Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses.

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.

Public health interventions successfully mitigated multiple incursions of SARS-CoV-2 Delta variant in the Australian Capital Territory.

The COVID-19 pandemic has presented a unique opportunity to understand how real-time pathogen genomics can be used for large-scale outbreak investigations. On 12 August 2021, the Australian Capital Territory (ACT) detected an incursion of the SARS-CoV-2 Delta (B.1.617.2) variant. Prior to this date, SARS-CoV-2 had been eliminated locally since 7 July 2020. Several public health interventions were rapidly implemented in response to the incursion, including a territory-wide lockdown and comprehensive contact tracing. The ACT has not previously used pathogen genomics at a population level in an outbreak response; therefore, this incursion also presented an opportunity to investigate the utility of genomic sequencing to support contact tracing efforts in the ACT. Sequencing of >75% of the 1793 laboratory-confirmed cases during the 3 months following the initial notification identified at least 13 independent incursions with onwards spread in the community. Stratification of cases by genomic cluster revealed that distinct cohorts were affected by the different incursions. Two incursions resulted in most of the community transmission during the study period, with persistent transmission in vulnerable sections of the community. Ultimately, both major incursions were successfully mitigated through public health interventions, including COVID-19 vaccines. The high rates of SARS-CoV-2 sequencing in the ACT and the relatively small population size facilitated detailed investigations of the patterns of virus transmission, revealing insights beyond those gathered from traditional contact tracing alone. Genomic sequencing was critical to disentangling complex transmission chains to target interventions appropriately.

Comparative Analysis of RNA Virome Composition in Rabbits and Associated Ectoparasites.

Ectoparasites play an important role in virus transmission among vertebrates. Little, however, is known about the nature of those viruses that pass between invertebrates and vertebrates. In Australia, flies and fleas support the mechanical transmission of two viral biological controls against wild rabbits-rabbit hemorrhagic disease virus (RHDV) and myxoma virus. We compared virome compositions in rabbits and these ectoparasites, sequencing total RNA from multiple tissues and gut contents of wild rabbits, fleas collected from these rabbits, and flies trapped sympatrically. Meta-transcriptomic analyses identified 50 novel viruses from multiple RNA virus families. Rabbits and their ectoparasites were characterized by markedly different viromes, with virus abundance greatest in flies. Although viral contigs from six virus families/groups were found in both rabbits and ectoparasites, they clustered in distinct host-dependent lineages. A novel calicivirus and a picornavirus detected in rabbit cecal content were vertebrate specific; the newly detected calicivirus was distinct from known rabbit caliciviruses, while the picornavirus clustered with sapeloviruses. Several picobirnaviruses were also identified that fell in diverse phylogenetic positions, compatible with the idea that they are associated with bacteria. Further comparative analysis revealed that the remaining viruses found in rabbits, and all those from ectoparasites, were likely associated with invertebrates, plants, and coinfecting endosymbionts. While no full genomes of vertebrate-associated viruses were detected in ectoparasites, small numbers of reads from rabbit astrovirus, RHDV, and other lagoviruses were present in flies. This supports a role for flies in the mechanical transmission of RHDV, while their involvement in astrovirus transmission merits additional exploration.IMPORTANCE Ectoparasites play an important role in the transmission of many vertebrate-infecting viruses, including Zika and dengue viruses. Although it is becoming increasingly clear that invertebrate species harbor substantial virus diversity, it is unclear how many of the viruses carried by invertebrates have the potential to infect vertebrate species. We used the European rabbit (Oryctolagus cuniculus) as a model species to compare virome compositions in a vertebrate host and known associated ectoparasite mechanical vectors, in this case, fleas and blowflies. In particular, we aimed to infer the extent of viral transfer between these distinct types of host. Our analysis revealed that despite extensive viral diversity in both rabbits and associated ectoparasites, and the close interaction of these vertebrate and invertebrate species, biological viral transmission from ectoparasites to vertebrate species is rare. We did, however, find evidence to support the idea of a role of blowflies in transmitting viruses without active replication in the insect.

Rabbit Hemorrhagic Disease Virus 2 (RHDV2; GI.2) Is Replacing Endemic Strains of RHDV in the Australian Landscape within 18 Months of Its Arrival.

Rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) is a pathogenic calicivirus that affects European rabbits (Oryctolagus cuniculus) and various hare (Lepus) species. GI.2 was first detected in France in 2010 and subsequently caused epidemics in wild and domestic lagomorph populations throughout Europe. In May 2015, GI.2 was detected in Australia. Within 18 months of its initial detection, GI.2 had spread to all Australian states and territories and rapidly became the dominant circulating strain, replacing Rabbit hemorrhagic disease virus (RHDV/GI.1) in mainland Australia. Reconstruction of the evolutionary history of 127 Australian GI.2 isolates revealed that the virus arrived in Australia at least several months before its initial description and likely circulated unnoticed in wild rabbit populations in the east of the continent prior to its detection. GI.2 sequences isolated from five hares clustered with sequences from sympatric rabbit populations sampled contemporaneously, indicating multiple spillover events into hares rather than an adaptation of the Australian GI.2 to a new host. Since the presence of GI.2 in Australia may have wide-ranging consequences for rabbit biocontrol, particularly with the release of the novel biocontrol agent GI.1a/RHDVa-K5 in March 2017, ongoing surveillance is critical to understanding the interactions of the various lagoviruses in Australia and their impact on host populations.IMPORTANCE This study describes the spread and distribution of Rabbit hemorrhagic disease virus 2 (GI.2) in Australia since its first detection in May 2015. Within the first 18 months following its detection, RHDV2 spread from east to west across the continent and became the dominant strain in all mainland states of Australia. This has important implications for pest animal management and for owners of pet and farmed rabbits, as there currently is no effective vaccine available in Australia for GI.2. The closely related RHDV (GI.1) is used to control overabundant wild rabbits, a serious environmental and agricultural pest in this country, and it is currently unclear how the widespread circulation of GI.2 will impact ongoing targeted wild rabbit management operations.

Detection and Circulation of a Novel Rabbit Hemorrhagic Disease Virus in Australia.

The highly virulent rabbit hemorrhagic disease virus (RHDV) has been widely used in Australia and New Zealand since the mid-1990s to control wild rabbits, an invasive vertebrate pest in these countries. In January 2014, an exotic RHDV was detected in Australia, and 8 additional outbreaks were reported in both domestic and wild rabbits in the 15 months following its detection. Full-length genomic analysis revealed that this virus is a recombinant containing an RHDVa capsid gene and nonstructural genes most closely related to nonpathogenic rabbit caliciviruses. Nationwide monitoring efforts need to be expanded to assess if the increasing number of different RHDV variants circulating in the Australian environment will affect biological control of rabbits. At the same time, updated vaccines and vaccination protocols are urgently needed to protect pet and farmed rabbits from these novel rabbit caliciviruses.

Identification of non-essential loci within the Meleagrid herpesvirus 1 genome.

BACKGROUND: Meleagrid herpesvirus 1 (MeHV-1) infectious bacterial artificial chromosomes (iBACs) are ideal vectors for the development of recombinant vaccines for the poultry industry. However, the full potential of iBACS as vectors can only be realised after thorough genetic characterisation, including identification of those genetic locations that are non-essential for virus replication. Generally, transposition has proven to be a highly effective strategy for rapid and efficient mutagenesis of iBAC clones. The current study describes the characterisation of 34 MeHV-1 mutants containing transposon insertions within the pMeHV1-C18 iBAC genome. METHODS: Tn5 and MuA transposition methods were used to generate a library of 76 MeHV-1 insertion mutants. The capacity of each mutant to facilitate the recovery of infectious MeHV-1 was determined by the transfection of clone DNA into chicken embryo fibroblasts. RESULTS: Attempts to recover infectious virus from the modified clones identified 14 genetic locations that were essential for MeHV-1 replication in cell culture. Infectious MeHV-1 was recovered from the remaining 14 intragenic insertion mutants and six intergenic insertion mutants, suggesting that the respective insertion locations are non-essential for MeHV-1 replication in cell culture. CONCLUSIONS: The essential and non-essential designations for those MeHV-1 genes characterised in this study were generally in agreement with previous reports for other herpesviruses homologues. However, the requirement for the mardivirus-specific genes LORF4A and LORF5 are reported for the first time. These findings will help direct future work on the development of recombinant poultry vaccines using MeHV-1 as a vector by identifying potential transgene insertion sites within the viral genome.

