Reduction of Chlamydia pecorum and Koala Retrovirus subtype B expression in wild koalas vaccinated with novel peptide and peptide/recombinant protein formulations.

Koalas are an endangered species under threat of extinction from several factors, including infections agents. Chlamydia pecorum infection results in morbidity and mortality from ocular and urogenital diseases while Koala Retrovirus (KoRV) infection has been linked to increased rates of cancer and chlamydiosis. Both C. pecorum and KoRV are endemic in many wild Australian koala populations, with limited treatment options available. Fortunately, vaccines for these pathogens are under development and have generated effective immune responses in multiple trials. The current study aimed to improve vaccine formulations by testing a novel peptide version of the Chlamydia vaccine and a combination Chlamydia - KoRV vaccine. Utilising a monitored wild population in Southeast Queensland, this trial followed koalas given either a 'Chlamydia only' vaccine (utilising four peptides from the chlamydial Major Outer Membrane Protein, MOMP), a combination 'Chlamydia and KoRV' vaccine (comprised of the chlamydial peptides plus a KoRV recombinant envelope protein (rEnv)) or no treatment. Clinical observations, C. pecorum and KoRV gene expression, serum IgG, and mucosal immune gene expression were assessed over a 17-month period. Overall, both vaccine formulations resulted in a decrease in chlamydiosis mortality, with decreases in C. pecorum, CD4, CD8ß and IL-17A gene expression observed. In addition, the combination vaccine group also showed an increase in anti-KoRV IgG production that corresponded to a decrease in detected KoRV-B expression. While these results are favourable, the chlamydial peptide vaccine did not appear to outperform the established recombinant chlamydial vaccine and suggests that a combination vaccine formulated with recombinant MOMP plus KoRV rEnv could capitalize on the demonstrated benefits of both for the betterment of koalas into the future.

Cryo-electron tomography of intact cardiac muscle reveals myosin binding protein-C linking myosin and actin filaments.

Myosin binding protein C (MyBP-C) is an accessory protein of the thick filament in vertebrate cardiac muscle arranged over 9 stripes of intervals of 430 Å in each half of the A-band in the region called the C-zone. Mutations in cardiac MyBP-C are a leading cause of hypertrophic cardiomyopathy the mechanism of which is unknown. It is a rod-shaped protein composed of 10 or 11 immunoglobulin- or fibronectin-like domains labelled C0 to C10 which binds to the thick filament via its C-terminal region. MyBP-C regulates contraction in a phosphorylation dependent fashion that may be through binding of its N-terminal domains with myosin or actin. Understanding the 3D organisation of MyBP-C in the sarcomere environment may provide new light on its function. We report here the fine structure of MyBP-C in relaxed rat cardiac muscle by cryo-electron tomography and subtomogram averaging of refrozen Tokuyasu cryosections. We find that on average MyBP-C connects via its distal end to actin across a disc perpendicular to the thick filament. The path of MyBP-C suggests that the central domains may interact with myosin heads. Surprisingly MyBP-C at Stripe 4 is different; it has weaker density than the other stripes which could result from a mainly axial or wavy path. Given that the same feature at Stripe 4 can also be found in several mammalian cardiac muscles and in some skeletal muscles, our finding may have broader implication and significance. In the D-zone, we show the first demonstration of myosin crowns arranged on a uniform 143 Å repeat.

A targeted approach to investigating immune genes of an iconic Australian marsupial.

Disease is a contributing factor to the decline of wildlife populations across the globe. Koalas, iconic yet declining Australian marsupials, are predominantly impacted by two pathogens, Chlamydia and koala retrovirus. Chlamydia is an obligate intracellular bacterium and one of the most widespread sexually transmitted infections in humans worldwide. In koalas, Chlamydia infections can present as asymptomatic or can cause a range of ocular and urogenital disease signs, such as conjunctivitis, cystitis and infertility. In this study, we looked at differences in response to Chlamydia in two northern populations of koalas using a targeted gene sequencing of 1209 immune genes in addition to genome-wide reduced representation data. We identified two MHC Class I genes associated with Chlamydia disease progression as well as 25 single nucleotide polymorphisms across 17 genes that were associated with resolution of Chlamydia infection. These genes are involved in the innate immune response (TLR5) and defence (TLR5, IFNγ, SERPINE1, STAT2 and STX4). This study deepens our understanding of the role that genetics plays in disease progression in koalas and leads into future work that will use whole genome resequencing of a larger sample set to investigate in greater detail regions identified in this study. Elucidation of the role of host genetics in disease progression and resolution in koalas will directly contribute to better design of Chlamydia vaccines and management of koala populations which have recently been listed as "endangered."

