Investigation of koala retrovirus in captive koalas with pneumonia and comparative analysis of subtype distribution.

This study focused on the involvement of koala retrovirus (KoRV) in pneumonia in koalas. Three deceased pneumonic koalas from a Japanese zoo were examined in this study. Hematological and histopathological findings were assessed, and KoRV proviral DNA loads in the blood and tissues were compared with those of eight other KoRV-infected koalas from different zoos. Demographic data and routine blood profiles were collected, and blood and tissue samples were analyzed to rule out concurrent infections in pneumonic koalas. KoRV subtyping and measurement of the KoRV proviral DNA load were performed by polymerase chain reaction (PCR) using specific primers targeting the pol and env genes. The results showed that the koalas had histopathologically suppurative and fibrinous pneumonia. Chlamydiosis was not detected in any of the animals. PCR analysis revealed KoRV-A, -B, and -C infections in all koalas, except for animals K10-11, which lacked KoRV-B. Significant variations in the proviral DNA loads of these KoRV subtypes were observed in all tissues and disease groups. Most tissues showed reduced KoRV loads in koalas with pneumonia, except in the spleen, which had significantly higher loads of total KoRV (2.54 × 107/µg DNA) and KoRV-A (4.74 × 107/µg DNA), suggesting potential immunosuppression. This study revealed the intricate dynamics of KoRV in various tissues, indicating its potential role in koala pneumonia via immunosuppression and opportunistic infections. Analysis of the levels of KoRV proviral DNA in different tissues will shed light on viral replication and the resulting pathogenesis in future studies.

Molecular Insights into Innate Immune Response in Captive Koala Peripheral Blood Mononuclear Cells Co-Infected with Multiple Koala Retrovirus Subtypes.

Koala retrovirus (KoRV) exists in both endogenous and exogenous forms and has appeared as a major threat to koala health and conservation. Currently, there are twelve identified KoRV subtypes: an endogenous subtype (KoRV-A) and eleven exogenous subtypes (KoRV-B to -I, KoRV-K, -L, and -M). However, information about subtype-related immune responses in koalas against multiple KoRV infections is limited. In this study, we investigated KoRV-subtype (A, B, C, D, and F)-related immunophenotypic changes, including CD4, CD8b, IFN-γ, IL-6, and IL-10 mRNA expression, in peripheral blood mononuclear cells (PBMCs) obtained from captive koalas (n = 37) infected with multiple KoRV subtypes (KoRV-A to F) reared in seven Japanese zoos. Based on KoRV subtype infection profiles, no significant difference in CD4 and CD8b mRNA expression was observed in the study populations. Based on the different KoRV subtype infections, we found that the IFN-γ mRNA expression in koala PMBCs differs insignificantly (p = 0.0534). In addition, IL-6 and IL-10 mRNA expression also did not vary significantly in koala PBMCs based on KoRV subtype differences. We also investigated the Toll-like receptors (TLRs) response, including TLR2-10, and TLR13 mRNA in koala PBMCs infected with multiple KoRV subtypes. Significant differential expression of TLR5, 7, 9, 10, and 13 mRNA was observed in the PBMCs from koalas infected with different KoRV subtypes. Therefore, based on the findings of this study, it is assumed that co-infection of multiple KoRV subtypes might modify the host innate immune response, including IFN-γ and TLRs responses. However, to have a more clear understanding regarding the effect of multiple KoRV subtypes on host cytokines and TLR response and pathogenesis, further large-scale studies including the koalas negative for KoRV and koalas infected with other KoRV subtypes (KoRV-A to -I, KoRV-K, -L and -M) are required.

Subtype distribution and expression of the koala retrovirus in the Japanese zoo koala population.

We investigated the proviral copies and RNA expression in koala retrovirus (KoRV)-infected koalas. To ascertain any variation in viral load by institution, age, sex, or body condition score, we quantified KoRV proviral DNA and RNA loads in captive koalas (n = 37) reared in Japanese zoos. All koalas were positive for KoRV genes (pol, LTRs, and env of KoRV-A) in genomic DNA (gDNA), and 91.89% were positive for the pol gene in RNA. In contrast, the distribution rates of KoRV-B, KoRV-C, KoRV-D, and KoRV-F env genes in gDNA were 94.59%, 27.03%, 67.57%, and 54.05%, respectively. A wide inter-individual variation and/or a significant inter-institutional difference in proviral DNA (p < 0.0001) and RNA (p < 0.001) amounts (copies/103 koala ß-actin copies) were observed in Awaji Farm England Hill Zoo koalas, which were obtained from southern koala populations, suggesting exogenous incorporation of KoRV in these koalas. Significant (p < 0.05) age differences were noted in KoRV RNA load (p < 0.05) and median total RNA load (p < 0.001), with loads higher in younger koalas (joeys and juveniles). Thus, the current study provides the distribution of KoRV subtypes in Japanese zoo koala populations and identifies several additional risk factors (sex, age, and body condition) associated with KoRV expression.

