Mx proteins are interferon-induced GTPases that inhibit a wide range of viruses. The loss of functional Mx genes in mice and other model species is associated with inferior innate immune responses and increased virus susceptibility. Here, we describe genetic variations in the Mx genes of sheep (Ovis aries). More than 700 single nucleotide polymorphisms within or adjacent to MX1 and MX2 were identified by analysing whole genome sequence data from 68 sheep, representing 43 breeds from 19 countries. Amongst those are two biologically significant variations in the ovine MX2 gene: a guanosine-to-adenosine transition that generates a stop at codon 166 (c.497G > A; p.W166*) and a single nucleotide deletion in codon 329 that creates a frameshift and a premature stop of translation eight codons later (c.985del; p.Q329Sfs7*). A subsequent genotyping of Australian Merino sheep identified animals with a stop codon at position 166 in two research flocks that have been kept as closed flocks since the 1970s. Immunoblotting, immunofluorescence and mass spectrometry assays show that animals homozygous for the defect do not express detectable amounts of Mx2 proteins, and quantitative PCR suggests that the premature stop codon destabilises Mx2 mRNA. Furthermore, we found that Mx2-negative ewes and rams are fertile and that Mx2-negative lambs are indistinguishable from heterozygotes and wild-type animals in appearance, birth weight and growth rate.

We describe isolation and characterization of a novel henipavirus, designated Salt Gully virus, from the urine of pteropid bats in Australia. We noted the virus to be most closely related to Angavokely virus, not reliant on ephrin receptors for cell entry, and of unknown risk for human disease.

The Rabbit hemorrhagic disease virus (RHDV) was discovered 40 years ago. This highly pathogenic virus threatens the integrity of ecosystems in the European rabbit's native range, while in Australia, it is used as a biocontrol tool to manage overabundant populations of feral European rabbits. Little is known about the life cycle of this virus due to the absence of a reliable cell culture system. In 2023, we developed a rabbit liver-derived organoid cell culture system that supports RHDV replication but is unable to sustain serial passaging in culture. Here, we report that the interferon signaling pathway inhibitor Ruxolitinib increases virus replication in organoid-derived monolayer cells and, for the first time, enables the serial passaging of RHDV in cell culture. Four consecutive passages were achieved with viral titers reaching the concentration of the initial virus stock as measured by real-time quantitative PCR. Immunofluorescence analysis showed that more cells are infected in the presence of Ruxolitinib. Furthermore, we noted that cells grew faster and formed healthier monolayers in the presence of the interferon inhibitor. To determine the cellular composition of the monolayers, we used single-cell RNA sequencing, revealing that our organoids consist largely of RHDV-permissive cholangiocytes. IMPORTANCE:In this work, we describe the use of an interferon inhibitor to enhance the permissiveness of our recently developed rabbit liver-derived organoid cell culture system for rabbit hemorrhagic disease viruses. The data show that interferon inhibitors offer a simple and cost-effective approach to increase the replication of difficult-to-grow viruses in culture systems that are not interferon-deficient.

Borrelia are tick-borne spirochetes that include important pathogens that cause Lyme borreliosis and relapsing fevers. While multiple Borrelia species have been identified in Australia, further research is needed to understand their ecological roles and potential zoonotic risks. This study aimed to isolate and characterise two Australian Borrelia species-Borrelia tachyglossi and Borrelia sp. HB-through in vitro culture, genomic sequencing, and molecular surveys of ticks and vertebrate hosts. Despite extensive efforts, Borrelia sp. HB could not be cultured from Haemaphysalis bancrofti ticks, and its prevalence in questing ticks was low (0.14 %). Additionally, molecular screening of 504 wildlife hosts found no evidence of Borrelia sp. HB infection, suggesting a cryptic or highly restricted enzootic cycle. In contrast, B. tachyglossi was successfully cultured from Bothriocroton concolor ticks collected from echidnas (Tachyglossus aculeatus), enabling complete genome sequencing. Complement-mediated bactericidal assays demonstrated that B. tachyglossi is highly susceptible to human innate immunity, indicating it is unlikely to be pathogenic. Molecular surveys of wildlife-associated ticks revealed a broad diversity of reptile-associated Borrelia species in monitor lizards and snakes, reinforcing the hypothesis that Australian Borrelia persist in host-specific enzootic cycles. Our findings confirm that Borrelia species in Australia belong exclusively to the relapsing fever and reptile-associated clades, with no evidence of B. burgdorferi sensu lato. These results improve our understanding of Borrelia diversity in Australia and highlight the need for further research into their ecology, vector competence, and evolutionary history.

