Recent Insights into the Molecular Mechanisms of the Toll-like Receptor Response to Influenza Virus Infection.

Influenza A viruses (IAVs) pose a significant global threat to human health. A tightly controlled host immune response is critical to avoid any detrimental effects of IAV infection. It is critical to investigate the association between the response of Toll-like receptors (TLRs) and influenza virus. Because TLRs may act as a double-edged sword, a balanced TLR response is critical for the overall benefit of the host. Consequently, a thorough understanding of the TLR response is essential for targeting TLRs as a novel therapeutic and prophylactic intervention. To date, a limited number of studies have assessed TLR and IAV interactions. Therefore, further research on TLR interactions in IAV infection should be conducted to determine their role in host-virus interactions in disease causation or clearance of the virus. Although influenza virus vaccines are available, they have limited efficacy, which should be enhanced to improve their efficacy. In this study, we discuss the current status of our understanding of the TLR response in IAV infection and the strategies adopted by IAVs to avoid TLR-mediated immune surveillance, which may help in devising new therapeutic or preventive strategies. Furthermore, recent advances in the use of TLR agonists as vaccine adjuvants to enhance influenza vaccine efficacy are discussed.

Effects of neddylation on viral infection: an overview.

Neddylation is a post-translational modification that plays an important role not only in cancer development but also in regulating viral infection and replication. Upregulation of neddylation occurs in viral infections, and inhibition of neddylation can suppress viral replication. Neddylation is thought to enhance viral protein stability and replication. Neddylation has been reported to enhance the stability of the regulatory hepatitis B virus (HBV) X protein, modulate viral replication, and enhance hepatocarcinogenesis. Inhibition of neddylation using the NEDD8-activating enzyme E1 inhibitor MLN4924 inhibits viral replication, including that of HBV. Understanding of the role of neddylation in viral infections is critical for developing new therapeutic targets and potential treatment strategies. In this review, we discuss recent progress in the understanding of the effects of neddylation during viral infection, particularly in HBV infection, and strategies for curing viral infection by targeting the neddylation pathway.

Toll-like Receptor Response to Human Immunodeficiency Virus Type 1 or Co-Infection with Hepatitis B or C Virus: An Overview.

Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that play important roles in the early detection of pathogen-associated molecular patterns and shaping innate and adaptive immune responses, which may influence the consequences of infection. Similarly to other viral infections, human immunodeficiency virus type 1 (HIV-1) also modulates the host TLR response; therefore, a proper understanding of the response induced by human HIV-1 or co-infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), due to the common mode of transmission of these viruses, is essential for understanding HIV-1 pathogenesis during mono- or co-infection with HBV or HCV, as well as for HIV-1 cure strategies. In this review, we discuss the host TLR response during HIV-1 infection and the innate immune evasion mechanisms adopted by HIV-1 for infection establishment. We also examine changes in the host TLR response during HIV-1 co-infection with HBV or HCV; however, this type of study is extremely scarce. Moreover, we discuss studies investigating TLR agonists as latency-reverting agents and immune stimulators towards new strategies for curing HIV. This understanding will help develop a new strategy for curing HIV-1 mono-infection or co-infection with HBV or HCV.

Mammalian animal models for dengue virus infection: a recent overview.

Dengue, a rapidly spreading mosquito-borne human viral disease caused by dengue virus (DENV), is a public health concern in tropical and subtropical areas due to its expanding geographical range. DENV can cause a wide spectrum of illnesses in humans, ranging from asymptomatic infection or mild dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue is caused by four DENV serotypes; however, dengue pathogenesis is complex and poorly understood. Establishing a useful animal model that can exhibit dengue-fever-like signs similar to those in humans is essential to improve our understanding of the host response and pathogenesis of DENV. Although several animal models, including mouse models, non-human primate models, and a recently reported tree shrew model, have been investigated for DENV infection, animal models with clinical signs that are similar to those of DF in humans have not yet been established. Although animal models are essential for understanding the pathogenesis of DENV infection and for drug and vaccine development, each animal model has its own strengths and limitations. Therefore, in this review, we provide a recent overview of animal models for DENV infection and pathogenesis, focusing on studies of the antibody-dependent enhancement (ADE) effect in animal models.