Koala retrovirus (KoRV) subtypes and their impact on captive koala (Phascolarctos cinereus) health.

Koala retrovirus (KoRV), a major pathogen of koalas, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-B to J). However, the impact of infection with multiple subtypes is not well understood. Accordingly, in this study, we surveyed a representative sample from a Japanese zoo population to determine the infection status for three KoRV subtypes (KoRV-A, B, and C) and to investigate the proviral and RNA load profiles in animals with single- and multiple-subtype infections, using peripheral blood mononuclear cells (PBMCs) and plasma. Six koalas were evaluated in the study; all were infected with KoRV-A, and two koalas were coinfected with non-A subtypes (KoRV-B and/or KoRV-C). The highest KoRV total RNA and viral loads in PBMCs and plasma were found in a koala infected with multiple subtypes (KoRV-A, -B and -C). The other koala infected with multiple subtypes (KoRV-A and B) showed the highest proviral PBMC load but the lowest RNA copy number in PBMC and plasma. PBMCs from this animal were cultured for further investigation, and KoRV RNA was detected in the cells and culture supernatant after 7 and/or 14 days. The koalas harboring multiple subtypes had a higher white blood cell count than those harboring only KoRV-A and were judged to be leukemic, and they subsequently died due to lymphoma. Accordingly, we conclude that coinfection with multiple KoRV subtypes may be linked to more-severe disease. In a sequence alignment, the detected KoRV-A env gene showed 100% sequence identity to the reference gene, whereas the KoRV-B and -C env genes varied from their reference sequences.

CD4, CD8b, and Cytokines Expression Profiles in Peripheral Blood Mononuclear Cells Infected with Different Subtypes of KoRV from Koalas (Phascolarctos cinereus) in a Japanese Zoo.

Koala retrovirus (KoRV) poses a major threat to koala health and conservation, and currently has 10 identified subtypes: an endogenous subtype (KoRV-A) and nine exogenous subtypes (KoRV-B to KoRV-J). However, subtype-related variations in koala immune response to KoRV are uncharacterized. In this study, we investigated KoRV-related immunophenotypic changes in a captive koala population (Hirakawa zoo, Japan) with a range of subtype infection profiles (KoRV-A only vs. KoRV-A with KoRV-B and/or -C), based on qPCR measurements of CD4, CD8b, IL-6, IL-10 and IL-17A mRNA expression in unstimulated and concanavalin (Con)-A-stimulated peripheral blood mononuclear cells (PBMCs). Although CD4, CD8b, and IL-17A expression did not differ between KoRV subtype infection profiles, IL-6 expression was higher in koalas with exogenous infections (both KoRV-B and KoRV-C) than those with the endogenous subtype only. IL-10 expression did not significantly differ between subtype infection profiles but did show a marked increase-accompanying decreased CD4:CD8b ratio-in a koala with lymphoma and co-infected with KoRV-A and -B, thus suggesting immunosuppression. Taken together, the findings of this study provide insights into koala immune response to multiple KoRV subtypes, which can be exploited for the development of prophylactic and therapeutic interventions for this iconic marsupial species.

Transmission of Koala Retrovirus from Parent Koalas to a Joey in a Japanese Zoo.

