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

P. K. Vaz,a A. R. Legione,a C. A. Hartley,a J. M. Devlina

aAsia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia

ABSTRACT The iconic koala (Phascolarctos cinereus) is host to two divergent gam-maherpesviruses, phascolarctid gammaherpesviruses 1 and 2 (PhaHV-1 and -2), butthe clinical significance of the individual viruses is unknown and current diagnosticmethods 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 studyof 810 koalas from disparate populations across Victoria, Australia, including isolatedisland populations. Animal and clinical data recorded at sampling were analyzed andcompared 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 to13 times more likely to be positive for PhaHV-1 than juveniles (P � 0.001), whereasPhaHV-2 detection did not change with age, suggesting differences in how thesetwo viruses are acquired over the life of the animal. PhaHV-1 detection was uniquelyassociated with the detection of koala retrovirus, particularly in females (P � 0.008).Both viruses were significantly associated (P � 0.05) with the presence of genitaltract abnormalities (uterine/ovarian cysts and testicular malformation), reduced fertil-ity in females, urinary incontinence, and detection of Chlamydia pecorum, althoughthe strength of these associations varied by sex and virus. Understanding the clinicalsignificance of these viruses and how they interact with other pathogens will informfuture management of threatened koala populations.

KEYWORDS Diagnostic, HRM, disease, herpesviruses, koala, marsupial, wildlife

Koalas (Phascolarctos cinereus) are iconic marsupials native to Australia. They are theonly extant member of the family Phascolarctidae. In the wild, these arboreal

herbivores inhabit coastal areas of the eastern and southern mainland. Captive popu-lations of koalas are found in zoological collections throughout Australia and aroundthe world. Wild koalas are vulnerable to a variety of known microbial and environmen-tal threats, and population declines have led to them being declared a vulnerablespecies in the central and northern coasts of eastern Australia. Environmental threatsinclude habitat fragmentation and destruction, human encroachment, and environ-mental change. Limited genetic diversity is also present in some populations due tohistorical population bottleneck events that have seen reduced population sizes re-sulting from environmental events or human activities (1). Key microbial threats includeinfection with Chlamydia pecorum and koala retrovirus (KoRV) (2–6), while less well-known microbial threats include the more recently identified koala herpesviruses,phascolarctid gammaherpesviruses 1 and 2 (PhaHV-1 and PhaHV-2, respectively) (7, 8).Herpesviruses have been studied in a number of different marsupial species, includingkoalas, but diagnostic tools are currently limited to serum virus neutralization assays

Citation Vaz PK, Legione AR, Hartley CA, DevlinJM. 2019. Detection and differentiation of twokoala gammaherpesviruses by use of high-resolution melt (HRM) analysis revealsdifferences in viral prevalence and clinicalassociations in a large study of free-rangingkoalas. J Clin Microbiol 57:e01478-18. https://doi.org/10.1128/JCM.01478-18.

Editor Brad Fenwick, University of Tennesseeat Knoxville

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to P. K. Vaz,pvaz@unimelb.edu.au.

Received 20 September 2018Returned for modification 6 October 2018Accepted 21 December 2018

Accepted manuscript posted online 9January 2019Published

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and PCR assays followed by amplicon cloning and sequencing (9–11). Serum virusneutralization assays have not been performed for detection of antibodies to PhaHV-1and -2, which are limited by the lack of availability of cells suitable for virus propagationand may also be complicated by cross-neutralization of these viruses, which is commonin herpesviruses (12, 13). PCR cloning and sequencing techniques are effective for thedetection and differentiation of PhaHV-1 and -2, but the process is inefficient and costlyfor large-scale population studies, particularly when resources for investigation ofinfectious diseases in wildlife is limited. Thus, prior studies examining virus prevalenceusing direct amplicon sequencing are likely to have underestimated the prevalence ofPhaHV-1 and PhaHV-2 within populations (11).