Current arbovirus surveillance strategies in Australia involve mosquito collection, species identification, and virus detection. These processes are labour-intensive, expensive, and time-consuming and can lead to delays in reporting. Mosquito excreta has been proposed as an alternative sample type to whole mosquito collection, with potential to streamline the virus surveillance pipeline. In this study, we investigated the feasibility of Aedes aegypti excreta as a sample type in the detection of Dengue virus serotype 2 (DENV2). DENV2 could be detected from as little as one DENV2-infected mosquito excreta spot, with virus levels in individual excreta spots varying within and between mosquitoes and depending highly on mosquito viral load. Detectability was improved by pooling up to 20 DENV2-infected mosquitoes and collecting excreta into liquid substrate, followed by virus concentration using magnetic nanoparticles. Virus concentration improves quantification accuracy in comparison to unconcentrated samples and increases the amount of material available for detection, expanding detection capabilities to techniques with higher limits of detection. Mosquito excreta as a sample type, coupled with magnetic virus concentration, expands the current detection toolbox for DENV2 and has the potential to improve arbovirus surveillance strategies in Australia.

Context Rabbit haemorrhagic disease virus 2 (RHDV2/b/GI.2) is the only representative of the genus Lagovirus that is known to fatally infect multiple lagomorph species. RHDV2 is the dominant lagovirus circulating in rabbits in Australia, where some lagoviruses are used for deliberate biological control of European rabbits, a major environmental and agricultural pest in this country. Evidence of exposure to lagoviruses has been reported for a range of species that feed on rabbits, and the reduced host specificity of RHDV2 compared with RHDV1 has occasionally raised concerns, especially in a biocontrol context. Aim We investigated evidence of exposure to RHDV2 in 99 individual feral foxes, cats, dogs and pigs and then we aimed to test these animals for evidence of a productive infection. Methods Sera were analysed for the presence of antibodies to RHDV2, and faeces and tissues for the presence of viral RNA. We made provisions for downstream analysis of liver tissues by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and histopathology should they return a positive RT-qPCR result, to further investigate any evidence of productive virus infection. We also infected RHDV2 in hepatobiliary organoids derived from cats and foxes to test for possible infection. Key results We detected serum antibodies and viral RNA in faeces indicative of ingestion of RHDV2-infected rabbits, but found no evidence for productive infection with RHDV2. Furthermore, no RHDV2 replication was seen in hepatobiliary organoids derived from foxes and cats after in vitro infection with RHDV2. Conclusion RHDV2 does not infect scavengers of rabbits, such as foxes, dogs, cats and pigs. Implications This study has provided insights into the safety of this strain.

Hendra virus (HeV) is a high consequence pathogen that causes severe respiratory and/or neurological disease in both horses and humans with high mortality. The equine vaccine, Equivac® HeV, is a recombinant soluble version of the G glycoprotein of HeV (HeV-sG) that elicits a potent HeV-neutralising antibody response in adult horses and also provides indirect protection for humans. Maternally derived HeV-G specific antibodies could interfere with subsequent foal vaccination, although other factors such as foal age and the suitability of vaccination protocols that have been optimised for adult horses may also be important. The aim of this study was to measure the HeV-G specific antibody levels of previously unvaccinated mares and their foals at key time points during the preliminary HeV vaccination schedule of three vaccinations. Previously unvaccinated mares and their foals were vaccinated according to the manufacturers recommendations and serum samples were tested for HeV-G specific antibodies using a microsphere-based immunoassay. There was no difference in the humoral response to vaccination between the mares and foals, and both mare and foal groups demonstrated a strong HeV-G specific antibody response after completion of the preliminary vaccination schedule. This study highlights the importance of the third vaccination in eliciting a strong antibody response in foals, as has been demonstrated previously for adult horses. However, the absence of passively acquired immunity and the limited response of foals to the initial two vaccinations does leave foals vulnerable to infection during the majority of the first year of life.

