Detection and Differentiation of Two Koala Gammaherpesviruses by Use of High-Resolution Melt (HRM) Analysis Reveals Differences in Viral Prevalence and Clinical Associations in a Large Study of Free-Ranging Koalas

The iconic koala () is host to two divergent gammaherpesviruses, phascolarctid gammaherpesviruses 1 and 2 (PhaHV-1 and -2), but the clinical significance of the individual viruses is unknown and current diagnostic methods are unsuitable for differentiating between the viruses in large-scale studies. To address this, we modified a pan-herpesvirus nested PCR to incorporate high-resolution melt analysis. We applied this assay in a molecular epidemiological study of 810 koalas from disparate populations across Victoria, Australia, including isolated island populations. Animal and clinical data recorded at sampling were analyzed and compared to infection status. Between populations, the prevalence of PhaHV-1 and -2 varied significantly, ranging from 1% to 55%. Adult and older animals were 5 to 13 times more likely to be positive for PhaHV-1 than juveniles ( < 0.001), whereas PhaHV-2 detection did not change with age, suggesting differences in how these two viruses are acquired over the life of the animal. PhaHV-1 detection was uniquely associated with the detection of koala retrovirus, particularly in females ( = 0.008). Both viruses were significantly associated ( < 0.05) with the presence of genital tract abnormalities (uterine/ovarian cysts and testicular malformation), reduced fertility in females, urinary incontinence, and detection of , although the strength of these associations varied by sex and virus. Understanding the clinical significance of these viruses and how they interact with other pathogens will inform future management of threatened koala populations.