wash detection of virus preceded viraemia in four out of five elephants with systemic EEHV5 infection, including two of the animals with low EEHV5A antibodies, indicating virus exposure or localised replication. Conclusions: Transport and relocation of the entire herd were not associated with widespread viral reactivation. In this herd, combined serology testing and trunk wash surveillance identified animals at risk of systemic EEHV infection and provided early evidence of viral exposure prior to viraemia. Integration of serology and trunk wash qPCR with routine haematology and blood qPCR screening may enhance early detection of EEHV infection in susceptible elephants.

Little is known about the baseline health status of Australia’s native rodents. The resultant knowledge gaps are problematic for wildlife translocations, as assessing disease risk is integral to translocation planning and outcomes. A disease risk analysis was conducted for the conservation translocation of native rodents to Dirk Hartog Island, Western Australia. This analysis resulted in a recommendation for a multidisciplinary project to reduce knowledge gaps about the health status of Shark Bay mice (Pseudomys gouldii) and greater-stick-nest rats (Leporillus conditor). Between 2021 and 2022, 130 Shark Bay mice and 122 greater stick-nest rats were translocated to Dirk Hartog Island. Mucosal swabs, whole blood, faeces, and ectoparasites were collected from a subset of these individuals. Molecular testing for four infectious agents was performed, parasite burdens and haematological parameters were examined, and relationships between these variables, health metrics and post-translocation short-term survival were assessed. Necropsies with routine histological assessment on translocation mortalities were performed where feasible. Several indicators of non-infectious factors contributing to morbidity or mortality were found, highlighting the need to iteratively evaluate translocation strategies. There was also an apparent absence of some host-specific parasites, suggesting that active approaches to parasite conservation during translocations should be considered. Molecular evidence of herpesviruses, a paramyxovirus, Bartonella sp., Trypanosoma sp. and Mycoplasma spp. were detected among the translocated animals. Although not associated with morbidity or short-term mortality in this study, the disease potential of these agents is still present. Overall, these results contribute important new data on health baselines for both species, which will assist in informing future conservation planning.

Elephant endotheliotropic herpesviruses (EEHV) pose a significant threat to the conservation of Asian elephants ( Elephas maximus ) worldwide, with a high mortality rate in young elephants. However, several components of EEHV virology remain underexplored, particularly for EEHV1B. This study describes a fatal case of EEHV1B infection in a nine-year-old Asian elephant from an ex situ conservation herd, examining herd viral dynamics, tissue viral loads and comparative genomics. This elephant succumbed to haemorrhagic disease within three days of developing clinical signs, despite therapeutic intervention. Quantitative PCR (qPCR) was performed on serial trunk washes and whole-blood surveillance samples collected before and after the clinical event, as well as on post-mortem tissues preserved in different storage media (DNA/RNA Shield, RNALater, and viral transport medium). Metagenomic next-generation sequencing of infected tissues was performed to characterise the complete viral genome, analyse variation from other published EEHV genomes and assess for evidence of viral recombination between EEHV subspecies. The affected elephant demonstrated a marked viraemia at onset of clinical disease, with viral load peaking at 5.47 x 10 ⁶ viral genome equivalents per mL of blood, one day after the onset of clinical signs. Samples stored in viral transport medium yielded the greatest viral and host DNA recovery by qPCR, although tissues stored at −80 °C without media were still suitable for molecular detection. Whole genome sequencing demonstrated 96.0% pairwise nucleotide identity between the assembled genome (EEHV1B_AUP_01_2023, GenBank accession: PX651398) and the previously reported EEHV1B sequence (KC462164), and a maximum of 90.9% identity to published EEHV1A genomes, with evidence of recombination between the viral subspecies at several genomic regions. Viral recombination between EEHV subspecies may have significant implications for the pathogenesis of EEHV disease, the reliability of molecular diagnostics and the efficacy of vaccinations and anti-viral therapy.