Archival specimens held in biorepositories (e.g. natural history collections) offer rare temporal snapshots of global biodiversity. These collections not only preserve species morphology and aspects of ecology, but increasingly provide access to historical molecular data, including insights into wildlife disease. As several pandemics have originated from animal viruses spilling over into the human population (i.e., SARS-CoV-2/COVID-19, 2009 H1N1 influenza, and HIV/AIDS), characterising the diversity of viruses circulating in wildlife populations is essential for proactive pandemic preparedness. Yet, current surveillance remains biased toward contemporary viruses of economic importance. One solution to bridging spatiotemporal gaps in wildlife virus knowledge is retrospective screening of vouchered wildlife specimens. However, such efforts have been hindered by formalin fixation of specimens, which degrades and cross-links nucleic acids. Here we demonstrate that formalin-fixed vouchered wildlife specimens retain both host and viral RNA fragments after being stored for up to sixty years. We recovered fragments of divergent strains of Rotavirus alphagastroenteritidis from two Australian microbat species; Nyctophilus geoffroyi (lesser long-eared bat ) and Rhinolophus megaphyllus (smaller horseshoe bat), representing the first characterisation of Rotavirus alphagastroenteritidis in Australian bats, and the oldest identification of the virus to date. Concurrently, we sequenced endogenous host RNA, providing a proof-of- concept for dual host-virus transcript recovery from vouchered specimens. This study highlights the role biorepositories can play in reconstructing historical viral landscapes and enabling spatiotemporal host-virus insight to advance both biodiversity science and global pandemic preparedness. ### Competing Interest Statement The authors have declared no competing interest. CSIRO Environomics Future Science Platform, R-10011 CSIRO Heath and Biosecurity Acorn Grant, R-16935 CSIRO Early Career Researcher Fellow Program, R-20592 National Research Collections Australia Strategic Operating, R-90579 Department of Agriculture, Fisheries and Forestry Bright Ideas Grant, R-19122

Pathogenic lagoviruses (Rabbit hemorrhagic disease virus, RHDV) are widely spread across the world and are used in Australia and New Zealand to control populations of feral European rabbits. The spread of the non-pathogenic lagoviruses, e.g., rabbit calicivirus (RCV), is less well studied as the infection results in no clinical signs. Nonetheless, RCV has important implications for the spread of RHDV and rabbit biocontrol as it can provide varying levels of cross-protection against fatal infection with pathogenic lagoviruses. In Chile, where European rabbits are also an introduced species, myxoma virus was used for localised biocontrol of rabbits in the 1950s. To date, there have been no studies investigating the presence of lagoviruses in the Chilean feral rabbit population. In this study, liver and duodenum rabbit samples from central Chile were tested for the presence of lagoviruses and positive samples were subject to whole RNA sequencing and subsequent data analysis. Phylogenetic analysis revealed a novel RCV variant in duodenal samples that likely originated from European RCVs. Sequencing analysis also detected the presence of a rabbit astrovirus in one of the lagovirus-positive samples.

Feral deer are widespread throughout Australia with the capacity to impact livestock production via transmission of parasites. Samples of Dama dama (fallow deer), Rusa unicolor (sambar deer), Cervus elaphus (red deer) and an unidentified deer were sourced from various locations in south-eastern Australia for examination for parasites. Adult nematodes were collected from the lungs of all deer species across four separate geographical locations. The nematodes were identified as species of Dictyocaulus through both morphological and molecular means. Species identification based on morphological features was difficult, with many measurements from described species overlapping . Molecular analyses targeting three markers, namely 18S rRNA, ITS2, and cox1 revealed the presence of two distinct species: Dictyocaulus cervi and Dictyocaulus skrjabini. These are the first genetically confirmed reports of species of Dictyocaulus in feral deer in Australia, and although cross-transmission of species of Dictyocaulus with livestock has not yet been reported, it cannot be completely discounted without further research.