The lack of cost-effective tools to detect and discriminate between the two koalagammaherpesviruses has limited our ability to understand the epidemiology andclinical significance of the two viruses. In a previous study of koalas, a strong association(odds ratio � 60; P � 0.001; 95% confidence interval [CI], 21 to 298) was reportedbetween the detection of herpesviruses and the detection of C. pecorum, as well as astrong association with the presence of urogenital disease (11). However, due to thesmall size of that study, only broad associations could be drawn and disentangling theimpact of individual viruses was not possible. In other marsupials, herpesvirus infectionhas been associated with fatal systemic disease and severe clinical signs in a number ofspecies, mostly in the form of respiratory disease and ulcerative cloacitis (12, 14–16),although these are mostly attributable to infection with members of the Alphaherpes-virinae subfamily. Less is known about the impact of gammaherpesviruses within theMarsupialia, although macropodid gammaherpesvirus 3 (MaHV-3) has been linked torespiratory disease and mammary gland tumors in eastern grey kangaroos (Macropusgiganteus) (12, 17).

To address the gap in our knowledge of herpesvirus infections in koalas, this studysought to develop an alternative method of koala herpesvirus detection and differen-tiation that could be used as a primary tool of herpesvirus surveillance in largepopulations. The assay was then applied in a molecular epidemiological study ofherpesviruses using an archived sample bank consisting of swab, blood, and tissuesamples collected from 810 koalas from disparate geographical regions and spanningthe years 2010 to 2015. Prior Chlamydia and KoRV detection data and samples fromthese animals were available from previous studies (11, 18–20) and were used in ouranalyis. The study aimed to assess the potential interaction of the koala herpesviruseswith other infectious agents and to identify any clinical variables that may be associ-ated with PhaHV-1 or PhaHV-2 infection.

MATERIALS AND METHODSSample collection from distinct Victorian koala populations. Over a period of 6 years (2010 to

2015), clinical samples were collected from geographically distinct Victorian (Australia) koala populationsduring various research field trips, management programs, and postmortem examinations. Additionalsamples were available from prior studies (7, 11, 18–20). Sample collection was approved by TheUniversity of Melbourne Animal Ethics Committee (approval numbers 1011687.1 and 1312813.2) andParks Victoria (Research Permits 10004605, 10006948, and 10005388).

Sampled animals were allocated into geographical regions based on boundaries that were estab-lished during prior studies investigating koala chlamydial and retrovirus prevalence. These differentgeographical populations are French Island, Raymond Island, South Coast, Far West, Gippsland, Morn-ington Peninsula, Zoo (captive animals), and Other (Fig. 1) (19, 20). French Island is a closed koalapopulation, with no recorded introductions for over 100 years (21, 22). Populations on Raymond Island,in eastern Victoria, and the South Coast and Far West populations in western Victoria, arose predomi-nantly through translocations from French Island over the last 50 years (21, 22). Large geographicaldistances separate the mainland populations. The distance between the major forested regions (GreatOtway National Park and Mt. Eccles National Park) in the two closest mainland populations (South Coastand Far West, respectively) is approximately 150 km, with limited connected corridor habitat spanningthis distance. Additional samples were sourced from clinical specimens submitted to the MelbourneVeterinary School for routine diagnostic purposes. Most samples were swabs collected from rostral sites(nasal, oropharyngeal, and ocular; n � 319) or caudal sites (cloacal and urogenital; n � 793). Bloodsamples were collected from 80 anaesthetized animals, and buffy coat fractions were separated wherepossible and added to RNAlater stabilization solution (Thermo Fisher Scientific, USA) (19). A small numberof animals included in this study (n � 41) were euthanized for animal welfare reasons unrelated to this

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study. These animals were necropsied and examined for the presence of genital or urinary tract grosspathology (11, 18–20).

At time of sampling, animals were examined by veterinary professionals and relevant signalment andclinical information was recorded. Age was estimated based on tooth wear class (where available) usinga previously described methodology (23), and animals were classified into juvenile, adult, and maturegroups to be consistent with previous studies on these populations (18–20). Clinical information includedthe body condition score (BCS) of the animal, based on palpation of the muscle mass over the scapula,from 1 to 5, where a score of 1 is emaciated, 3 is healthy, and 5 is obese (20). The presence ofreproductive abnormalities (uterine/ovarian cysts and testicular malformation) and urinary tract abnor-

FIG 1 (A) Map of Victoria, Australia, showing the geographical regions where koala populations were sampled from 2010 to 2015. (B) Distributionof sampling sites for those animals for which a sampling location was known. Relative sampling size per site is indicated by the size of the circle.Base maps used for panel B and the inset (Australia), and state boundaries were sourced from Geoscience Australia (accessed 8 April 2009) andreproduced under Creative Commons 4.0 (https://researchdata.ands.org.au/geodata-topo-250k-file-format/1278865).