Elephant endotheliotropic herpesviruses (EEHV) pose a significant threat to the conservation of Asian elephants ( Elephas maximus ) worldwide, with a high mortality rate in young elephants. However, several components of EEHV virology remain underexplored, particularly for EEHV1B. This study describes a fatal case of EEHV1B infection in a nine-year-old Asian elephant from an ex situ conservation herd, examining herd viral dynamics, tissue viral loads and comparative genomics. This elephant succumbed to haemorrhagic disease within three days of developing clinical signs, despite therapeutic intervention. Quantitative PCR (qPCR) was performed on serial trunk washes and whole-blood surveillance samples collected before and after the clinical event, as well as on post-mortem tissues preserved in different storage media (DNA/RNA Shield, RNALater, and viral transport medium). Metagenomic next-generation sequencing of infected tissues was performed to characterise the complete viral genome, analyse variation from other published EEHV genomes and assess for evidence of viral recombination between EEHV subspecies. The affected elephant demonstrated a marked viraemia at onset of clinical disease, with viral load peaking at 5.47 × 10 ⁶ viral genome equivalents per mL of blood, one day after the onset of clinical signs. Samples stored in viral transport medium yielded the greatest viral and host DNA recovery by qPCR, although tissues stored at -80 °C without media were still suitable for molecular detection. Whole genome sequencing demonstrated 96.0% pairwise nucleotide identity between the assembled genome (EEHV1B_AUP_01_2023, GenBank accession: PX651398 ) and the previously reported EEHV1B sequence ( KC462164 ), and a maximum of 90.9% identity to published EEHV1A genomes, with evidence of recombination between the viral subspecies at several genomic regions. Viral recombination between EEHV subspecies may have significant implications for the pathogenesis of EEHV disease, the reliability of molecular diagnostics and the efficacy of vaccinations and anti-viral therapy.

Coronaviruses evolve rapidly, with recombination and mutation fostering the emergence of variant strains. The avian coronavirus infectious bronchitis virus (IBV) is an important poultry pathogen and a valuable natural model for studying coronaviruses. Australian strains have evolved independently of those infecting chickens elsewhere in the world, so understanding the biology and evolution of these strains can further our understanding of factors driving the emergence of novel coronaviruses. We infected groups of specific pathogen-free Leghorn chickens with six Australian IBVs (from five distinct genotypes) isolated between 1962 and 2013. All six affected the respiratory tract, but only one was nephropathogenic (N1/62). All six induced significant lesions and actively replicated in the upper respiratory tract, but they had lower levels of replication and induced less severe lesions in the middle and lower trachea. There were significant differences between the six strains in the severity of the lesions they induced and in their tissue tropism and effect on tracheal ciliary motility. Strains N1/62 (strain T) and N1/03 caused the most severe tracheal ciliostasis and replicated to the highest levels in tissues. Strain N1/03 caused the most severe lesions at 9 days post-infection. Only strain N1/03 caused lesions in the lower trachea. Overall, strains N1/03 and N1/62 were the most virulent. This study is the first to characterize the histological changes induced by the recently isolated Australian IBVs and compare them directly with older strains. Recombination between field and vaccine strains of IBV has yielded emergent IBVs in Australia that appear to have enhanced virulence for the respiratory tract.

The realm Duplodnaviria includes viruses of archaea, bacteria and eukaryotes, with linear dsDNA genomes. Duplodnavirians share a distinct morphogenetic module of four hallmark genes encoding the HK97-fold major capsid protein, a genome packaging ATPase-nuclease (large terminase subunit), a portal protein and a capsid maturation protease. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the realm Duplodnaviria, which is available at ictv.global/report/duplodnaviria.

Coinfections are known to play an important role in disease progression and severity. Coinfections are common in cats, but no coinfection studies have investigated the in vitro dynamics between feline viral and bacterial pathogens. In this study, we performed co-culture and invasion assays to investigate the ability of common feline bacterial respiratory pathogens, Chlamydia felis and Mycoplasma felis , to replicate in and invade into Crandell–Rees feline kidney cells. We subsequently investigated how coinfection of these feline cells with each bacterium ( C. felis or M. felis ) and the common feline viral pathogen, felid alphaherpesvirus 1 (FHV-1), affects replication of each agent in this cell culture system. We also investigated the metabolic impact of each co-pathogen using metabolomic analysis of infected and coinfected cells. C. felis was able to invade and replicate in CRFKs, while M. felis had little capacity to invade. During coinfection, FHV-1 replication was minimally affected by the presence of either bacterial pathogen, but bacterial replication kinetics were more affected, particularly in M. felis . Both C. felis and M. felis replicated to higher levels in the presence of a secondary pathogen. Coinfections resulted in reprogramming of the glycolysis pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle. The distinct metabolic profiles of coinfected cells compared to those of cells infected with just one of these three pathogens, as well as the impact of coinfections on viral or bacterial load, suggest strong interactions between these three pathogens and possible synergistic mechanisms enhancing virulence that need further investigation. IMPORTANCE: In the natural world, respiratory pathogens coexist within their hosts, but their dynamics and interactions remain largely unexplored. Herpesviruses, mycoplasmas, and chlamydias are common and significant causes of acute and chronic respiratory and system disease in animals and people, and these diseases are increasingly found to be polymicrobial. This study investigates how coinfection of feline cells between three respiratory pathogens of cats impact each other as well as the host innate metabolic response to infection. Each of these pathogens have been implicated in the induction of feline upper respiratory tract disease in cats, which is the leading cause of euthanasia in shelters. Understanding how coinfection impacts co-pathogenesis and host responses is critical for improving disease management.