Ticks are important medical and veterinary parasites that represent a substantial health threat to humans, companion animals, and livestock. Ixodiphagus wasps (Hymenoptera; Encyrtidae) are known endoparasitoids of ixodid (hard) and argasid (soft) ticks, with potential utility as natural biocontrol agents. Two species, Ixodiphagus brunneus and Ixodiphagus mysorensis, are previously recorded from Australia, however, the genus lacks formal revisionary work in Australia, and the validity and host ranges of these species remain uncertain. This work aimed to investigate the diversity of Ixodiphagus in Australasia and provide a molecular data resource for future work on these understudied endoparasitoids. We extracted DNA from archival Ixodiphagus specimens from Australian and New Zealand insect collections and performed high-throughput sequencing which resulted in complete or mostly complete mitochondrial genome sequences from 11 specimens, including I. brunneus, Ixodiphagus taiaroaensis, and a novel Ixodiphagus sp. reared from Rhipicephalus linnaei from Townsville, Australia. In addition, approximately 70% of the genome of the Wolbachia endosymbiont of I. brunneus was recovered. Finally, we screened 178 recently collected pooled tick samples from southern New South Wales, Australia, for Ixodiphagus spp. using 28S rRNA and cytochrome c oxidase subunit 1(COI) gene PCR, and recovered 14 positive samples. Phylogenetic analysis of Australasian Ixodiphagus spp. based on 28S rRNA and complete mitochondrial genome sequences determined that members of the Australasian fauna are distinct from Ixodiphagus hookeri (the only other Ixodiphagus species for which genetic data exists), and that at least two distinct species are present in Australia; I. brunneus identified from Ixodes holocyclus and Haemaphysalis bancrofti ticks, and an uncharacterised Ixodiphagus sp. found in Rhipicephalus linnaei ticks from northern Queensland. Furthermore, there was substantial genetic diversity at the 28S rRNA loci among I. brunneus samples, which may represent normal genetic variability or a secondary cryptic species. The molecular data generated here represents the first known for the genus Ixodiphagus in Australasia, doubling that of the world fauna, and provides the first known complete mitochondrial genomes for these important tick parasitoids.

Abstract Lyssaviruses are the causative agent for rabies, a disease that is uniformly fatal in humans. Once infection reaches the central nervous system (CNS), currently approved therapies are ineffective. Using luminescence-based longitudinal tracing of lyssavirus infection in a mouse model of lethal rabies disease, we have shown that peripheral administration of a single dose of human monoclonal antibody (mAb) F11 protects animals from lethality, even when virus is already robustly replicating in the CNS. Additionally, behavioral analyses demonstrate improved motor function of the infected mAb-treated animals compared to infected, untreated controls. Interestingly, A6, another huIgG1 mAb that targets the same lyssavirus G epitope and exhibits similar in vitro neutralization efficiency, is inferior to F11 when used for in vivo therapy. Importantly, functional inactivation of the F11 Fc domain significantly impairs protection against mortality. Thus, Fc effector function is a major determinant of mAb in vivo efficacy. Histology and flow cytometry analysis shows that mAb therapy leads to a shift in leukocyte populations that infiltrate the brain, with significant increases in MHCII+ myeloid cells and CD4+ T cells, consistent with our demonstration that CD4+ T cells are essential for mAb efficacy. Collectively, these data suggest that Fc-mediated effector functions in the periphery yield a potent antiviral immune response in the CNS.

Hendra virus (HeV) is lethal to horses and a zoonotic threat to humans in Australia, causing severe neurological and/or respiratory disease with high mortality. An equine vaccine has been available since 2012. Foals acquire antibodies from their dams by ingesting colostrum after parturition, therefore it is assumed that foals of mares vaccinated against HeV will have passive HeV antibodies circulating during the first several months of life until they are actively vaccinated. However, no studies have yet examined passive or active immunity against HeV in foals. Here, we investigated anti-HeV antibody levels in vaccinated mares and their foals. Testing for HeV neutralising antibodies is cumbersome due to the requirement for Biosafety level 4 (BSL-4) containment to conduct virus neutralisation tests (VNT). For this study, a subset of samples was tested for HeV G-specific antibodies by both an authentic VNT with infectious HeV and a microsphere-based immunoassay (MIA), revealing a strong correlation. An indicative neutralising level was then applied to the results of a larger sample set tested using the MIA. Mares had high levels of HeV-specific neutralising antibodies at the time of parturition. Foals acquired high levels of maternal antibodies which then waned to below predictive protective levels in most foals by 6 months old when vaccination commenced. Foals showed a suboptimal response to vaccination, suggesting maternal antibodies may interfere with active vaccination. The correlation analysis between the authentic HeV VNT and HeV MIA will enable further high throughput serological studies to inform optimal vaccination protocols for both broodmares and foals.

