Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population

Abstract

Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala retrovirus (KoRV) is a gammaretrovirus present in most wild koala populations and captive colonies. Five subtypes of KoRV (A to E) have been identified based on amino acid sequence divergence in a hypervariable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has been conducted primarily on KoRV in captive koalas housed in zoos in Japan, the United States, and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from southeast Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into three major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hypervariable region subtypes (C, D, and E) as well as four hypervariable region subtypes that we newly define here (F, G, H, and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D, and F were found to be at high prevalence, while subtypes G, H, and I were present in a smaller number of animals. IMPORTANCE Koala retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence among a wild koala population, and it significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance.

Differential and defective expression of Koala Retrovirus indicate complexity of host and virus evolution

“…Pseudomapping of the sequence reads to the KoRV A genome (complete gag, pro-pol and env genes) and type sequences of the hypervariable region of the env gene (base pairs 6000-6575 of KoRV A) of each of the previously identified KoRV subtypes (KoRV A to I as per the classification scheme used in Chappell et.al. 2016 21 ) demonstrated that while QLD koalas had multiple subtypes within individuals, SA animals had far lower KoRV subtype diversity. Significantly different expression was observed for KoRV A,B,D,E and G variants between QLD and SA samples (unpaired t-test with unequal variance, P<0.05) (Figure 3).…”

“…A number of sequence variants of the env gene region, which encodes the surface unit (SU) of the envelope protein (Env), have also been identified (Figure 1). These vary between individuals and resemble the viral quasispecies common to infectious retroviruses 21 . There has been debate as to whether the KoRV B/J variant, which displays a different receptor usage to KoRV A, is an exogenous virus as it has been epidemiologically linked with clinical disease, however this is still unresolved 22 23 .…”

Assessing the significance of endemic disease in conservation—koalas, chlamydia, and koala retrovirus as a case study

“…Ishida, Zhao, Greenwood, and Roca (2015) estimated a maximum age for KoRV-A invasion of the koala germ line as 22,200-49,900 years ago, reflecting a lengthy association. Other subtypes are found with fewer copies and lower prevalence and can be geographically restricted in their distribution; these other subtypes appear to be primarily horizontally transmitted (Chappell et al, 2017).…”

“…Nine KoRV subtypes in three clades have thus far been described from wild or captive koala populations (Chappell et al., ). One of these, KoRV‐A, appears to be have been integrated into the genome of some koala populations (endogenized), and is found as multiple copies at 100% prevalence in some populations (Queensland and New South Wales) (Legione et al., ; Oliveira, Satija, Kouwenhoven, & Eiden, ).…”

The piRNA Response to Retroviral Invasion of the Koala Genome

“…Analysis of hybrid dysgenesis in Drosophila has provided insights into piRNA pathway adaptation to DNA transposon invasion in insects [16][17][18] , but the response to invasions of the mammalian genome has not been examined. KoRV-A is associated with leukemia, immunodeficiency and chlamydia infection in koala, and characterization of proviral integration led to the surprising discovery that the virus had entered the koala germline in the last 50,000 years and is spreading by an unusual combination of vertical and horizontal transfers 4,5,19,20 . The virus is believed to have entered the wild population at the northern end of Australia and swept south, where a few naive populations persist 5 .…”