Koala Retrovirus in Northern Australia Shows a Mixture of Stable Endogenization and Exogenous Lineage Diversification within Fragmented Koala Populations.

The koala population in northern Australia has become increasingly fragmented due to natural and man-made barriers and interventions. This situation has created a unique opportunity to study both endogenous and exogenous koala retrovirus (KoRV). To determine the impact that population isolation has had on KoRV diversity in Queensland, 272 koalas from six fragmented koala populations were profiled for their KoRV provirus across two natural biogeographical barriers (the St Lawrence Gap and the Brisbane Valley Barrier), one man-made geographical barrier (the city of Brisbane) and two translocation events (the single movement of koalas to an island and the repeated movement of koalas into a koala sanctuary). Analysis revealed that all koalas tested were KoRV-A positive, with 90 - 96% of the detected KoRV provirus from each koala representing a single, likely endogenous, KoRV-A strain. The next most abundant proviral sequence was a defective variant of the dominant KoRV-A strain, accounting for 3 - 10% of detected provirus. The remaining KoRV provirus represented expected exogenous strains of KoRV and included geographically localized patterns of KoRV-B, -C, -D, -F, -G, and -I. These results indicate that lineage diversification of exogenous KoRV is actively ongoing. In addition, comparison of KoRV provirus within known dam-sire-joey family groups from the koala sanctuary revealed that joeys consistently had KoRV proviral patterns more similar to their dams than their sires in KoRV-B, -C and -D provirus composition. Collectively, this study highlights both the consistency of endogenous KoRV and the diversity of exogenous KoRV across the fragmented koala populations in northern Australia.IMPORTANCE KoRV infection has become a permanent part of koalas in northern Australia. With KoRV presence and abundance linked to more severe chlamydial disease and neoplasia in these koalas, understanding how KoRV exists throughout an increasingly fragmented koala population is a key first step in designing conservation and management strategies. This survey of KoRV provirus in Queensland koalas indicates that endogenous KoRV provirus is ubiquitous and consistent throughout the state while exogenous KoRV provirus is diverse and distinct in fragmented koala populations. Understanding the prevalence and impact of both endogenous and exogenous KoRV will be needed to ensure a future for all koala populations.

Phylogenetic and geographical analysis of a retrovirus during the early stages of endogenous adaptation and exogenous spread in a new host.

Most retroviral endogenization and host adaptation happened in the distant past, with the opportunity to study these processes as they occurred lost to time. An exception exists with the discovery that koala retrovirus (KoRV) has recently begun its endogenization into the koala (Phascolarctos cinereus) genome. What makes this opportunity remarkable is the fact that Northern Australian koalas appear to be undergoing endogenization with one KoRV subtype (KoRV-A), while all subtypes (KoRV-A-I) coexist exogenously, and Southern Australian koalas appear to carry all KoRV subtypes as an exogenous virus. To understand the distribution and relationship of all KoRV variants in koalas, the proviral KoRV envelope gene receptor binding domain was assessed across the koala's natural range. Examination of KoRV subtype-specific proviral copy numbers per cell found that KoRV-A proviral integration levels were consistent with endogenous incorporation in Northern Australia (southeast Queensland and northeast New South Wales) while revealing lower levels of KoRV-A proviral integration (suggestive of exogenous incorporation) in southern regions (southeast New South Wales and Victoria). Phylogeographical analysis indicated that several major KoRV-A variants were distributed uniformly across the country, while non-KoRV-A variants appeared to have undergone lineage diversification in geographically distinct regions. Further analysis of the major KoRV-A variants revealed a distinct shift in variant proportions in southeast New South Wales, suggesting this as the geographical region where KoRV-A transitions from being predominantly endogenous to exogenous in Australian koalas. Collectively, these findings advance both our understanding of KoRV in koalas and of retroviral endogenization and diversification in general.