Tick-borne haemoparasites, including piroplasms and trypanosomes, are almost ubiquitous in Australian wildlife, with some associated with health impacts to individual animals and declining wildlife populations. An array of ecologically distinct piroplasm and trypanosome species occur throughout Australia although many of these species and their sylvatic ecologies are poorly characterised. Between May 2022 and October 2023, an anecdotally reported localised eastern grey kangaroo (Macropus giganteus) morbidity/mortality event occurred in coastal southern New South Wales, Australia, characterised by animals presenting with blindness, emaciation, lethargy, ataxia, and astasia. Here we used molecular techniques to identify tick-borne piroplasms (Babesia and Theileria) and trypanosomes in affected animals. Blood (n=89) and liver (n=19) samples were collected after the humane euthanasia of wild animals due to welfare concerns, and brief notes on the animal’s health were recorded. In total, 20 (22.5%) animals were infected with tick-borne haemoparasites, including a novel Theileria sp. nov. (14, 15.7%), Babesia macropus (2, 2.2%), Trypanosoma gilletti (5, 5.6%), and Trypanosoma vegrandis (1, 1.1%). Liver samples were also screened for Wallal and Warego viruses due to animals’ blindness, but were negative. This is the first report of T. gilletti and T. vegrandis in eastern grey kangaroos, although they have been previously reported in high numbers in ticks which commonly parasites this host. The novel Theileria sp. was previously reported in questing Ixodes holocyclus and in ticks from an opportunistically collected eastern grey kangaroo and red-necked wallaby (Notamacropus rufogriseus). However, we show for the first time this Theileria sp. can occur widely in eastern grey kangaroos. Ultimately, this small study did not intend, and is not able to draw inference regarding the pathogenicity of these haemoparasites to eastern grey kangaroos and it is likely that other factors, such as chronic Phalaris grass toxicity, had a role in this localised mortality/morbidity event.

Prediction and management of zoonotic pathogen spillover requires an understanding of infection dynamics within reservoir host populations. Transmission risk is often assessed using prevalence of infected hosts, with infection status based on the presence of genomic material. However, detection of viral genomic material alone does not necessarily indicate the presence of infectious virus, which could decouple prevalence from transmission risk. We undertook a multi-faceted investigation of Hendra virus shedding in Pteropus bats, combining insights from virus isolation, viral load proxies, viral prevalence, and longitudinal patterns of shedding, from 6,151 samples. In addition to seasonal and interannual fluctuation in prevalence, we found evidence for periodic shifts in the distribution of viral loads. The proportion of bats shedding high viral loads was higher during peak prevalence periods during which spillover events were observed, and lower during non-peak periods when there were no spillovers. We suggest that prolonged periods of low viral load and low prevalence reflect prolonged shedding of non-infectious RNA, or viral loads that are insufficient or unlikely to overcome dose barriers to spillover infection. These findings show that incorporating viral load (or proxies of viral load) into longitudinal studies of virus excretion will better inform predictions of spillover risk than prevalence alone. Significance statement We present a comprehensive analysis of a high-profile bat-virus system (Hendra virus in Australian flying-foxes) to demonstrate that both prevalence and viral loads can shift systematically over time, resulting in concentrated periods of increased spillover risk when prevalence and viral loads are high. We further suggest that prolonged periods of low-prevalence, low-load shedding may not reflect excretion of infectious virus, resolving the outstanding puzzle of why spillovers have not been observed during periods of low off-season prevalence in subtropical Australia, or more frequently in tropical Australia despite consistent low-prevalence shedding. The consideration of viral loads (or proxies of viral load) along with prevalence may improve risk inference from longitudinal surveys of zoonotic viruses across wildlife reservoir hosts.

To examine protective and risk factors for Buruli ulcer (BU), we conducted a case-control study of 245 adult BU cases and 481 postcode-matched controls across BU-endemic areas of Victoria, Australia. We calculated age- and sex-adjusted odds ratios for socio-environmental, host, and behavioral factors associated with BU by using conditional logistic regression. Odds of BU were >2-fold for persons with diabetes mellitus and persons working outdoors who had soil contact in BU-endemic areas (compared with indoor work) but were lower among persons who had bacillus Calmette-Guerin vaccinations. BU was associated with increasing numbers of possums and with ponds and bore water use at residences. Using insect repellent, covering arms and legs outdoors, and immediately washing wounds were protective; undertaking multiple protective behaviors was associated with the lowest odds of BU. Skin hygiene/ protection behaviors and previous bacillus Calmette-Guerin vaccination might provide protection against BU in BU-endemic areas.