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malities (cystitis) was detected via ultrasound or gross pathology (24). Fecundity in females wasmeasured based on the presence of back or pouch young, and lymph node enlargement was used as aproxy for potential lymphoid leukemias. Also recorded was the presence of urogenital disease, specifi-cally wet bottom, a condition indicating urinary incontinence.

Detection and differentiation of PhaHV-1 and PhaHV-2 using PCR and HRM analysis. To detectand differentiate PhaHV-1 and PhaHV-2, a pan-herpesvirus nested PCR assay that amplifies a conservedregion of the herpesvirus DNA polymerase gene (10, 25) was modified to incorporate high-resolutionmelt (HRM) curve analysis. Used in its unmodified form, the final nested PCR product from the assay isvisualized using agarose gel electrophoresis and has a lower limit of detection at or below 100 copies per100 ng of carrier DNA (25). In the present study, the assay was modified to include SYTO9 in the reactionand incorporate an HRM component. These allow amplification to be detected by fluorescence and thespecificity of the amplicon to be determined by the specific HRM pattern resulting from the uniquesequence of each of these herpesviruses.

Briefly, for the modified assay, DNA was extracted from clinical samples using VX universal liquidsample DNA extraction kits (Qiagen, Germany) and a Corbett X-tractor Gene Robot (Corbett Robotics,Australia). This extraction protocol used 200 �l of supernatant from swabs suspended in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 [pH 7.4]), Tris-EDTA buffer(10 mM Tris, 1 mM EDTA [pH 8.0]), or RLT buffer (Qiagen, Germany) and stored at �20°C. DNA wassimilarly extracted from 200 �l of whole-blood or buffy coat samples stored in RNAlater (Sigma-Aldrich,USA). Some nucleic acid extractions were originally performed for previous studies (18, 19) and wererepurposed for this study.

Five microliters of extracted DNA was used as the template in the first round of this nested PCR, with400 nM primers DFA, ILK, and KGI (5=-GAYTTYGCIAGYYTITAYC, 5=-TCCTGGACAAGCAGCARIYSGCIMTIAA,and 5=-GTCTTGCTCACCAGITCIACICCYTT, respectively) (10, 25) and 200 �M each deoxynucleosidetriphosphate (dNTP), 1 mM MgCl2, 5� GoTaq colorless Flexi buffer, and 1 U of GoTaq Flexi DNApolymerase (Promega, USA). Samples were incubated at 95°C for 5 min, followed by 45 cycles of 95°C for30 s, 46°C for 30 s, and 72°C for 90 s. In the second round, 5 �l of the first-round product was used as thetemplate, including 800 nM primers TGV and IYG (5=-TGTAACTCGGTGTAYGGITTYACIGGIGT and 5=-CACAGAGTCCGTRTCICCRTAIAT, respectively) (10, 25), 8 �M SYTO9 green fluorescent nucleic acid stain(Thermo Fisher Scientific, USA), 200 �M each dNTP, 1 mM MgCl2, 5� GoTaq colorless Flexi buffer, and 1U of GoTaq Flexi DNA polymerase (Promega, USA). Reaction mixtures were incubated at 95°C for 5 min,followed by 45 cycles of 95°C for 30 s, 46°C for 30 s, and 72°C for 60 s. In order to differentiate ampliconsfrom PhaHV-1 and PhaHV-2, the products from the second round of the PCR assay were subjected totemperature increments of 0.1, 0.15, 0.2, and 0.3 between 70°C and 95°C. High-resolution melt analysiswas applied to the PCR amplicons using a Rotor-Gene 6000 or Rotor-Gene-Q (Qiagen, Germany).Positive herpesvirus HRM controls were PhaHV-1 or PhaHV-2 (220 to 230 bp) products from the finalround of the nested PCR cloned into plasmids (pGEM-T; Promega, USA), constructed as part of a priorstudy (7), specifically, 0.01 ng of each plasmid clone was used in each control reaction in thesecond-round PCR. Template-free and negative extraction controls consisted of sterile PBS or sterilewater. Positive extraction controls used supernatant from cell cultures infected with the infectiouslaryngotracheitis herpesvirus.