Koalas vaccinated against Koala retrovirus respond by producing increased levels of interferon-gamma.

Koala retrovirus (KoRV) is believed to be in an active state of endogenization into the koala genome. KoRV is present as both an endogenous and exogenous infection in all koalas in northern Australia. KoRV has been linked to koala pathologies including neoplasia and increased susceptibility to Chlamydia. A KoRV vaccine recently trialled in 10 northern koalas improved antibody response and reduced viral load. This communication reports the expression of key immune genes underlining the innate and adaptive immune response to vaccination in these northern koalas. The results showed that prior to vaccination, IL-8 was expressed at the highest levels, with at least 200-fold greater expression compared to other cytokines, while CD8 mRNA expression was significantly higher than CD4 mRNA expression level. Interferon-γ was up-regulated at both 4- and 8-weeks post-vaccination while IL-8 was down-regulated at 8-weeks post-vaccination.

High-throughput immunogenetic typing of koalas suggests possible link between MHC alleles and cancers.

Characterizing the allelic diversity within major histocompatibility complex (MHC) genes is an important way of determining the potential genetic resilience of a population to infectious and ecological pressures. For the koala (Phascolarctos cinereus), endemic diseases, anthropogenic factors and climate change are all placing increased pressure on this vulnerable marsupial. To increase the ability of researchers to study MHC genetics in koalas, this study developed and tested a high-throughput immunogenetic profiling methodology for targeting MHC class I UA and UC genes and MHC class II DAB, DBB, DCB and DMB genes in a population of 82 captive koalas. This approach was validated by comparing the determined allelic profiles from 36 koala family units (18 dam-sire-joey units and 18 parent-joey pairs), finding 96% overall congruence within family profiles. Cancers are a significant cause of morbidity in koalas and the risk factors remain undetermined. Our analysis of this captive population revealed several novel MHC alleles, including a potential link between the DBB*03 allele and a risk of developing cancer. This method offers a reliable, high-throughput protocol for expanded study into koala immunogenetics.

Koala immunogenetics and chlamydial strain type are more directly involved in chlamydial disease progression in koalas from two south east Queensland koala populations than koala retrovirus subtypes.

Chlamydial disease control is increasingly utilised as a management tool to stabilise declining koala populations, and yet we have a limited understanding of the factors that contribute to disease progression. To examine the impact of host and pathogen genetics, we selected two geographically separated south east Queensland koala populations, differentially affected by chlamydial disease, and analysed koala major histocompatibility complex (MHC) genes, circulating strains of Chlamydia pecorum and koala retrovirus (KoRV) subtypes in longitudinally sampled, well-defined clinical groups. We found that koala immunogenetics and chlamydial genotypes differed between the populations. Disease progression was associated with specific MHC alleles, and we identified two putative susceptibility (DCb 03, DBb 04) and protective (DAb 10, UC 01:01) variants. Chlamydial genotypes belonging to both Multi-Locus Sequence Typing sequence type (ST) 69 and ompA genotype F were associated with disease progression, whereas ST 281 was associated with the absence of disease. We also detected different ompA genotypes, but not different STs, when long-term infections were monitored over time. By comparison, KoRV profiles were not significantly associated with disease progression. These findings suggest that chlamydial genotypes vary in pathogenicity and that koala immunogenetics and chlamydial strains are more directly involved in disease progression than KoRV subtypes.

Vaccination of koalas during antibiotic treatment for Chlamydia-induced cystitis induces an improved antibody response to Chlamydia pecorum.