Blocking neddylation elicits antiviral effect against hepatitis B virus replication.

BACKGROUND: Hepatitis B Virus (HBV) is the most common cause of chronic liver disease worldwide. The mechanisms that regulate HBV viral replication remain poorly defined. Here, we show that blocking of the neddylation elicits antiviral effect against HBV replication, indicating that NEDD8 supports viral production. METHODS AND RESULTS: To explore role of neddylation, HBV-replicating HepG2.2.15.7 cells and HBV-infected HepG2-hNTCP-30 cells were treated with siNEDD8 and MLN4924, a potent and selective NEDD8-activating enzyme inhibitor. Cell viability, intracellular and extracellular HBV DNA, covalently closed circular DNA (cccDNA), HBsAg, HBeAg, and HBcrAg were measured to assess the consequences of the various treatments on viral replication. Our data showed that HBV infection increased NEDD8 expression in human liver cell lines. Symmetrically, NEDD8 knockdown by siRNA or MLN4924 treatments decreased HBV replication in HepG2.2.15.7 and HepG2-hNTCP-30 cells. Notably, HBsAg, and HBeAg secretions were strongly suppressed in the culture supernatants, but not the HBcrAg. These results indicate that the suppression of NEDD8 decreases HBV replication. However, cccDNA steady level confirms once again its persistence and longevity in chronic infection. CONCLUSION: The manipulation of the neddylation pathway can thus provide new tools interfering with HBV persistence as well as novel therapeutic strategies against chronic hepatitis B.

Toll-like Receptor Response to Hepatitis C Virus Infection: A Recent Overview.

Hepatitis C virus (HCV) infection remains a major global health burden, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that detect pathogen-associated molecular patterns and activate downstream signaling to induce proinflammatory cytokine and chemokine production. An increasing number of studies have suggested the importance of TLR responses in the outcome of HCV infection. However, the exact role of innate immune responses, including TLR response, in controlling chronic HCV infection remains to be established. A proper understanding of the TLR response in HCV infection is essential for devising new therapeutic approaches against HCV infection. In this review, we discuss the progress made in our understanding of the host innate immune response to HCV infection, with a particular focus on the TLR response. In addition, we discuss the mechanisms adopted by HCV to avoid immune surveillance mediated by TLRs.

Toll-Like Receptor and Cytokine Responses to Infection with Endogenous and Exogenous Koala Retrovirus, and Vaccination as a Control Strategy.

Koala populations are currently declining and under threat from koala retrovirus (KoRV) infection both in the wild and in captivity. KoRV is assumed to cause immunosuppression and neoplastic diseases, favoring chlamydiosis in koalas. Currently, 10 KoRV subtypes have been identified, including an endogenous subtype (KoRV-A) and nine exogenous subtypes (KoRV-B to KoRV-J). The host's immune response acts as a safeguard against pathogens. Therefore, a proper understanding of the immune response mechanisms against infection is of great importance for the host's survival, as well as for the development of therapeutic and prophylactic interventions. A vaccine is an important protective as well as being a therapeutic tool against infectious disease, and several studies have shown promise for the development of an effective vaccine against KoRV. Moreover, CRISPR/Cas9-based genome editing has opened a new window for gene therapy, and it appears to be a potential therapeutic tool in many viral infections, which could also be investigated for the treatment of KoRV infection. Here, we discuss the recent advances made in the understanding of the immune response in KoRV infection, as well as the progress towards vaccine development against KoRV infection in koalas.

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.

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.

Toll-Like Receptor Response to Hepatitis B Virus Infection and Potential of TLR Agonists as Immunomodulators for Treating Chronic Hepatitis B: An Overview.

Chronic hepatitis B virus (HBV) infection remains a major global health problem. The immunopathology of the disease, especially the interplay between HBV and host innate immunity, is poorly understood. Moreover, inconsistent literature on HBV and host innate immunity has led to controversies. However, recently, there has been an increase in the number of studies that have highlighted the link between innate immune responses, including Toll-like receptors (TLRs), and chronic HBV infection. TLRs are the key sensing molecules that detect pathogen-associated molecular patterns and regulate the induction of pro- and anti-inflammatory cytokines, thereby shaping the adaptive immunity. The suppression of TLR response has been reported in patients with chronic hepatitis B (CHB), as well as in other models, including tree shrews, suggesting an association of TLR response in HBV chronicity. Additionally, TLR agonists have been reported to improve the host innate immune response against HBV infection, highlighting the potential of these agonists as immunomodulators for enhancing CHB treatment. In this study, we discuss the current understanding of host innate immune responses during HBV infection, particularly focusing on the TLR response and TLR agonists as immunomodulators.

