Koala study population. In total, 126 koalas were available for our study, of which 27 (21.4%) were from two Australian zoos (24 were from Taronga Zoo and 3 from a Queensland zoo), 78 (61.9%) were from ten US zoos, and 21 (16.7%) were wild-caught at St. Bees Island (Table 1). Age for the wild koalas was estimated from their physical characteristics, including length and comparisons with their physical characteristics at first capture 8-13 years ago. Using these estimates and animal records for the zoo koalas, the mean age was 6.2 years old (yo) with a median of 5 yo and 25th and 75th percentiles of 2 and 9 yo, respectively. There was almost an equal number of females (n=56, 44.4%) and males (n=58, 46.0%) and 12 (9.5%) joeys. Koalas were considered joeys if they were younger than or equal to 12 months old, the age when they are considered fully weaned. Among the 126 koalas, 66 (52.4%) were alive at the conclusion of the study and generally healthy, 16 (12.7%) had died from leukemia-lymphoma, 7 (5.6%) had died from other cancers (osteoma, metastatic sarcoma, osteosarcoma, hemangiosarcoma, and mammary carcinoma), and 37 (29.4%) had died from other causes, including anemia, old age, euthanasia, acute respiratory illness, pulmonary interstitial fibrosis, intestinal volvulus, torsion, intussusception, early young pouch death, or the cause was not determined (Table 1). Chlamydiosis was not reported for any koala.
Blood specimens were available from 92 adult koalas (45 males, 47 females) and from 8 joeys with 118 total blood specimens tested when including multiple specimens from eight koalas collected longitudinally. Only tissues (skin, and/or brain and muscle) were available for two joeys that died while in the pouch and only liver and spleen tissues were available for a joey that died from anemia. Multiple tissues (spleen, lymph node, bone marrow, intestine, liver, lungs, nodules, thymus, heart, kidney, and/or brain) were also available from 28 adults at two US zoos for a total of 77 tissue specimens tested. Of these 28 adult koalas, five also had blood specimens available
qPCR assay validation. The KoRV generic and type-specific and the koala β–actin assay could each reliably detect 10 copies of DNA or RNA targets/reaction in each replicate. Each assay could detect five copies of DNA or RNA target/reaction except the KoRV-B and KoRV-E RNA specific tests that only detected 90% and 70%, respectively, of the replicates containing five DNA or RNA copies per/reaction. Hence, the assay cutoff for generic and KoRV subtype-specific detection was between 5-10 DNA and RNA copies/reaction. The koala β–actin assay could detect 90% of the DNA and RNA replicates with one copy/reaction and 100% of the DNA and RNA replicates at five copies/reaction, and we thus set the cutoff for that assay at 1-5 copies/reaction.
The linear dynamic range and specificity of each qPCR assay was measured by testing ten replicates of ten-fold dilutions (100 to 107) of the KoRV-mosaic plasmid control. Each assay could detect between 100-107 copies/reaction. Testing of the different KoRV subtype controls gave quantification cycle (Cq) assay values that averaged 20 for 107 copies, 23 for 106 copies, 26 for 105 copies, 29 for 104 copies, 32 for 103 copies, 35 for 102 copies, 39 for 101 copies, and 42 for 100 copies with an average correlation coefficient (R2) of 0.995 for the replicates. Each assay also showed 100% specificity for detection of the KoRV-specific (A, B, E, F and J) env sequences, except for the KoRV-B test which also detects KoRV-J. Nonetheless, the KoRV-J-specific assay did not detect KoRV-B env sequences and can be used to distinguish these two genotypes.
Distribution of KoRV variants and VLs in wild and zoo koalas. Koalas were considered infected with a specific subtype if any specimen was positive for either KoRV-specific DNA or RNA sequences. For koalas with multiple blood specimens, a KoRV subtype was considered present if any specimen was positive for that subtype and the VL was averaged over the specimens. Koala β-actin sequences were detected at expected levels in all specimens with a mean and median of 6.39 and 6.47 log10 copies/ug PBMC DNA, 5.29 and 5.19 log10 copies/mL plasma RNA, 6.73 and 6.87 log10 copies/ug tissue DNA, and 5.53 and 5.66 log10 copies/ug tissue RNA, respectively. Table 2 provides a summary of the distribution of KoRV subtypes in koalas by institution location. The generic KoRV qPCR assay identified infection in specimens from all 126 koalas. Testing of PBMC and tissue DNA using the type-specific qPCR assays for detection of KoRV provirus found that all koalas in our study were infected with KoRV-A.