Chlamydia infection and disease are endemic in free-ranging koalas. Antibiotics remain the front line treatment for Chlamydia in koalas, despite their rates of treatment failure and adverse gut dysbiosis outcomes. A Chlamydia vaccine for koalas has shown promise for replacing antibiotic treatment in mild ocular Chlamydia disease. In more severe disease presentations that require antibiotic intervention, the effect of vaccinating during antibiotic use is not currently known. This study investigated whether a productive immune response could be induced by vaccinating koalas during antibiotic treatment for Chlamydia-induced cystitis. Plasma IgG antibody levels against the C. pecorum major outer membrane protein (MOMP) dropped during antibiotic treatment in both vaccinated and unvaccinated koalas. Post-treatment, IgG levels recovered. The IgG antibodies from naturally-infected, vaccinated koalas recognised a greater proportion of the MOMP protein compared to their naturally-infected, unvaccinated counterparts. Furthermore, peripheral blood mononuclear cell gene expression revealed an up-regulation in genes related to neutrophil degranulation in vaccinated koalas during the first month post-vaccination. These findings show that vaccination of koalas while they are being treated with antibiotics for cystitis can result in the generation of a productive immune response, in the form of increased and expanded IgG production and host response through neutrophil degranulation.

Helping koalas battle disease - Recent advances in Chlamydia and koala retrovirus (KoRV) disease understanding and treatment in koalas.

The iconic Australian marsupial, the koala (Phascolarctos cinereus), has suffered dramatic population declines as a result of habitat loss and fragmentation, disease, vehicle collision mortality, dog attacks, bushfires and climate change. In 2012, koalas were officially declared vulnerable by the Australian government and listed as a threatened species. In response, research into diseases affecting koalas has expanded rapidly. The two major pathogens affecting koalas are Chlamydia pecorum, leading to chlamydial disease and koala retrovirus (KoRV). In the last eight years, these pathogens and their diseases have received focused study regarding their sources, genetics, prevalence, disease presentation and transmission. This has led to vast improvements in pathogen detection and treatment, including the ongoing development of vaccines for each as a management and control strategy. This review will summarize and highlight the important advances made in understanding and combating C. pecorum and KoRV in koalas, since they were declared a threatened species. With complementary advances having also been made from the koala genome sequence and in our understanding of the koala immune system, we are primed to make a significant positive impact on koala health into the future.

Antibiotic treatment of Chlamydia-induced cystitis in the koala is linked to expression of key inflammatory genes in reactive oxygen pathways.

Chlamydial-induced cystitis in the koala (Phascolarctos cinereus) is currently treated by antibiotics. However, while reducing the chlamydial load, this treatment can also lead to gastrointestinal complications and death. Development of alternative treatments, such as a therapeutic chlamydial vaccine, are hindered by the lack of detailed understanding of the innate immune response to chlamydial clearance and disease regression during antibiotic treatment. Through clinical, microbiological and transcriptomic approaches, disease regression, bacterial clearance and innate immune responses were mapped in koalas with signs of chlamydial-induced cystitis while receiving anti-chlamydial antibiotics. Significant reduction in the signs of cystitis were observed during and post antibiotic treatment. This was observed as a thinning of the bladder wall and complete reversal of urinary incontinence. Transcriptomic analysis before treatment, at the end of treatment and prior to release identified significant down-regulation of specific genes involved in 21 biological pathways. Of these, the chemokine receptor signalling and NOD-like receptor signalling pathways where identified as important markers of inflammation. Specific genes within these pathways (NCF1 and NOX2) were significantly down-regulated, suggesting a decrease in reactive oxygen species production. Through the monitoring of specific clinical and transcriptomic markers, these findings allow detailed profiling of the clinical response to therapeutic vaccination in koalas with current signs of disease. This also adds to our understanding of innate immune responses to chlamydial infections and indicates that chlamydial-induced cystitis in the koala is linked to the regulation of reactive oxygen pathways.

Changes in Endogenous and Exogenous Koala Retrovirus Subtype Expression over Time Reflect Koala Health Outcomes.