Genomic deletions and mutations resulting in the loss of eight genes reduce the in vivo replication capacity of Meleagrid herpesvirus 1.

Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek's disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.

First Detection and Circulation of RHDV2 in New Zealand.

Rabbit haemorrhage disease virus 2 (RHDV2) is a highly pathogenic lagovirus that causes lethal disease in rabbits and hares (lagomorphs). Since its first detection in Europe in 2010, RHDV2 has spread worldwide and has been detected in over 35 countries so far. Here, we provide the first detailed report of the detection and subsequent circulation of RHDV2 in New Zealand. RHDV2 was first detected in New Zealand in 2018, with positive samples retrospectively identified in December 2017. Subsequent time-resolved phylogenetic analysis suggested a single introduction into the North Island between March and November 2016. Genetic analysis identified a GI.3P-GI.2 variant supporting a non-Australian origin for the incursion; however, more accurate identification of the source of the incursion remains challenging due to the wide global distribution of the GI.3P-GI.2 variant. Furthermore, our analysis suggests the spread of the virus between the North and South Islands of New Zealand at least twice, dated to mid-2017 and around 2018. Further phylogenetic analysis also revealed a strong phylogeographic pattern. So far, no recombination events with endemic benign New Zealand rabbit caliciviruses have been identified. This study highlights the need for further research and surveillance to monitor the distribution and diversity of lagoviruses in New Zealand and to detect incursions of novel variants.

Complete Mitochondrial Genome for Lucilia cuprina dorsalis (Diptera: Calliphoridae) from the Northern Territory, Australia.

The Australian sheep blowfly, Lucilia cuprina dorsalis, is a major sheep ectoparasite causing subcutaneous myiasis (flystrike), which can lead to reduced livestock productivity and, in severe instances, death of the affected animals. It is also a primary colonizer of carrion, an efficient pollinator, and used in maggot debridement therapy and forensic investigations. In this study, we report the complete mitochondrial (mt) genome of L. c. dorsalis from the Northern Territory (NT), Australia, where sheep are prohibited animals, unlike the rest of Australia. The mt genome is 15,943 bp in length, comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. The gene order of the current mt genome is consistent with the previously published L. cuprina mt genomes. Nucleotide composition revealed an AT bias, accounting for 77.5% of total mt genome nucleotides. Phylogenetic analyses of 56 species/taxa of dipterans indicated that L. c. dorsalis and L. sericata are the closest among all sibling species of the genus Lucilia, which helps to explain species evolution within the family Luciliinae. This study provides the first complete mt genome sequence for L. c. dorsalis derived from the NT, Australia to facilitate species identification and the examination of the evolutionary history of these blowflies.

Comment on Shah et al. Genetic Characteristics and Phylogeographic Dynamics of Lagoviruses, 1988-2021. Viruses 2023, 15, 815.

Shah and colleagues [...].

The Meleagrid herpesvirus 1 genome is partially resistant to transposition.

The propagation of herpesvirus genomes as infectious bacterial artificial chromosomes (iBAC) has enabled the application of highly efficient strategies to investigate gene function across the genome. One of these strategies, transposition, has been used successfully on a number of herpesvirus iBACs to generate libraries of gene disruption mutants. Gene deletion studies aimed at determining the dispensable gene repertoire of the Meleagrid herpesvirus 1 (MeHV-1) genome to enhance the utility of this virus as a vaccine vector have been conducted in this report. A MeHV-1 iBAC was used in combination with the Tn5 and MuA transposition systems in an attempt to generate MeHV-1 gene interruption libraries. However, these studies demonstrated that Tn5 transposition events into the MeHV-1 genome occurred at unexpectedly low frequencies. Furthermore, characterization of genomic locations of the rare Tn5 transposon insertion events indicated a nonrandom distribution within the viral genome, with seven of the 24 insertions occurring within the gene encoding infected cell protein 4. Although insertion events with the MuA system occurred at higher frequency compared with the Tn5 system, fewer insertion events were generated than has previously been reported with this system. The characterization and distribution of these MeHV-1 iBAC transposed mutants is discussed at both the nucleotide and genomic level, and the properties of the MeHV-1 genome that could influence transposition frequency are discussed.