Most of the samples used in this study had previously been tested for C. pecorum (18, 20) and KoRV(19). Briefly, to detect C. pecorum, DNA extracted from rostral or caudal swabs using the above-describedmethods was used as the template in a quantitative PCR (qPCR) targeting the 16S rRNA gene (26). Todetect KoRV, DNA extracted from blood or other samples, as described previously (19), was used as thetemplate in a qPCR targeting the KoRV pol gene as previously described (4, 19).

Statistical analyses. Univariable and multivariable statistical analyses were conducted to identifyany variables associated with the detection of PhaHV-1 and/or PhaHV-2 using Minitab version 18 (MinitabInc.) as per reference 19. Univariable binary logistic regression was performed to estimate the associationof each explanatory variable with each outcome variable, that is, with the presence or absence of eachherpesvirus. Variables with P values of �0.05 was considered significant based on the likelihood ratiotest. Any variables with P values of �0.25 were also considered for the multivariable model, as were anypotential confounders.

Multivariable logistic regression models were fitted to the data using a backwards-eliminationstepwise approach. At each step, all eligible candidate variables were individually tested and removedfrom the model if they had a P value of �0.1 based on the likelihood ratio test.

Explanatory variables included sex, age, location (pooled into seven broader regions, excluding Other[Fig. 1]), wet-bottom presence/absence, lymphoid node enlargement, C. pecorum presence/absence,KoRV presence/absence, the presence of a second herpesvirus, BCS, reproductive and urinary tractabnormality, presence/absence of pouch young in females, swab site (caudal or rostral), and samplingyear. Univariable and multivariable analyses were also repeated using male-only and female-onlyinfection data to determine which associations may be influenced by the sex of the animal. Sensitivityanalysis of population data was performed for each variable to identify skew due to bias introduced atsampling.

RESULTSHRM curve analysis. Both PhaHV-1 and PhaHV-2 final amplicons produced a

single-peak melt curve, with a 4°C separation between the melt profiles of the twoviruses (Fig. 2). The average peaks for the PhaHV-1 and PhaHV-2 replicates were 83°C

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and 87°C, respectively. The melt curves of the amplicons from clinical samples knownto be positive for PhaHV-1 (koalas A, B, and C) and PhaHV-2 (koalas C, D, and E) wereconsistent with PhaHV-1 and -2 plasmid controls (Fig. 2). The melting profile of theamplicon derived from a clinical sample that was sequence confirmed positive for bothPhaHV-1 and -2 (koala C) showed two melt curves in the HRM analysis, correspondingwith that of PhaHV-1 and PhaHV-2 (Fig. 2).

PCR and HRM analysis were applied to the koala sample collection, where theamplicons derived from each sample were classified as PhaHV-1 or PhaHV-2 or boththrough visual inspection of melt profiles. Amplicons from five animal samples in whichviruses were codetected were cloned and sequenced to confirm that they representedtrue dual infections. Ambiguous peaks (due to low amplification) were confirmedthrough visualization of the amplicons in an agarose gel. If the viral status of a samplewas not able to be resolved (n � 9 samples), those samples were excluded from furtheranalysis. Ultimately, samples from a total of 810 animals were tested for viral DNA andsuccessfully genotyped for viral species. An animal was considered positive for thatvirus if any one or more of the multiple samples from a single animal were positive byPCR HRM.

Prevalence of PhaHV-1 and PhaHV-2 in Victorian koala populations. The prev-alence of PhaHV-1 detection in the different populations ranged from 7.4% to 45.5%,and PhaHV-2 detection ranged from 0.9% to 54.6% (Fig. 3; see also Table S1 in thesupplemental material). The South Coast population was selected as the reference dueto the large number of animals tested (204 animals). Koalas in each of the MorningtonPeninsula, Raymond Island, Gippsland, and Far West populations were significantlymore likely to be detected with PhaHV-1 (21.8% to 45.5%) than the reference popula-tion (7.4% [Fig. 3]). Comparatively, for PhaHV-2, the only population that was signifi-cantly different to the reference population (30.4%) was French Island (0.9% [Fig. 3;Table S1]).

Herpesvirus prevalence was compared across multiple years in four populations:South Coast, Far West, French Island, and Raymond Island (Fig. 3; Table S2). A significant

FIG 2 Normalized HRM curves of the plasmid standards containing the nested pan-herpesvirus inserts of PhaHV-1 and PhaHV-2, as well as HRM curves fromclinical samples known to be positive for PhaHV-1 and/or PhaHV-2. Amplicons shown were dissociated at a temperature increment of 0.2°C. Koalas A and Bwere sequence confirmed to be positive for PhaHV-1 DNA, koalas D and E were sequence confirmed to be positive for PhaHV-2 DNA, and koala C was sequenceconfirmed to be positive for PhaHV-1 and PhaHV-2 DNA.

