Meeting report: 36th International Conference on Antiviral Research in Lyon, France - March 13-17, 2023.

The 36th International Conference on Antiviral Research (ICAR), sponsored by the International Society for Antiviral Research (ISAR), was held March 13-17, 2023, in Lyon, France, and concurrently through an interactive remote meeting platform. Here we provide a report summarizing the presentations at the 36th ICAR, including the ISAR speaker awards. We also detail special events, sessions, and additional awards conferred at the meeting. ICAR returned to in-person meetings in 2022, convening in Seattle, WA, USA. The 36th ICAR is the first in-person meeting of the society in Europe since the beginning of the COVID-19 pandemic, which restricted most events to virtual attendance to help mitigate the spread and subsequent public health impact of SARS-CoV-2. An exceptionally high number of registrants and record attendance at this year's ICAR, along with a vast array of demonstrable expertise in a variety of antiviral research-related fields, reflected a strong and growing antiviral research community committed to improving health outcomes from viral diseases, including SARS-CoV-2, and to future pandemic preparedness. This report highlights the breadth of expertise, quality of research, and notable advancements that were contributed by members of ISAR and other participants at the meeting. ICAR aims to continue to provide a platform for sharing information, fostering collaborations, and supporting trainees in the field of antiviral research. The 37th ICAR will be held in Gold Coast, Australia, May 20-24, 2024.

Detection of Leishmania (Mundinia) macropodum (Kinetoplastida: Trypanosomatidae) and heterologous Leishmania species antibodies among blood donors in a region of Australia with marsupial Leishmania endemicity.

OBJECTIVES: The Australian Leishmania (Mundinia) macropodum parasite causes cutaneous leishmaniasis among marsupial species. Although cutaneous leishmaniasis is a major public health burden worldwide, it is not clear if humans are naturally exposed to the unique L. macropodum. To assess whether humans have an immunoglobulin (Ig) G response to L. macropodum, we examined anti-Leishmania antibodies among humans residing in a region of marsupial Leishmania endemicity in Australia. METHODS: Using a serological enzyme-linked immunosorbent assay, we characterized Leishmania-specific IgG and IgG subclass responses to soluble Leishmania antigen from L. macropodum, and other Leishmania species (L. donovani, L. major, and L. mexicana) in 282 blood donor samples. RESULTS: We found that 20.57% of individuals demonstrated a positive total IgG response to L. macropodum. For individuals with antibodies to soluble Leishmania antigen from one Leishmania species, there was no increased likelihood of recognition to other Leishmania species. For samples with detectable L. macropodum IgG, IgG1 and IgG2 were the prevalent subclasses detected. CONCLUSION: It is not yet clear whether the IgG antibody detection in this study reflects exposure to Leishmania parasites or a cross-reactive immune response that was induced against an unrelated immunogen. Future studies should investigate whether L. macropodum can result in a viable infection in humans.

Identification of transient receptor potential melastatin 3 proteotypic peptides employing an efficient membrane protein extraction method for natural killer cells.

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders, hence they make suitable targets in proteomic studies. However, extraction of membrane proteins is challenging due to their low abundance, stability, and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction, the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Methods: Two protocols, internally identified as Protocol 1 and 2, were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation, while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer's recommendation. Using Protocol 2, protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150-200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples. Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients. Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships, particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states, such as ME/CFS.

Human Seroprevalence for Dengue, Ross River, and Barmah Forest viruses in Australia and the Pacific: A systematic review spanning seven decades.

BACKGROUND: Dengue (DENV), Ross River (RRV) and Barmah Forest viruses (BFV) are the most common human arboviral infections in Australia and the Pacific Island Countries and Territories (PICTs) and are associated with debilitating symptoms. All are nationally notifiable in Australia, but routine surveillance is limited to a few locations in the PICTs. Understanding the level of human exposure to these viruses can inform disease management and mitigation strategies. To assess the historic and current seroprevalence of DENV, RRV and BFV in Australia and the PICTs we conducted a systematic literature review of all published quantitative serosurveys. METHODOLOGY AND PRINCIPAL FINDINGS: The Preferred Reporting of Items for Systematic Reviews and Meta-Analyses procedures were adopted to produce a protocol to systematically search for published studies reporting the seroprevalence of DENV, RRV and BFV in Australia and the PICTs. Data for author, research year, location, study population, serosurvey methods and positive tests were extracted. A total of 41 papers, reporting 78 serosurveys of DENV, RRV and BFV including 62,327 samples met the inclusion criteria for this review. Seroprevalence varied depending on the assay used, strategy of sample collection and location of the study population. Significant differences were observed in reported seropositivity depending on the sample collection strategy with clinically targeted sampling reporting the highest seroprevalence across all three viruses. Non-stratified seroprevalence showed wide ranges in reported positivity with DENV 0.0% - 95.6%, RRV 0.0% - 100.0%, and BFV 0.3% - 12.5%. We discuss some of the causes of variation including serological methods used, selection bias in sample collection including clinical or environmental associations, and location of study site. We consider the extent to which serosurveys reflect the epidemiology of the viruses and provide broad recommendations regarding the conduct and reporting of arbovirus serosurveys. CONCLUSIONS AND SIGNIFICANCE: Human serosurveys provide important information on the extent of human exposure to arboviruses across: (1) time, (2) place, and (3) person (e.g., age, gender, clinical presentation etc). Interpreting results obtained at these scales has the potential to inform us about transmission cycles, improve diagnostic surveillance, and mitigate future outbreaks. Future research should streamline methods and reduce bias to allow a better understanding of the burden of these diseases and the factors associated with seroprevalence. Greater consideration should be given to the interpretation of seroprevalence in studies, and increased rigour applied in linking seroprevalence to transmission dynamics.