A quantitative risk assessment for the incursion of lumpy skin disease virus into Australia via long-distance windborne dispersal of arthropod vectors.

Lumpy skin disease (LSD) is an infectious disease of cattle and water buffalo caused by lumpy skin disease virus (LSDV). It is primarily transmitted mechanically by biting insects. LSDV has spread from Africa to the Middle-East, the Balkans, Caucasus, Russia, Kazakhstan, China, Asia and India, suggesting that a wide variety of arthropod vectors are capable of mechanical transmission. In 2022, LSD was detected in Indonesia, heightening awareness for Australia's livestock industries. To better understand the risk of LSDV incursion to Australia we undertook a quantitative risk assessment (QRA) looking at windborne dispersal of arthropod vectors, assuming a hypothetical situation where LSD is endemic in south-east Asia and Papua New Guinea. We estimated the risk of LSDV incursion to be low, with a median incursion rate of one incursion every 403 years, based on a model where several infectious insects (i.e. a 'small batch' of 3-5) must bite a single bovine to transmit infection. The incursion risk increases substantially to one incursion every 7-8 years if a bite from a single insect is sufficient for transmission. The risk becomes negligible (one incursion every 20,706 years) if bites from many insects (i.e. a 'large batch' of 30-50 insects) are necessary. Critically, several of our parameter estimates were highly uncertain during sensitivity analyses. Thus, a key outcome of this QRA was to better prioritise surveillance activities and to understand the key research gaps associated with LSDV in the Australasian context. The current literature shows that multiple vectors are required for successful bovine-to-vector transmission of LSDV, suggesting that our estimate of one outbreak every 403 years more accurately represents the risk to Australia; however, the role of single insects in transmission has not yet been evaluated. Similarly, attempts to transmit LSDV between bovines by Culicoides have not been successful, although midges were the highest risk vector category in our model due to the high vector-to-host ratio for midges compared to other vector categories. Our findings provide further insight into the risk of LSD to Australian cattle industries and identify the Tiwi Islands and areas east of Darwin as priority regions for LSDV surveillance, especially between December and March.

Sustained Impact of RHDV2 on Wild Rabbit Populations across Australia Eight Years after Its Initial Detection.

Following the arrival of rabbit haemorrhagic disease virus 2 (RHDV2) in Australia, average rabbit population abundances were reduced by 60% between 2014 and 2018 based on monitoring data acquired from 18 sites across Australia. During this period, as the seropositivity to RHDV2 increased, concurrent decreases were observed in the seroprevalence of both the previously circulating RHDV1 and RCVA, a benign endemic rabbit calicivirus. However, the detection of substantial RHDV1 seropositivity in juvenile rabbits suggested that infections were continuing to occur, ruling out the rapid extinction of this variant. Here we investigate whether the co-circulation of two pathogenic RHDV variants was sustained after 2018 and whether the initially observed impact on rabbit abundance was still maintained. We monitored rabbit abundance and seropositivity to RHDV2, RHDV1 and RCVA at six of the initial eighteen sites until the summer of 2022. We observed sustained suppression of rabbit abundance at five of the six sites, with the average population reduction across all six sites being 64%. Across all sites, average RHDV2 seroprevalence remained high, reaching 60-70% in adult rabbits and 30-40% in juvenile rabbits. In contrast, average RHDV1 seroprevalence declined to <3% in adult rabbits and 5-6% in juvenile rabbits. Although seropositivity continued to be detected in a low number of juvenile rabbits, it is unlikely that RHDV1 strains now play a major role in the regulation of rabbit abundance. In contrast, RCVA seropositivity appears to be reaching an equilibrium with that of RHDV2, with RCVA seroprevalence in the preceding quarter having a strong negative effect on RHDV2 seroprevalence and vice versa, suggesting ongoing co-circulation of these variants. These findings highlight the complex interactions between different calicivirus variants in free-living rabbit populations and demonstrate the changes in interactions over the course of the RHDV2 epizootic as it has moved towards endemicity. While it is encouraging from an Australian perspective to see sustained suppression of rabbit populations in the eight years following the arrival of RHDV2, it is likely that rabbit populations will eventually recover, as has been observed with previous rabbit pathogens.