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decrease in PhaHV-1 prevalence was identified only in the Raymond Island population,from 45% in 2010 to 17% in 2013, while the prevalence of PhaHV-2 decreasedsignificantly in the South Coast koalas, from 39% in 2013 to 22% in 2015.

Relationship between PhaHV-1 and PhaHV-2 detection and animal age andsex. All univariable analyses were performed using female-only and male-only data, aswell as combined animal data (both sexes) where possible (Fig. 3 to 6; Tables S3 to S5).Detection of PhaHV-1 increased with age in both sexes (Fig. 4), with adult and olderanimals 5 and 13 times more likely to be positive for PhaHV-1 than juveniles, respec-tively, ranging from 5.1% to 45.1% (P � 0.001) (Fig. 4; Table S1). Conversely, PhaHV-2detection did not change significantly across age groups, regardless of sex (19.5% to27.6% [Fig. 4; Table S1]). Females without young were 1.7, 1.8, and 2.5 times more likelyto be positive for PhaHV-1 or PhaHV-2 or coinfected with PhaHV-1 and PhaHV-2,respectively, than females with young (Fig. 5; Table S4).

Relationship between PhaHV-1 and PhaHV-2 detection and clinical observa-tions. (i) Site of PhaHV-1 and PhaHV-2 detection. The detection of PhaHV-1 andPhaHV-2 at different anatomical sites, specifically rostral sites (eye/nose/oral) or caudalsites (urogenital/cloacal), was investigated by comparing the results from 272 animalsfrom which swabs were available from both sites (Fig. S1). Detection of PhaHV-1 andPhaHV-2 DNA occurred more frequently in caudal swabs (36.4%) than in rostral swabs

FIG 3 (A) Output of univariable logistic regression analysis assessing geographical region as a variable for association with the presence of herpesvirus DNA,using the South Coast region as the reference (i.e., odds ratio � 1.0). Odds ratios and 95% confidence intervals are shown. Odds ratios are statistically significantwhere 95% confidence intervals do not cross the reference line (1.0). (B) Percentage prevalence of each herpesvirus in each geographical region across thesampled years. Error bars indicate 95% confidence intervals, and asterisks indicate P values of �0.05 by Fisher’s exact test (two-tailed). “Either” indicatesdetection of PhaHV-1 and/or PhaHV-2 DNA.

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(16.5%; P � 0.001, Fisher’s exact test). There was no difference in the proportion ofswabs that were positive for PhaHV-1 from the proportion of swabs that were positivefor PhaHV-2 at each anatomical site.

(ii) Coinfections with other pathogens (C. pecorum and KoRV). The presence ofone herpesvirus was strongly associated with the detection of a second herpesvirus, inboth females and males (Table S6). Additionally, detection of both PhaHV-1 andPhaHV-2 was consistently and strongly associated with C. pecorum regardless of sex.Animals positive for C. pecorum were 6.7 times more likely to be positive for PhaHV-1,4.3 times more likely to be positive for PhaHV-2, and 8.3 times more likely to be infectedwith both herpesviruses (Table S3). Females positive for KoRV were also 2.0 times morelikely to be positive for PhaHV-1. This relationship with KoRV was not identified in malesor with PhaHV-2.

(iii) Reproductive and urinary tract abnormalities. Females with abnormalities inthe reproductive tract, such as reproductive cysts, were 2.1 times more likely to bepositive for PhaHV-1 and 2.4 times more likely to be positive for PhaHV-2. There werefew males with reproductive tract abnormalities, such as testicular hypoplasia; however,80% (4/5) were positive for PhaHV-2 (P � 0.007). Additionally, animals with wet bottomwere 2.0 and 4.5 times more likely to be positive for PhaHV-1 in the case of females

FIG 4 (A) Outputs of univariable logistic regression analysis assessing age group as a variable for association with the presence of herpesvirus DNA, using theadult age group as the reference (i.e., odds ratio � 1.0). Odds ratios and 95% confidence intervals are shown. Odds ratios are statistically significant where 95%confidence intervals do not cross the reference line (1.0). (B) Percentage prevalence of each herpesvirus in koalas by age group in combined (male and female),female-only, and male-only data. Asterisks indicate P values of �0.05 by Fisher’s exact test (two-tailed). Error bars indicate 95% confidence intervals.