Most koalas in the 10 U.S. zoos were infected with KoRV-A only (70/78, 89.7%) (Table 2). A small number of koalas (10.3%) at the LA Zoo were infected with non-A subtypes. A wider diversity of KoRV was seen in 16/27 (59.3%) koalas at the Australian zoos and were co-infected with at least one additional non-KoRV-A genotype, compared to those at US zoos. Of 24 koalas infected with KoRV-B, four were KoRV-J-infected, 15 were KoRV-F-infected, and 14 had KoRV-E. The most common mixed subtype infection was KoRV-A, -B, -F, -E (n=9). Two koalas were infected with subtypes A, B, J, E, and F, one was infected with KoRV-A, -B, -J, and -F, and three koalas each were infected with KoRV-A, -B, -E or KoRV-A, -B -F. One koala was infected with subtypes A, B, and J. In contrast, all 21 wild koalas from St. Bees Island were infected with only KoRV-A.
The distribution of VLs for each detected KoRV subtype is shown in the box plots in Figs. 1–4. Fig. 1 shows the overall distribution in blood and tissue specimens, while Figs. 2–4 show the distribution by institution location, gender, and health status, respectively. In each case, distributions are shown separately for each specimen type (blood and tissue). For blood samples, VLs from animals with multiple collection dates were averaged for a total of 100 koalas with blood specimens. Tissues were only available from koalas at US zoos and were not averaged when multiple tissues were from the same animal. Plasma and tissue RNA levels were not directly comparable since the copies per unit differ.
Although the KoRV-A median log10 pVLs (7.43) were higher for blood PBMCs than non-A subtypes (range 2.26-6.36) for all koala characteristics, the KoRV-A median log10 blood plasma VLs (4.62) were lower than the median log10 plasma VLs for KoRV-B, -E, and -F (6.26, 5.39, and 6.145, respectively) (Fig. 1). Similarly, the median plasma VLs were higher than KoRV-A for subtype F at US zoos (5.76 vs 5.04), subtypes B and E at Australian zoos (5.35 and 5.41 vs 4.00), subtype E for males (5.48 vs 4.41), and subtype B and E for females (5.93 and 5.40 vs 4.61) (Figs. 2-3). Median subtype B and -F plasma VLs were about two log10 copies higher for female than male koalas (5.93 and 3.63 vs 4.18 and 1.82, respectively) (Fig. 3). The median KoRV-B, -E, and -F plasma VLs (5.07, 5.48, and 5.15, respectively) were at least one log10 higher than those for KoRV-A (3.78) in koalas with leukemia/lymphoma (Fig. 4). Similarly, the median KoRV-B and KoRV-E plasma VLs (6.38 and 5.78, respectively) were at least one log10 higher than those for KoRV-A (4.53) in koalas with other cancers. For koalas that died from other causes or were alive the differences in the median plasma VLs were than less than one log10 for KoRV-B (5.70 or 5.30, respectively) and KoRV-E (5.39 or 5.37, respectively) compared to KoRV-A (5.01 or 4.48, respectively) (Fig. 4).
As for the koala blood DNA specimens, KoRV-A in general showed the highest median pVLs in tissue gDNA for all koala characteristics (Figs. 1-4). Unlike plasma samples, male tissues had almost twice the median gDNA levels for subtype B compared to females (7.17 vs 4.49, respectively) (Fig. 3). Subtype B gDNA pVLs were only slightly higher than those for KoRV-A (5.00 vs 4.91, respectively) (Fig. 4). KoRV-A was the only subtype found in tissues from koalas with other cancers (Fig. 4).
The generic KoRV qPCR test for total KoRV expression gave nearly equivalent median pVLs as for KoRV-A using the type specific assay with non-A subtypes having at least 1.5-fold less median pVLs. In contrast, plasma viral expression was greater for at least one of the non-A KoRVs compared to the KoRV-A specific assay, except for female tissues, tissues from koalas that died from other cancers, and joey specimens or when a koala was infected with only KoRV-A (Figs. 1 - 4).