Koala retrovirus epidemiology, transmission mode, pathogenesis, and host immune response in koalas (Phascolarctos cinereus): a review.

Koala retrovirus (KoRV) is a major threat to koala health and conservation. It also represents a series of challenges across the fields of virology, immunology, and epidemiology that are of great potential interest to any researcher in the field of retroviral diseases. KoRV is a gammaretrovirus that is present in both endogenous and exogenous forms in koala populations, with a still-active endogenization process. KoRV may induce immunosuppression and neoplastic conditions such as lymphoma and leukemia and play a role in chlamydiosis and other diseases in koalas. KoRV transmission modes, pathogenesis, and host immune response still remain unclear, and a clear understanding of these areas is critical for devising effective preventative and therapeutic strategies. Research on KoRV is clearly critical for koala conservation. In this review, we provide an overview of the current understanding and future challenges related to KoRV epidemiology, transmission mode, pathogenesis, and host immune response and discuss prospects for therapeutic and preventive vaccines.

Intranasal vaccination with HBs and HBc protein combined with carboxyl vinyl polymer induces strong neutralizing antibody, anti-HBs IgA, and IFNG response.

Hepatitis B virus (HBV) infection causes acute and chronic hepatitis, which is a major public health concern worldwide. Immunization methods incorporating hepatitis B surface-small (HBs-S) antigen and hepatitis B core antigen (HBc) have been proposed as candidate therapeutic vaccines, but the elimination of existing HBV infection remains a challenge. To enhance the efficacy of HBs and HBc vaccination, we investigated HBs-large (HBs-L) as an immunogen, and carboxyl vinyl polymer (CVP) as an excipient. HBs-S or HBs-L, in combination with HBc antigen, was administered subcutaneously (without CVP) or intranasally (with or without CVP) for the evaluation of immune response in the tree shrew, which is considered to be a suitable small animal model of HBV infection. Immunization with HBs-L antigen by either route induced a rapid IgG response. Intranasal immunization with HBs-S or HBs-L and HBc formulated with CVP strongly induced neutralizing antibody activity, IgA response, and HBc-specific expression of the interferon gamma-encoding gene. These data indicated the potential of HBs-L and HBc intranasal immunization with CVP, not only as a therapeutic vaccine, but also as a prophylactic vaccine candidate.

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.

Development of membrane protein-based vaccine against lumpy skin disease virus (LSDV) using immunoinformatic tools.

INTRODUCTION: Lumpy skin disease, an economically significant bovine illness, is now found in previously unheard-of geographic regions. Vaccination is one of the most important ways to stop its further spread. AIM: Therefore, in this study, we applied advanced immunoinformatics approaches to design and develop an effective lumpy skin disease virus (LSDV) vaccine. METHODS: The membrane glycoprotein was selected for prediction of the different B- and T-cell epitopes by using the immune epitope database. The selected B- and T-cell epitopes were combined with the appropriate linkers and adjuvant resulted in a vaccine chimera construct. Bioinformatics tools were used to predict, refine and validate the 2D, 3D structures and for molecular docking with toll-like receptor 4 using different servers. The constructed vaccine candidate was further processed on the basis of antigenicity, allergenicity, solubility, different physiochemical properties and molecular docking scores. RESULTS: The in silico immune simulation induced significant response for immune cells. In silico cloning and codon optimization were performed to express the vaccine candidate in Escherichia coli. This study highlights a good signal for the design of a peptide-based LSDV vaccine. CONCLUSION: Thus, the present findings may indicate that the engineered multi-epitope vaccine is structurally stable and can induce a strong immune response, which should help in developing an effective vaccine towards controlling LSDV infection.

TLR agonists as vaccine adjuvants in the prevention of viral infections: an overview.