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(Table S4) and males (Table S5), respectively, but wet bottom was only associated withPhaHV-2 detection in females (P � 0.036).

Koalas with the lowest BCS (BCS 1, emaciated) were 6.2, 5.8, and 11.1 times morelikely to be positive for PhaHV-1 or PhaHV-2 or coinfected, respectively, than thehealthy animals (BCS 3), although animals with BCS 4 were also 1.8 and 1.9 times morelikely to be positive for PhaHV-1 and PhaHV-2, respectively (Fig. 6; Table S3).

Multivariable logistic regression analyses. As PhaHV-1 and PhaHV-2 were eachcorrelated with C. pecorum detection, detection of a second herpesvirus was excludedas a variable from each of the multivariable analyses. Instead, a multivariable analysiswith coinfection (with both PhaHV-1 and PhaHV-2) as an outcome variable wasperformed that included biologically plausible variables that were associated witheither PhaHV-1 or PhaHV-2 infection by univariable analysis.

(i) Sex-dependent multivariable analyses. In females, reproductive tract abnor-malities were excluded in the PhaHV-1 multivariable logistic regression model, as theirinclusion resulted in insufficient data points. The final variables significantly associatedwith PhaHV-1 detection in females were the presence of C. pecorum (P � 0.001) and theage of the animal (P � 0.029) (Table 1; Table S7). In males, BCS was excluded from

FIG 5 Outputs of univariable logistic regression analysis assessing clinical variables for association with the presence of herpesvirus DNA, using the absenceof each variable as the reference, in females, males, and the two sexes combined. Odds ratios and 95% confidence intervals are shown. Odds ratios arestatistically significant where 95% confidence intervals do not cross the reference line (1.0). No PY, no pouch young; UT Abnorm, urinary tract abnormality; GTAbnorm, reproductive abnormality; Wet Bottom �, wet bottom positive; KoRV �, koala retrovirus positive; Chlamydia � � Chlamydia pecorum positive.

FIG 6 Outputs of univariable logistic regression analysis assessing body condition scores (BCS) for association with the presence of herpesvirus DNA, using thehealthy animals (BCS 3) as the reference, in females, males, and the two sexes combined. Odds ratios and 95% confidence intervals are shown. Odds ratios arestatistically significant where 95% confidence intervals do not cross the reference line (1.0). An insufficient number of males with a BCS of 1 were available, sothese were excluded from the analysis.

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the multivariable logistic regression model, as there was insufficient data for BCS in thedifferent age groupings. This enabled age, which was more strongly associated in theunivariable analysis (Table S5), to be included. In the final model, only the presence ofC. pecorum (P � 0.001) and increased age (P � 0.042) were identified as significant riskfactors for the detection of PhaHV-1 in males.

Reproductive tract abnormalities in females were excluded from the PhaHV-2multivariable logistic regression model, as described above, to enable inclusion of BCSas a variable. In the final model, the variables significantly associated with PhaHV-2detection in females were the presence of C. pecorum (P � 0.003) and BCS 1 and BCS4 (P � 0.015) (Table 1; Table S7). Possible interactions were identified between C.pecorum and reproductive tract abnormalities, but repeating the multivariable analysistreating reproductive tract abnormality as the outcome variable and including C.pecorum, KoRV, and the two herpesviruses as explanatory variables only identifiedPhaHV-2 as a predictor (P � 0.008) (Table S8). In males, no multivariable logisticregression was performed with PhaHV-2 as an outcome, as univariable analysis iden-tified only C. pecorum, reproductive abnormality, and the presence of PhaHV-1 aspossible factors, and thus, mutual correlation between variables confounded theconstruction of a model.

A multivariable logistic regression model was performed using coinfection (PhaHV-1and PhaHV-2) as the outcome variable, because clinical observations may be linked tocoinfection status. In females, in the final multivariable logistic regression model (Table1; Table S7), the variables identified as significant risk factors for detection of coinfec-tion included C. pecorum, wet bottom, and increasing age (P � 0.05), while in males,only the presence of C. pecorum was identified as a significant predictor (P � 0.001[Table 1]).