Australia has multiple lagoviruses with differing pathogenicity. The circulation of these viruses was traditionally determined through opportunistic sampling events. In the lead up to the nationwide release of RHDVa-K5 (GI.1aP-GI.1a) in 2017, an existing citizen science program, RabbitScan, was augmented to allow members of the public to submit samples collected from dead leporids for lagovirus testing. This study describes the information obtained from the increased number of leporid samples received between 2015 and 2022 and focuses on the recent epidemiological interactions and evolutionary trajectory of circulating lagoviruses in Australia between October 2020 and December 2022. A total of 2771 samples were tested from January 2015 to December 2022, of which 1643 were lagovirus-positive. Notable changes in the distribution of lagovirus variants were observed, predominantly in Western Australia, where RHDV2-4c (GI.4cP-GI.2) was detected again in 2021 after initially being reported to be present in 2018. Interestingly, we found evidence that the deliberately released RHDVa-K5 was able to establish and circulate in wild rabbit populations in WA. Overall, the incorporation of citizen science approaches proved to be a cost-efficient method to increase the sampling area and enable an in-depth analysis of lagovirus distribution, genetic diversity, and interactions. The maintenance of such programs is essential to enable continued investigations of the critical parameters affecting the biocontrol of feral rabbit populations in Australia, as well as to enable the detection of any potential future incursions.

The genus Lagovirus of the family Caliciviridae contains some of the most virulent vertebrate viruses known. Lagoviruses infect leporids, such as rabbits, hares and cottontails. Highly pathogenic viruses such as Rabbit haemorrhagic disease virus 1 (RHDV1) cause a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 h of infection, killing over 95 % of susceptible animals. Research into the pathophysiological mechanisms that are responsible for this extreme phenotype has been hampered by the lack of a reliable culture system. Here, we report on a new ex vivo model for the cultivation of lagoviruses in cells derived from the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus). We show that three different lagoviruses, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids, but not in monolayer cultures derived from cat (Felis catus) or mouse (Mus musculus) organoids. Virus multiplication was demonstrated by (i) an increase in viral RNA levels, (ii) the accumulation of dsRNA viral replication intermediates and (iii) the expression of viral structural and non-structural proteins. The establishment of an organoid culture system for lagoviruses will facilitate studies with considerable implications for the conservation of endangered leporid species in Europe and North America, and the biocontrol of overabundant rabbit populations in Australia and New Zealand.

Infections with rabies virus (RABV) and related lyssaviruses are uniformly fatal once virus accesses the central nervous system (CNS) and causes disease signs. Current immunotherapies are thus focused on the early, pre-symptomatic stage of disease, with the goal of peripheral neutralization of virus to prevent CNS infection. Here, we evaluated the therapeutic efficacy of F11, an anti-lyssavirus human monoclonal antibody (mAb), on established lyssavirus infections. We show that a single dose of F11 limits viral load in the brain and reverses disease signs following infection with a lethal dose of lyssavirus, even when administered after initiation of robust virus replication in the CNS. Importantly, we found that F11-dependent neutralization is not sufficient to protect animals from mortality, and a CD4 T cell-dependent adaptive immune response is required for successful control of infection. F11 significantly changes the spectrum of leukocyte populations in the brain, and the FcRγ-binding function of F11 contributes to therapeutic efficacy. Thus, mAb therapy can drive potent neutralization-independent T cell-mediated effects, even against an established CNS infection by a lethal neurotropic virus.

Rabies virus and related lyssaviruses cause uniformly fatal disease, once the infection progresses to the central nervous system. Current lyssavirus immunotherapies are directed toward peripheral neutralization of virus to prevent CNS infection during the pre-symptomatic stage of disease. In this study, using an anti-lyssavirus human monoclonal antibody (mAb), F11, we evaluated the efficacy of immunotherapy on established lyssavirus infections in mice. Remarkably, using luminescence-based longitudinal tracing of virus infection in a mouse model of lethal disease, we found that a single dose of F11 reverses clinical signs of disease and protects animals from lethality following lyssavirus infection, even when administered after initiation of virus replication in the CNS. Investigation of the mechanisms of F11 efficacy revealed that F11-dependent neutralization is insufficient to protect animals from mortality. Control of infection requires an intact adaptive immune response, particularly CD4 T cells. Additionally, in vivo analysis of the F11-N297G mutant, which has defective FcRγ binding, showed a transiently increased viral load and increased morbidity when compared to F11 treated mice. Despite long-term survival and absence of clinical signs of disease in F11-treated animals, we found that lyssavirus infection persists chronically, concomitant with elevated expression of cellular immune response genes. These findings demonstrate that single-dose mAb therapy can stimulate an adaptive, T cell-dependent response that is durable and highly effective against an established CNS infection by a lethal neurotropic virus. Supported by grants from the National Institute of Health (U01GM109887, R01AI125552), and a USU Program Project Grant (MIC-732515) and a USU Center for Global Health Engagement grant (HU00011920118)