Tol-like receptor (TLR) agonists, as potent adjuvants, have gained attention in vaccine research for their ability to enhance immune responses. This study focuses on their application in improving vaccine efficacy against key viral infections, including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), SARS-CoV-2, influenza virus, and flaviviruses, including West Nile virus, dengue virus, and chikungunya virus. Vaccines are crucial in preventing microbial infections, including viruses, and adjuvants play a vital role in modulating immune responses. However, there are still many diseases for which effective vaccines are lacking or have limited immune response, posing significant threats to human health. The use of TLR agonists as adjuvants in viral vaccine formulations holds promise in improving vaccine effectiveness. By tailoring adjuvants to specific pathogens, such as HBV, HCV, HIV, SARS-CoV-2, influenza virus, and flavivirus, protective immunity against chronic and emerging infectious disease can be elicited.

Increasing Dengue Burden and Severe Dengue Risk in Bangladesh: An Overview.

Dengue is a prevalent and rapidly spreading mosquito-borne viral disease affecting humans. The geographic range of dengue is expanding, and much like in many other tropical regions of the world, dengue has become a major public health issue in Bangladesh. Until a large epidemic dengue outbreak in 2000, sporadic outbreaks have occurred in Bangladesh since 1964. After 2000, varying intensities of dengue activity were observed each year until 2018. However, in 2019, Bangladesh experienced the largest dengue epidemic in its history, with 101,354 dengue cases and 164 dengue-related deaths. Notably, this outbreak occurred in many regions that were previously considered free of the disease. As of 10 December 2022, a total of 60,078 dengue cases and 266 dengue-related deaths were reported in Bangladesh, with the 2022 outbreak being the second largest since 2000. There is an increased genetic diversity of the dengue virus (DENV) in Bangladesh and all four DENV serotypes are prevalent and co-circulating, which increases the risk for severe dengue owing to the antibody-dependent enhancement effect. Vector control remains the mainstay of dengue outbreak prevention; however, the vector control programs adopted in Bangladesh seem inadequate, requiring improved vector control strategies. In this review, we provide an overview of the epidemiology of DENV infection and the risks for a severe dengue outbreak in Bangladesh. Additionally, we discuss different dengue vector control strategies, from which the most suitable and effective measures can be applied in the context of Bangladesh for tackling future dengue epidemics.

Epidemiology and Risk Factors for Acute Viral Hepatitis in Bangladesh: An Overview.

Viral infections by hepatotropic viruses can cause both acute and chronic infections in the liver, resulting in morbidity and mortality in humans. Hepatotropic viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), are the major pathogens that cause acute and chronic infections in humans. Although all of these viruses can cause acute hepatitis in humans, HAV and HEV are the predominant causative agents in Bangladesh, where the occurrence is sporadic throughout the year. In this review, we provide an overview of the epidemiology of hepatotropic viruses that are responsible for acute hepatitis in Bangladesh. Additionally, we focus on the transmission modes of these viruses and the control and prevention of infections.

In vivo Delivery Tools for Clustered Regularly Interspaced Short Palindromic Repeat/Associated Protein 9-Mediated Inhibition of Hepatitis B Virus Infection: An Update.

Chronic hepatitis B virus (HBV) infection remains a major global health problem despite the availability of an effective prophylactic HBV vaccine. Current antiviral therapies are unable to fully cure chronic hepatitis B (CHB) because of the persistent nature of covalently closed circular DNA (cccDNA), a replicative template for HBV, which necessitates the development of alternative therapeutic approaches. The CRISPR/Cas system, a newly emerging genome editing tool, holds great promise for genome editing and gene therapy. Several in vitro and/or in vivo studies have demonstrated the effectiveness of HBV-specific clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) systems in cleaving HBV DNA and cccDNA. Although recent advances in CRISPR/Cas technology enhance its prospects for clinical application against HBV infection, in vivo delivery of the CRISPR/Cas9 system at targets sites remains a major challenge that needs to be resolved before its clinical application in gene therapy for CHB. In the present review, we discuss CRISPR/Cas9 delivery tools for targeting HBV infection, with a focus on the development of adeno-associated virus vectors and lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery to treat CHB. In addition, we discuss the importance of delivery tools in the enhancement of the antiviral efficacy of CRISPR/Cas9 against HBV infection.

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.