(ii) Sex-independent multivariable analyses. To identify clinical observations thatmay be independent of sex, multivariable logistic regression analysis was performedusing combined (male and female) animal data with PhaHV-1, PhaHV-2, and coinfectionwith the two herpesviruses as outcome variables (Table 1; Table S7). The explanatoryvariables identified as significant predictors for detection of PhaHV-1 included increas-ing age (P � 0.001) and C. pecorum (P � 0.001). Detection of PhaHV-2 was associatedwith C. pecorum (P � 0.001) and poor BCS (P � 0.03), while herpesvirus coinfection wasassociated with increasing age (P � 0.006), C. pecorum (P � 0.001), and wet bottom(P � 0.028).

TABLE 1 Summary of epidemiological variables significantly associated with the detection of herpesvirus DNA in samples collected fromkoalas in 2010 to 2015, as determined using multivariable analysis

Virus Sexa Variable Odds ratio(s) (95% CI)b P value

PhaHV-1 Both sexes (n � 444) Increased age: adult or mature 4.8 (1.8, 13.2) or 13.1 (3.5, 49.4) �0.001Chlamydia pecorum 5.0 (2.8, 9.0) �0.001

Females (n � 302) Increased age: adult or mature 3.4 (1.1, 10.4) or 8.1 (1.8, 37.1) 0.012Chlamydia pecorum 4.4 (2.1, 9.2) �0.001

Males (n � 155) Increased age: adult or mature 8.7 (1.1, 69.5) or 30.6 (2.4, 398.6) 0.005Chlamydia pecorum 6.2 (2.5, 15.3) �0.001

PhaHV-2 Both sexes (n � 646) Chlamydia pecorum 4.6 (2.8, 7.3) �0.001Poor body condition (BCS 1 and BCS 2) 6.3 (1.5, 26.4) and 2.0 (1.1, 3.6) 0.030

Females (n � 452) Chlamydia pecorum 2.8 (1.4, 5.5) 0.003Emaciated body condition and BCS 4 21.3 (2.1, 212.5) and 1.8 (1.0, 3.3) 0.015

PhaHV-1 and PhaHV-2 Both sexes (n � 444) Increased age: adult or mature 7.6 (1.0, 57.6) or 18.7 (2.0, 177.1) 0.006Chlamydia pecorum 4.9 (2.3, 10.5) �0.001Wet bottom 2.4 (1.1, 5.1) 0.028

Females (n � 298) Increased age: mature 15.9 (1.4, 174.1) 0.033Chlamydia pecorum 4.4 (1.7, 11.5) 0.003Wet bottom 3.4 (1.3, 8.7) 0.009

Males (n � 171) Chlamydia pecorum 10.2 (3.0, 34.6) �0.001an � total number of animals.bCI, confidence interval.

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DISCUSSION

We used high-resolution melt analysis of amplicons generated by a widely usedpan-herpesvirus nested PCR to differentiate between the two koala gammaherpesvi-ruses, PhaHV-1 and PhaHV-2. HRM analysis has been used previously to differentiatebetween related strains of other veterinary and human pathogens (27–30). As this assaytargets a conserved region of the herpesvirus DNA polymerase gene, similar ap-proaches could also be suitable for other marsupial herpesviruses in other host species.

The targeted region of the koala gammaherpesvirus genomes shared only 60%pairwise nucleotide identity (7); thus, they were easily differentiated by HRM. The assaywas an efficient tool for the high-throughput identification and typing of herpesviruses,and as a result, we were able to conduct a large molecular epidemiological study. Wedetected both PhaHV-1 and PhaHV-2 across all regional populations and observeddifferences between populations and between the herpesvirus species. While mostpopulations maintained a relatively stable viral prevalence over time, changes overtime were detected in three regions, possibly due to external factors such as environ-mental change, a general change in the health status of the animals, or bias introducedat sampling, particularly in those populations (French Island and Far West) in whichmost of the sampled animals were females. The French Island population is a unique,closed, and inbred population (21, 22). In this population 98% of the tested animalswere females. Differences in clinical associations for each virus were also detected,including associations with reproductive abnormalities (stronger association withPhaHV-2), wet bottom (stronger association with PhaHV-1), C. pecorum detection(stronger association with PhaHV-1), and koala retrovirus detection (associated withPhaHV-1).