Cohorting children in a childcare setting: a strategy to reduce SARS-CoV-2 Delta transmission, August-September 2021.

Background: Childcare centres can be high-risk settings for SARS-CoV-2 transmission due to age, vaccination status, and infection control challenges. We describe the epidemiology and clinical characteristics of a childcare SARS-CoV-2 Delta outbreak. When the outbreak occurred, little was known about the transmission dynamics of SARS-CoV-2 ancestral and Delta strains among children. Vaccinations for coronavirus disease 2019 (COVID-19) were not mandatory for childcare staff, and children (< 12 years) were ineligible. Methods: A retrospective cohort design of childcare attendees was used to investigate age-cohorts exposure and transmission of SARS-CoV-2. We defined a case as a person who tested positive to SARS-CoV-2; we defined a close contact as a person who attended the childcare during 16-20 August 2021. Childcare centre exposures were defined by three cohorts: younger children (0-< 2.5 years) with designated staff; older children (2.5-5 years) with designated staff; and a staff-only group that moved between both age cohorts. We calculated the number and proportion of SARS-CoV-2 Delta infections, symptom profile and severity in children and adults, secondary attack rates, and relative risks (RR) with 95% confidence intervals (CIs) to compare age-cohort exposures and SARS-CoV-2 infection. Results: There were 38 outbreak cases that tested positive to SARS-CoV-2 Delta infection, comprising one primary case, 11 childcare attendees and 26 household members. Child attendees were in two non-interacting groups, 0-< 2.5 years and 2.5-5 years, with designated staff, separate rooms, and independent ventilation. The greatest risk of infection to childcare attendees was in the < 2.5 years age cohort which had a secondary attack rate of 41% and were five times more likely to be infected with SARS-CoV-2 (RR = 5.73; 95% CI: 1.37-23.86; p ≤ 0.01). No identified transmission (n = 0/21) occurred in the ≥ 2.5 years age cohort. Conclusion: Young children play an important role in SARS-CoV-2 Delta transmission to their peers and staff in childcare settings and to household members. Cohorting may be effective at limiting the propagation of SARS-CoV-2 in childcare settings. These findings highlight a need for multi-layered mitigation strategies and implementation support to manage respiratory infection control challenges at childcares. If prevention measures are not in place, this may facilitate ongoing transmission in these settings and into the broader community.

Does 2x2 airplane passenger contact tracing for infectious respiratory pathogens work? A systematic review of the evidence.

We critically appraised the literature regarding in-flight transmission of a range of respiratory infections to provide an evidence base for public health policies for contact tracing passengers, given the limited pathogen-specific data for SARS-CoV-2 currently available. Using PubMed, Web of Science, and other databases including preprints, we systematically reviewed evidence of in-flight transmission of infectious respiratory illnesses. A meta-analysis was conducted where total numbers of persons on board a specific flight was known, to calculate a pooled Attack Rate (AR) for a range of pathogens. The quality of the evidence provided was assessed using a bias assessment tool developed for in-flight transmission investigations of influenza which was modelled on the PRISMA statement and the Newcastle-Ottawa scale. We identified 103 publications detailing 165 flight investigations. Overall, 43.7% (72/165) of investigations provided evidence for in-flight transmission. H1N1 influenza A virus had the highest reported pooled attack rate per 100 persons (AR = 1.17), followed by SARS-CoV-2 (AR = 0.54) and SARS-CoV (AR = 0.32), Mycobacterium tuberculosis (TB, AR = 0.25), and measles virus (AR = 0.09). There was high heterogeneity in estimates between studies, except for TB. Of the 72 investigations that provided evidence for in-flight transmission, 27 investigations were assessed as having a high level of evidence, 23 as medium, and 22 as low. One third of the investigations that reported on proximity of cases showed transmission occurring beyond the 2x2 seating area. We suggest that for emerging pathogens, in the absence of pathogen-specific evidence, the 2x2 system should not be used for contact tracing. Instead, alternate contact tracing protocols and close contact definitions for enclosed areas, such as the same cabin on an aircraft or other forms of transport, should be considered as part of a whole of journey approach.