Association of KoRV subtypes with koala demographic and clinical variables. We next investigated potential associations between KoRV subtype, in both RNA and gDNA for tissue (Table 3A) and blood samples (Table 3B), and age in years, gender, and final health status classification. Associations between subtype and koala locations were only presented in Table 3B for blood samples, as tissue samples were available only for koalas at US zoos. The outcome variable was defined as infection with KoRV-B, -E, and -F for any specimen measurement of each individual koala. The p values were determined comparing the distributions of variables in koalas infected with each KoRV subtype separately and did not include KoRV-A since all koalas in our study population had the endogenous KoRV-A. KoRV subtypes B, E, and F were observed to have similar associations with age, sex and final health status classification in both RNA and DNA samples. For tissue samples, we observed that the positive koalas were significantly younger than the negative koalas in both RNA and DNA measurements (Table 3A). However, for blood samples, of these four koala characteristics only the presence of health status classification and cause of death and animal location showed significance for subtypes B and F. For example, for koalas infected with subtype B by RNA testing, 37.5% (6/16) died from leukemia-lymphoma and other cancers, compared to 7.1% (6/84) of koalas infected with only KoRV-A. No significant age difference was found comparing the negative (KoRV-A-only) and positive (also KoRV-B infected) populations. The presence of the non-A KoRV subtypes in both RNA and DNA specimens was also significantly different by koala location for blood samples (p<0.0001, Table 3B) with the majority of subtype-B-positive RNA (81.3%, 13/16) and subtype-B-positive DNA (83.3%, 15/18) koalas being from the Australian zoos (Table 3B).
Model-based analysis of KoRV subtype VLs and koala demographic and clinical characteristics. We then combined all available KoRV plasma RNA and gDNA VL blood-sample results, except for subtype J which was only detected in a few animals, to explore potential associations between VL and the available koala characteristics (Table 4). In this analysis, we included all specimen measurements, including those that were not detectable (below the assay detection limit, BLD). Results are not reported for tissue samples as the Tobit model did not converge in that analysis. For the blood sample analysis, we included subtype comparisons with subtype B as the reference and compared the variables age at testing, sex, final health status classification, and animal location. Koala age and sex were not significantly associated with VL levels after controlling for other variables.
Overall, we found that different subtypes showed significantly different VLs compared to subtype B. For example, for plasma RNA the mean log10 VLs for KoRV-A and KoRV-F were significantly higher than that for KoRV-B (mean difference = 4.47, 95% CI = 3.71, 5.22, p<0.0001 and mean difference = 2.12, 95% CI= 0.93, 3.32, p=0.0007, respectively). The mean log10 VLs for KoRV-E was significantly lower than that for subtype B (mean difference = -2.80, 95% CI = -3.79, -1.81, p<0.0001). Compared to other causes, koalas that died from leukemia-lymphoma or other cancers had significantly higher VLs compared to those that died from other causes (mean difference = 1.47, 95% CI 0.14, 2.81, p=0.03) and compared to those alive (mean difference = 2.01, 95% CI = 0.89, 3.13, p=0.0006).
When controlling for other covariates including subtype, koalas at the Australian zoos were found to have the highest mean VLs. When compared to wild koalas the mean difference was 1.87 (95% CI = 0.59, 3.15), while compared to koalas in US zoos the mean difference was 1.31 (95% CI = 0.42, 2.19). Analysis of the pVLs gave results similar to the plasma RNA VLs with koalas with leukemia-lymphoma or other cancers again having significantly higher pVLs compared to those that died from other causes (mean difference = 2.18, 95% CI = 0.93, 3.42, p=0.0008) or that were alive (mean difference = 2.01, 95% CI = 0.94, 3.07, p=0.0003).
Longitudinal KoRV blood levels, diversity, and antiretroviral treatment. Longitudinal blood specimens were available for eight koalas (three males, four females, one joey) at six zoos. Of these eight, five adult koalas were infected with only KoRV-A, which showed fluctuating KoRV levels over time. For example, plasma VLs decreased 0.8 and 1.2 logs over 8 months and about 0.4 logs over 2 years for two KoRV-A-infected male koalas at US zoos. In contrast, plasma KoRV-A VLs increased 0.7 logs over 19 days for a female at the Taronga Zoo. The one joey was infected with low levels of KoRV-B in the PBMCs and KoRV-E in the plasma at 9 months of age but both KoRV-B and -E were undetectable 19 days later. Two adults with longitudinal specimens died from leukemia/lymphoma. The male housed at the Taronga Zoo, had two specimens collected 19 days apart and was infected with KoRV-A, -B, -F and –E at both time points and had DNA and RNA levels within 0.5 logs, except for KoRV-E DNA levels that increased by almost 2.5 logs at the later time point. The female (named Brooklyn) housed at the LA Zoo had 12 samples collected every 3-4 days over a month. KoRV-A, -B, -F were detected at each time point, while KoRV-J was detected intermittently with 3/12 plasma RNA specimens testing positive while all matching PBMC DNA specimens were KoRV-J-negative.