Koala retrovirus (KoRV) is unique in that it exists as both an exogenous and actively endogenizing gamma retrovirus of koalas. While nine subtypes of KoRV have been recognized, focused study of these subtypes in koalas over time and with different health outcomes has been lacking. Therefore, in this study, three wild koala cohorts were established and monitored to examine KoRV proviral and expression data from koalas that either remained healthy over time, began healthy before developing chlamydial cystitis, or presented with chlamydial cystitis and were treated with antibiotics. Deep sequencing of the proviral KoRV envelope gene revealed KoRV-A, -B, -D, and -F to be the major subtypes in this population and allowed for subtype-specific assays to be created. Quantification of KoRV transcripts revealed that KoRV-D expression mirrored the total KoRV expression levels (106 copies/ml of plasma), with KoRV-A and KoRV-F expression being ∼10-fold less and KoRV-B expression being ∼100-fold less, when detected. Strikingly, there was significantly higher expression of KoRV-D in healthy koalas than in koalas that developed chlamydial cystitis, with healthy koalas expressing a major KoRV-D/minor KoRV-A profile, whereas koalas that developed cystitis had variable KoRV expression profiles. Total anti-KoRV IgG antibody levels were found not to correlate with the expression of total KoRV or any individual KoRV subtype. Finally, KoRV expression was consistent between systemic and mucosal body sites and during antibiotic treatment. Collectively, this gives a comprehensive picture of KoRV dynamics during several important koala health states.IMPORTANCE The long-term survival of the koala is under serious threat, with this iconic marsupial being declared "vulnerable" by the Australian Government and officially listed as a threatened species. KoRV is clearly contributing to the overall health status of koalas, and research into this virus has been lacking detailed study of the multiple subtypes at both the proviral and expressed viral levels over time. By designing new subtype-specific assays and following well-defined koala cohorts over time, this study has generated a new more complete picture of KoRV and its relationship to koala health outcomes in the wild. Only by building a comprehensive picture of KoRV during both koala health and disease can we bring meaningful koala health interventions into better focus.

Molecular Dynamics and Mode of Transmission of Koala Retrovirus as It Invades and Spreads through a Wild Queensland Koala Population

The recent acquisition of a novel retrovirus (KoRV) by koalas (Phascolarctos cinereus) has created new opportunities for retroviral research and new challenges for koala conservation. There are currently two major subtypes of KoRV: KoRV-A, which is believed to be endogenous only in koalas from the northern part of Australia, and KoRV-B, which appears to be exogenous. Understanding and management of these subtypes require population level studies of their prevalence and diversity, especially when coinfected in the same population, and investigations of their modes of transmission in the wild. Toward this end, we studied a wild Queensland koala population of 290 animals over a 5-year period and investigated the prevalence, diversity and mode of transmission of KoRV-A and KoRV-B. We found KoRV-A to have an infection level of 100% in the population, with all animals sharing the same dominant envelope protein sequence. In contrast, the KoRV-B infection prevalence was only 24%, with 21 different envelope protein sequence variants found in the 83 KoRV-B-positive animals. Linked to severe disease outcomes, a significant association between KoRV-B positivity and both chlamydial disease and neoplasia was found in the population. Transmission of KoRV-B was found at a rate of 3% via adult-to-adult contact per year, while there was a 100% rate of KoRV-B-positive mothers transmitting the virus to their joeys. Collectively, these findings demonstrate KoRV-B as the pathogenic subtype in this wild koala population and inform future intervention strategies with subtype variation and transmission data. IMPORTANCE KoRV represents a unique opportunity to study a relatively young retrovirus as it goes through its molecular evolution in both an endogenous form and a more recently evolved exogenous form. The endogenous form, KoRV-A, now appears to have stably and completely established itself in Northern Australian koala populations and is progressing south. Conversely, the exogenous form, KoRV-B, is undergoing continuous mutation and spread in the north and, as yet, has not reached all southern koala populations. We can now link KoRV-B to neoplasia and chlamydial disease in both wild and captive koalas, making it an imminent threat to this already vulnerable species. This work represents the largest study of koalas in a wild population with respect to KoRV-A/KoRV-B-infected/coinfected animals and the linkage of this infection to chlamydial disease, neoplasia, viral evolution, and spread.