Koala retrovirus (KoRV) is of an interest to virologists due to its currently active endogenization into the koala (Phascolarctos cinereus) genome. Although KoRV has frequently been isolated in wild and captive koala populations, its pathogenesis and transmission remain to be fully characterized, and most previous research has concentrated on adult koalas rather than on joeys. Here, we characterized KoRV isolates obtained from a deceased male joey and its parents (animals reared in a Japanese zoo) to investigate KoRV transmission mode and pathogenesis. We sequenced the KoRV long terminal repeat (LTR) and envelope genes isolated from the joey and its parents and found KoRV-A and KoRV-C in genomic DNA from both the parents and the joey. Notably, both parents were also positive for KoRV-B, whereas the joey was KoRV-B negative, further confirming that KoRV-B is an exogenous strain. The KoRV LTR sequence of the joey was considerably closer to that of its sire than its dam. For further characterization, total KoRV, KoRV-A, KoRV-B, and KoRV-C proviral loads were quantified in peripheral blood mononuclear cells from the parents and in blood samples from the joey. Total KoRV, KoRV-A, and KoRV-C proviral loads were also quantified for different tissues (bone, liver, kidney, lung, spleen, heart, and muscle) from the joey, revealing differences suggestive of a distinct tissue tropism (highest total KoRV proviral load in the spleen and lowest in bone). The amount of KoRV-C in the parents was less than that in the joey. Our findings contribute to an improved understanding of KoRV pathogenesis and transmission mode and highlight useful areas for future research.IMPORTANCE KoRV is unique among retroviruses in that one strain (KoRV-A) is undergoing endogenization, whereas the other main subtype (KoRV-B) and another subtype (KoRV-C) are reportedly exogenous strains. Its transmission and pathogenesis are of interest in the study of retroviruses and are crucial for any conservation strategy geared toward koala health. This study provides new evidence on the modes of KoRV transmission from parent koalas to their joey. We found vertical transmission of KoRV-A, confirming its endogenization, but with closer conservation between the joey and its sire than its dam (previous reports on joeys are rare but have postulated dam-to-joey vertical transmission). This is also the first report of a KoRV-B-negative joey from KoRV-B-positive parents, contrasting with the few previous reports of 100% transmission of KoRV-B from dams to joeys. Thus, the results in this study give some novel insights for the transmission mode of KoRV.

Coinfection with koala retrovirus subtypes A and B and its impact on captive koalas in Japanese zoos.

Koala retrovirus (KoRV) is unique among endogenous retroviruses because its endogenization is still active. Two major KoRV subtypes, KoRV-A and B, have been described, and KoRV-B is associated with disease and poses a health threat to koalas. Here, we investigated the co-prevalence of KoRV-A and KoRV-B, detected by type-specific PCR and sequencing, and their impact on the health of koalas in three Japanese zoos. We also investigated KoRV proviral loads and found varying amounts of genomic DNA (gDNA) in peripheral blood mononuclear cells (PBMCs). We found that 100% of the koalas examined were infected with KoRV-A and 60% (12/20) were coinfected with KoRV-B. The KoRV-A sequence was highly conserved, whereas the KoRV-B sequence varied among individuals. Interestingly, we observed possible vertical transmission of KoRV-B in one offspring in which the KoRV-B sequence was similar to that of the father but not the mother. Moreover, we characterized the KoRV growth patterns in concanavalin-A-stimulated PBMCs isolated from KoRV-B-coinfected or KoRV-B-uninfected koalas. We quantified the KoRV provirus in gDNA and the KoRV RNA copy numbers in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-seeding. As the study population is housed in captivity, a longitudinal study of these koalas may provide an opportunity to study the transmission mode of KoRV-B. In addition, we characterized KoRV isolates by infecting tupaia cells. The results suggested that tupaia may be used as an infection model for KoRV. Thus, this study may enhance our understanding of KoRV-B coinfection and transmission in the captive koalas.

Molecular dynamics of koala retrovirus infection in captive koalas in Japan.

Koala retrovirus (KoRV) is a gammaretrovirus that is becoming endogenous in koalas. Here, we explored the dynamics of KoRV infection in captive koalas in Japan. We isolated peripheral blood mononuclear cells (PBMCs) from 11 koalas, from which we extracted the KoRV genome. We found the prevalence of KoRV provirus in the koalas to be 100%, and the copy number of KoRV proviral DNA in genomic DNA isolated from PBMCs was variable. The KoRV envelope genes from 11 koalas were sequenced and all were found to be KoRV type A. Nucleotide substitution analysis revealed differences in the KoRV env gene sequences of parents and their offspring. Although no viral KoRV RNA was detected in plasma of healthy koalas, a high copy number was found in plasma of a diseased koala (#6). Hematological analysis showed a high white blood cell (WBC) count in the blood of koala #6. Notably, when retested ~ 5 months later, koala #6 was found to be negative for KoRV in plasma, and the WBC count was within the normal range. Therefore, KoRV in the plasma could be a possible indicator of koala health. We also investigated KoRV growth in concanavalin-A-stimulated koala PBMCs by measuring the KoRV provirus copy number in gDNA and the KoRV RNA copy number in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-culture. We also observed that KoRV isolates were able to infect HEK293T cells. These findings could enhance our understanding of the dynamics of KoRV and its pathogenesis in koalas.