Mycoplasma bovis is an important emerging pathogen of cattle and bison, but our understanding of the genetic basis of its interactions with its host is limited. The aim of this study was to identify genes of M. bovis required for interaction and survival in association with host cells. One hundred transposon-induced mutants of the type strain PG45 were assessed for their capacity to survive and proliferate in Madin-Darby bovine kidney cell cultures. The growth of 19 mutants was completely abrogated, and 47 mutants had a prolonged doubling time compared to the parent strain. All these mutants had a similar growth pattern to the parent strain PG45 in the axenic media. Thirteen genes previously classified as dispensable for the axenic growth of M. bovis were found to be essential for the growth of M. bovis in association with host cells. In most of the mutants with a growth-deficient phenotype, the transposon was inserted into a gene involved in transportation or metabolism. This included genes coding for ABC transporters, proteins related to carbohydrate, nucleotide and protein metabolism, and membrane proteins essential for attachment. It is likely that these genes are essential not only in vitro but also for the survival of M. bovis in infected animals. IMPORTANCE: Mycoplasma bovis causes chronic bronchopneumonia, mastitis, arthritis, keratoconjunctivitis, and reproductive tract disease in cattle around the globe and is an emerging pathogen in bison. Control of mycoplasma infections is difficult in the absence of appropriate antimicrobial treatment or effective vaccines. A comprehensive understanding of host-pathogen interactions and virulence factors is important to implement more effective control methods against M. bovis . Recent studies of other mycoplasmas with in vitro cell culture models have identified essential virulence genes of mycoplasmas. Our study has identified genes of M. bovis required for survival in association with host cells, which will pave the way to a better understanding of host-pathogen interactions and the role of specific genes in the pathogenesis of disease caused by M. bovis .

Mycoplasma felis has been isolated from diseased cats and horses, but to date only a single fully assembled genome of this species, of an isolate from a horse, has been characterized. This study aimed to characterize and compare the completely assembled genomes of four clinical isolates of M. felis from three domestic cats, assembled with the aid of short- and long-read sequencing methods. The completed genomes encoded a median of 759 ORFs (range 743–777) and had a median average nucleotide identity of 98.2 % with the genome of the available equid origin reference strain. Comparative genomic analysis revealed the occurrence of multiple horizontal gene transfer events and significant genome reassortment. This had resulted in the acquisition or loss of numerous genes within the Australian felid isolate genomes, encoding putative proteins involved in DNA transfer, metabolism, DNA replication, host cell interaction and restriction modification systems. Additionally, a novel mycoplasma phage was detected in one Australian felid M. felis isolate by genomic analysis and visualized using cryo-transmission electron microscopy. This study has highlighted the complex genomic dynamics in different host environments. Furthermore, the sequences obtained in this work will enable the development of new diagnostic tools, and identification of future infection control and treatment options for the respiratory disease complex in cats.

A domestic short hair cat (Felis catus) suffering from a purulent wound infection resulting from a dog bite was sampled for bacterial culture and isolation as the wound had been unresponsive to prolonged antimicrobial treatment. A mycoplasma was isolated from the wound. Whole genome sequencing of the isolate was performed using short-read Illumina and long-read Oxford Nanopore chemistry, and the organism was identified as Mycoplasma edwardii . Comparison of the genome sequence of the isolate to a reference M. edwardii genome sequence (canid isolate) identified the loss of several key bacterial factors involved in genome editing, as well the insertion of several novel ORFs most closely related to those found in other canine mycoplasmas, specifically Mycoplasma canis, M. cynos, M. molare and M. maculosa. This is only the second known report of disease caused by M. edwardii in a non-canid species, and the first report of it infecting and causing clinical disease in a cat.