Rabbit haemorrhagic disease virus 2 from Singapore 2020 outbreak revealed an Australian recombinant variant.

Rabbit haemorrhagic disease (RHD) is a significant and debilitating viral disease affecting lagomorphs. In September 2020, Singapore reported its first cases of RHD virus (RHDV) infection in domesticated rabbits. The initial findings reported that the outbreak strain belonged to genotype GI.2 (RHDV2/RHDVb), and epidemiological investigations could not identify the definitive source of the virus origin. Further recombination detection and phylogenetic analyses of the Singapore outbreak strain revealed that the RHDV was a GI.2 structural (S)/GI.4 non-structural (NS) recombinant variant. Sequence analyses on the National Centre for Biotechnology Information (NCBI) database showed high homology to recently emerged Australian variants, which were prevalent in local Australian lagomorph populations since 2017. Time-structured and phylogeographic analyses for the S and NS genes revealed a close genetic relationship between the Singapore RHDV strain and the Australian RHDV variants. More thorough epidemiological inquiries are necessary to ascertain how an Australian RHDV was introduced into the Singapore rabbit population, and opportune development of RHDV diagnostics and vaccines will be important to safeguard lagomorphs from future RHDV infection and disease management.

Attenuated IL-2 muteins leverage the TCR signal to enhance regulatory T cell homeostasis and response in vivo.

Interleukin-2 (IL-2), along with T-cell receptor (TCR) signaling, are required to control regulatory T cell (Treg) homeostasis and function in vivo. Due to the heightened sensitivity to IL-2, Tregs retain the ability to respond to low-dose or attenuated forms of IL-2, as currently being developed for clinical use to treat inflammatory diseases. While attenuated IL-2 increases Treg selectivity, the question remains as to whether a weakened IL-2 signal sufficiently enhances Treg suppressive function(s) toward disease modification. To understand this question, we characterized the in vivo activity and transcriptomic profiles of two different attenuated IL-2 muteins in comparison with wildtype (WT) IL-2. Our study showed that, in addition to favoring Tregs, the attenuated muteins induced disproportionately robust effects on Treg activation and conversion to effector Treg (eTreg) phenotype. Our data furthermore suggested that Tregs activated by attenuated IL-2 muteins showed reduced dependence on TCR signal, at least in part due to the enhanced ability of IL-2 muteins to amplify the TCR signal in vivo. These results point to a new paradigm wherein IL-2 influences Tregs' sensitivity to antigenic signal, and that the combination effect may be leveraged for therapeutic use of attenuated IL-2 muteins.

Utilizing Molecular Epidemiology and Citizen Science for the Surveillance of Lagoviruses in Australia.

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.

Pathogen profiling of Australian rabbits by metatranscriptomic sequencing.

Australia is known for its long history of using biocontrol agents, such as myxoma virus (MYXV) and rabbit haemorrhagic disease virus (RHDV), to manage wild European rabbit populations. Interestingly, while undertaking RHDV surveillance of rabbits that were found dead, we observed that approximately 40% of samples were negative for RHDV. To investigate whether other infectious agents are responsible for killing rabbits in Australia, we subjected a subset of these RHDV-negative liver samples to metatranscriptomic sequencing. In addition, we investigated whether the host transcriptome data could provide additional differentiation between likely infectious versus non-infectious causes of death. We identified transcripts from several Clostridia species, Pasteurella multocida, Pseudomonas spp., and Eimeria stiedae, in liver samples of several rabbits that had died suddenly, all of which are known to infect rabbits and are capable of causing disease and mortality. In addition, we identified Hepatitis E virus and Cyniclomyces yeast in some samples, both of which are not usually associated with severe disease. In one-third of the sequenced total liver RNAs, no infectious agent could be identified. While metatranscriptomic sequencing cannot provide definitive evidence of causation, additional host transcriptome analysis provided further insights to distinguish between pathogenic microbes and commensals or environmental contaminants. Interestingly, three samples where no pathogen could be identified showed evidence of up-regulated host immune responses, while immune response pathways were not up-regulated when E. stiedae, Pseudomonas, or yeast were detected. In summary, although no new putative rabbit pathogens were identified, this study provides a robust workflow for future investigations into rabbit mortality events.