Brooklyn received antiretroviral treatment with integrase (raltegravir) and nucleotide reverse transcriptase (tenofovir) inhibitors for 33 days for experimental treatment of her leukemia/lymphoma. While KoRV-A levels in Brooklyn were relatively stable over time averaging 7.10 and 7.59 log10 copies for plasma and PBMCs, respectively, the KoRV-B and -F averages were 1.24 (8.05 vs 6.81) and 0.67 (7.54 vs 6.87) logs higher in plasma than in PBMC DNA (Fig. S1), respectively. Mean KoRV-B and -F plasma VLs were also 0.95 and 0.44 logs higher, respectively, than KoRV-A plasma levels. There was about a one log decrease in all KoRV plasma levels except for KoRV-F at day 7 post treatment (pt) that rebounded to pre-treatment levels four days later followed by another drop in plasma VLs at 21 – 28 days pt and for KoRV-A at 33 days pt. KoRV-J plasma levels were low with a mean of 1.3 log10 copies/ml when detected. After not responding to treatment and continued poor health, she was euthanized on day 33 pt.
Transmission of KoRV subtypes and associated diseases. To investigate possible transmission patterns of KoRV variants and disease associations in koalas we mapped detection of each subtype in offspring and their recorded parents when those specimens were available. From animal records we identified 66 offspring with KoRV test results for both parents (n=40) or from the dam (n=15) or sire (n=11) only. To evaluate dam-to-offspring transmission we examined the KoRV profiles and disease status of 55 offspring and their dams (Table 5). The age at testing of the 55 offspring ranged from 1-262 months with a mean and median of 53.7 and 40 months, respectively.
We found within the 55 complete pairs of dam and offspring data, 36 pairs were negative for subtype B. For three pairs, the dam was infected with subtype B but not the offspring and for five offspring with subtype B infection, their dam was only infected with KoRV-A. For 11 dam-offspring pairs, both koalas were infected with at least subtype B. Overall, we found that the dam KoRV subtype was significantly associated with the offspring subtypes (p<0.0001). Within the group of dams infected with subtype B and other non-A subtypes, 11/14 (78.6%) of the offspring were also infected with subtype B and other non-A subtypes. Within the subtype B-negative dams, 5/41 (12.2%) of the offspring were at least subtype B-positive. These results indicate that dams with KoRV-B alone or with other non-A subtypes were more likely to transmit their non-A KoRV subtypes to their offspring, but that discordant transmission profiles exist.
We identified eight offspring (7 adults, 1 joey) with discordant KoRV infections from the dam, including one KoRV-A, -B and -E-infected dam that birthed three KoRV-A-only infected offspring (two females, one male). The sire of two of these offspring tested KoRV-A-positive only. One female offspring with KoRV-B, -F, and –E tested at 72 months was born to a dam with only KoRV-A; the sire’s name was not recorded. Of the four other offspring (three males, one joey) with discordant KoRV infections, the dams were all KoRV-A-positive only while the one male sire of three of these was infected with KoRV-B and other non-A variants. The sire’s name for the fourth offspring was not recorded.
We also evaluated if subtype transmission was associated with disease in the offspring. Of the 55 dam/offspring pairs, neoplasia was identified in 10 (18.2%) pairs with leukemia/lymphoma in 7 pairs and other cancers in three (Table 5). Of these 10 pairs, three were infected with KoRV-A only including two with leukemia/lymphoma and one with another cancer. Interestingly, of these 10 dam/offspring pairs, two sires (1 KoRV-B-infected, 1 KoRV-A-infected only) of four offspring had leukemia/lymphoma and for one dam/offspring pair with other cancers the sire had leukemia/lymphoma. Discordant results were observed for 15 dam/offspring pairs (27.3%) with four dams giving birth to six offspring having leukemia/lymphoma and four dams giving birth to 9 offspring having other cancers while all 15 offspring were neoplasia-free at time of sampling. Of these 8 dams, five were KoRV-A-infected only and three were also infected with KoRV-B. We found that the odds of death from leukemia/lymphoma or other cancers for the offspring was higher in the multi-infection group compared to the KoRV-A only infection group (OR=9.87, 95% CI=1.63, 59.74, p=0.01) though the 95% CI is large.