Retroviral invasion of the koala genome

Rachael opened her talk by outlining koala ecology and natural history. Koalas are restricted to the eastern seaboard of Australia, primarily because this is where their food is located. They spend most of their time up large trees and they are fussy eaters, only eating eucalyptus leaves - which are actually very toxic, and therefore koalas require specialised gut microflora for their digestion. Young koalas are born very immature and, as marsupials, the last two-thirds of their gestation occurs in the mother's pouch. Rachael explained that there are two key considerations about koala ecology and natural history for this talk - firstly, the fact that they spend most of their lives up tall trees means that they are difficult to work with scientifically. Secondly, Southern koalas were almost hunted to extinction in the late 1800s/early 1900s, and were then restocked from small island populations, meaning that the population became, and still is, very genetically restricted.

In terms of koala conservation, Rachael described how we face a number of problems for its successful implementation. For starters, even the number of koalas is disputed, with a wide predicted range between 40,000 and 400,000; even so, and despite past intensive hunting, these numbers are "really drastically reduced compared to where they were". As a result, there is a big argument over the ICUN protected status. The biggest threat to the population is habitat loss due to deforestation for house building in urban areas. Rachael further explained how the species distribution is unbalanced, with the Northern populations doing less well in terms of numbers compared to the Southern population. With overabundance in some areas, such as Kangaroo island, culling and desexing of koalas is often performed to manage overbreeding and population numbers. Other major causes of their death include cars, dogs, and disease.

Focusing on koala diseases, there is a high incidence of Chlamydia infection in the population. As urinogenitary tract infections are one result of this disease, this impacts their ability to breed. Another result is eye infection, and if they cannot see then they cannot eat. However, even though there is a high incidence of disease, actual clinical, symptomatic disease is not always present, as opposed to an asymptomatic carrier status. What is the difference between those koalas which contract clinical disease versus those that remain asymptomatic?

There is also very high incidence of leukaemia and lymphoma in koalas - up to 40% in captive koalas, compared to an average incidence of 1% in wild koalas. This is typically a retroviral disease, whereby leukaemia/lymphoma is thought to result from retroviral-mediated immunosuppression. Yet Rachael explained how "retroviruses are strange creatures"; the ones that infect koalas are different to the ones that we are more familiar with, like HIV. Retroviruses integrate into the host cell's genome and if these cells are germ cells, then they become inheritable. About 8% of the human genome consists of integrated ancient retroviruses. Rachael said that it is not great having them "hopping around", causing cancer, so the host genome has a lot of mechanisms to inactivate them. In the long term, retrotransposition usually gets shut down by mutation, and the virus no longer functions as an infectious virus.

Koala retrovirus (KoRV) is an unusual endogenous (inherited) retrovirus which is able to integrate into the koala genome. KoRV is still a relatively new virus - it is a new entrant into the koala genome (originally integrating <49,000 years ago), and it is highly polymorphic. It is very similar to pathogenic exogenous viruses that infect Gibbons (GaLV); Rebecca described how KoRV was transferred to Gibbons by accident in the laboratory from infected rats. Similar retroviruses have been found in other animal species, such as in flying foxes, and other animals which are generally unrelated to each other; it is not known how these viruses are able to transfer between each other.

Rachael described the varying prevalence of KoRV in koala populations - there is 100% prevalence in Northern koalas, yet not all koalas in Southern Australia are infected. In fact, there is a gradient of decreasing prevalence from the North to South, which looks much like the spread of an infectious disease. Rachael asked - is this variability due to different strains infecting the populations? Also, how do we vaccinate against it? Delivery of a vaccine is "not a very practical solution for a wildlife problem", as the koalas live high up in trees.

There are pathology differences between the Southern Australian and Queensland populations. Much higher KoRV loads are presnt in the North compared to the South; disease is associated with decreased load. The disease oxalate nephrosis is only detected in Southern koalas, probably due to to genetic restriction as opposed to viral loads. On the other hand, neoplasias and chlamydia are far more common in Northern animals. Rachael described how short-read sequencing of koala populations identified the presence of KoRV in the Northern koalas, yet identified that Southern koalas also had the virus. However, the sequencing reads were only mapping to the viral LTRs, which are present at either ends of the viral genome, meaning that the middle section of the viral genome was missing. Rachael explained that this has important implications for viral biology - having a mutated or truncated form of a retrovirus can protect from the infectious form. For example, if the env genes are present and expressed, viral envelope proteins can bind to cell receptors and prevent the infectious form entering the host cell. She stated how therefore there could be positive selection for the truncated form of KoRV for disease prevention.

Rachael next described her application of nanopore technology to sequence KoRV in the Southern koala population. This arose because sequencing across the entire viral genome was required, according to publication reviewers, to validate her findings and hypothesis, yet short sequencing reads had mapping problems and PCR was unsuccessful in amplifying a genome flanked by problematic repeats. Therefore, Rachael turned to long-read nanopore sequencing for resolution of the entire KoRV genome. Using the PromethION platform, one Southern koala genome has been resequenced; even though this koala had in fact resided in Longleat, UK, it was originally from the Southern Australian population. Sequencing of this koala's genome on the PromethION was only achieved earlier this week! The run produced >3 million reads with read length N50 of >38 kb; from this, 94 reads matched the KoRV reference sequence AF151794. Such a low read number was expected as it is known that the retrovirus is present at low copy number in the genome. Rachael stated how, thanks to nanopore long-read sequencing, they managed to achieve their goal of sequencing across the entire retroviral genome. Both the full KoRV genome, as well as combined 5' and 3' LTRs, were identified in the sequencing data. Rachael also described how she would like to apply CRISPR targeting for KoRV sequence enrichment, followed by nanopore sequencing, which would susbtantially reduce the cost and time of sequencing compared to sequencing the whole koala genome.

Rachael concluded her talk by describing the impact of this research on koala conservation. As vaccination against KoRV is impractical, other solutions need to be used; if the truncated form of the virus does protect animals from disease, then selective breeding can be performed. She said that "this will be: watch this space!"