Our findings support prior studies that have shown C. pecorum detection to be astrong predictor for detection of a koala gammaherpesvirus (11). C. pecorum infectionis associated with mortality and morbidity in wild koalas. Clinical disease linked to thisbacterium includes keratoconjunctivitis (3), and urogenital infections and infertility (31,32). Associations between different herpesviruses and Chlamydia infection have alsobeen described epidemiologically for humans and cats (33–36). In vitro coinfectionstudies of Chlamydia trachomatis and human herpesviruses have indicated complicatedvirus-host-bacterium interactions, including virus-induced chlamydial persistence (37,38) and an increase in viral entry into normally nonsusceptible cells infected withChlamydia (37). Future research into the relationship between C. pecorum and koalagammaherpesviruses is indicated.

Some clinical variables were identified as significant predictors of both gammaher-pesvirus detection and C. pecorum detection; thus, it is difficult to disentangle theclinical variables associated with each individual pathogen. For example, wet bottomwas associated with detection of PhaHV-1, PhaHV-2, and Chlamydia and so may beconfounded by any viral-bacterial synergy or interaction, although no interactions weredetected in multivariable analysis. Similarly, reproductive abnormalities were goodpredictors for the detection of C. pecorum (in females) (18), PhaHV-2 (in both males andfemales), and PhaHV-1 (when data from both sexes were combined). However, ourmultivariable logistic regression model that included all known koala pathogens withreproductive tract abnormalities as the outcome variable identified only PhaHV-2 as asignificant predictor. It is likely that in all these cases there are interactions betweeneach of these infectious agents that increase the likelihood of disease susceptibility intheir host. These interactions may also increase the persistence of both viral andbacterial species, leading to increased codetection.

The koala gamma retrovirus KoRV (39) is another key infectious threat to wild koalapopulations. In northeastern populations, KoRV has been associated with immunosup-pressive disease and lymphoid neoplasia (4); however, in southern populations KoRVinfections appear to be less pathogenic, possibly due to the absence of more patho-genic strains of KoRV (19). In our study, female koalas positive for KoRV were twice aslikely to be positive for PhaHV-1 but not PhaHV-2. Associations between gammaher-

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pesviruses and retrovirus infections have been observed and widely investigated inhumans, in whom these associations often result in increased disease risk, with theeffect typically through immunosuppressive pathways (40, 41). It is possible that asimilar biological interaction between PhaHV-1 and KoRV resulted in an increased riskof opportunistic infections causing disease. Further studies are needed to clarify thislink with PhaHV-1 and what significance it may have for animal health.

PhaHV-1 detection increased with increasing age. This is not unexpected for her-pesvirus infections, as these viruses establish latent infections that persist over the lifeof the host (42); thus, there is a logically cumulative effect, with older animals increas-ingly likely to be virus positive. The increase in PhaHV-1 detection with increasing agesuggests that it may be acquired through activity related to maturation, either sexualor through aggressive behavior in males. In contrast, PhaHV-2 prevalence did notchange with age. It is possible that PhaHV-2 is acquired while in the pouch throughclose contact with the mother.

The assay is able to detect latent virus, as well as replicating virus (11), but only if thecollected samples contain cells harboring latent infections. As the sites of latency forPhaHV-1 and PhaHV-2 are not known, this uncertainty should be taken into consider-ation when interpreting the results. The possibility of reactivation and subsequentdetection of herpesvirus secondary to other disease processes, or due to general poorhealth of the host, should also be considered when interpreting the results (43, 44).Serological assays would help to further understand the biology and epidemiology ofPhaHV-1 and -2, but such assays currently do not exist. The future development ofserological tests for PhaHV-1 and -2, combined with the HRM assay developed in thisstudy, represents a promising approach to better understanding these two gamma-herpesviruses. It is recommended that future studies of PhaHV-1 and PhaHV-2 incor-porate koalas from northern populations, in which differences in infection and diseaseprogression have been observed for other pathogens (18–20).

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/JCM

.01478-18.SUPPLEMENTAL FILE 1, PDF file, 0.6 MB.

ACKNOWLEDGMENTSThis work was supported by the efforts of many veterinary clinicians and past

wildlife research students from 2010 to 2015. We especially thank Jade Patterson, MegCurnick, Kathryn Stalder, Pam Whiteley, Michael Lynch, Kate Bodley, and JemimaAmery-Gale for the use of their collections.

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