TIR-Domain-Containing Adapter-Inducing Interferon-ß (TRIF)-Dependent Antiviral Responses Protect Mice against Ross River Virus Disease.

Ross River virus (RRV) is the major mosquito-borne virus in the South Pacific region. RRV infections are characterized by arthritic symptoms, which can last from several weeks to months. Type I interferon (IFN), the primary antiviral innate immune response, is able to modulate adaptive immune responses. The relationship between the protective role of type I IFN and the induction of signaling proteins that drive RRV disease pathogenesis remains poorly understood. In the present study, the role of TIR-domain-containing adapter-inducing interferon-ß (TRIF), an essential signaling adaptor protein downstream of Toll-like receptor (TLR) 3, a key single-stranded RNA (ssRNA)-sensing receptor, was investigated. We found that TRIF-/- mice were highly susceptible to RRV infection, with severe disease, high viremia, and a low type I IFN response early during disease development, which suggests the TLR3-TRIF axis may engage early in response to RRV infection. The number and the activation level of CD4+ T cells, CD8+ T cells, and NK cells were reduced in TRIF-/- mice compared to those in infected wild-type (WT) mice. In addition, the number of germinal center B cells was lower in TRIF-/- mice than WT mice following RRV infection, with lower titers of IgG antibodies detected in infected TRIF-/- mice compared to WT. Interestingly, the requirement for TRIF to promote immunoglobulin class switch recombination was at the level of the local immune microenvironment rather than B cells themselves. The slower resolution of RRV disease in TRIF-/- mice was associated with persistence of the RRV genome in muscle tissue and a continuing IFN response. IMPORTANCE RRV has been prevalent in the South Pacific region for decades and causes substantial economic and social costs. Though RRV is geographically restricted, a number of other alphaviruses have spread globally due to expansion of the mosquito vectors and increased international travel. Since over 30 species of mosquitoes have been implicated as potent vectors for RRV dissemination, RRV has the potential to further expand its distribution. In the pathogenesis of RRV disease, it is still not clear how innate immune responses synergize with adaptive immune responses. Type I IFN is crucial for bridging innate to adaptive immune responses to viral invasion. Hence, key signaling proteins in type I IFN induction pathways, which are important for type I IFN modulation, may also play critical roles in viral pathogenesis. This study provides insight into the role of TRIF in RRV disease development.

Pathways Activated by Infected and Bystander Chondrocytes in Response to Ross River Virus Infection.

Old world alphaviruses, such as Ross River virus (RRV), cause debilitating arthralgia during acute and chronic stages of the disease. RRV-induced cartilage degradation has been implicated as a cause of joint pain felt by RRV patients. Chondrocytes are a major cell type of cartilage and are involved in the production and maintenance of the cartilage matrix. It is thought that these cells may play a vital role in RRV disease pathogenesis. In this study, we used RNA-sequencing (RNA-Seq) to examine the transcriptomes of RRV-infected and bystander chondrocytes in the same environment. RRV containing green fluorescent protein (GFP) allowed for the separation of RRV-infected (GFP+) and bystander uninfected cells (GFP-). We found that whereas GFP+ and GFP- populations commonly presented similar gene expression profiles during infection, there were also unique signatures. For example, RIMS2 and FOXJ1 were unique to GFP+ cells, whilst Aim2 and CCL8 were only found in bystander chondrocytes. This indicates that careful selection of potential therapeutic targets is important to minimise adverse effects to the neighbouring uninfected cell populations. Our study serves as a resource to provide more information about the pathways and responses elicited by RRV in cells which are both infected and stimulated because of neighbouring infected cells.

Pentosan polysulfate sodium prevents functional decline in chikungunya infected mice by modulating growth factor signalling and lymphocyte activation.

Chikungunya virus (CHIKV) is an arthropod-borne virus that causes large outbreaks world-wide leaving millions of people with severe and debilitating arthritis. Interestingly, clinical presentation of CHIKV arthritides have many overlapping features with rheumatoid arthritis including cellular and cytokine pathways that lead to disease development and progression. Currently, there are no specific treatments or vaccines available to treat CHIKV infections therefore advocating the need for the development of novel therapeutic strategies to treat CHIKV rheumatic disease. Herein, we provide an in-depth analysis of an efficacious new treatment for CHIKV arthritis with a semi-synthetic sulphated polysaccharide, Pentosan Polysulfate Sodium (PPS). Mice treated with PPS showed significant functional improvement as measured by grip strength and a reduction in hind limb foot swelling. Histological analysis of the affected joint showed local inflammation was reduced as seen by a decreased number of infiltrating immune cells. Additionally, joint cartilage was protected as demonstrated by increased proteoglycan staining. Using a multiplex-immunoassay system, we also showed that at peak disease, PPS treatment led to a systemic reduction of the chemokines CXCL1, CCL2 (MCP-1), CCL7 (MCP-3) and CCL12 (MCP-5) which may be associated with the reduction in cellular infiltrates. Further characterisation of the local effect of PPS in its action to reduce joint and muscle inflammation was performed using NanoString™ technology. Results showed that PPS altered the local expression of key functional genes characterised for their involvement in growth factor signalling and lymphocyte activation. Overall, this study shows that PPS is a promising treatment for alphaviral arthritis by reducing inflammation and protecting joint integrity.

[Delayed administration of thrombolytic therapy in minor stroke]. / Retraso en la administración de tratamiento trombolítico en el ictus minor.

INTRODUCTION: Minor strokes are considered to be those that present with few symptoms, although up to 40% of them entail long-term disability. The rate of thrombolysis in these patients is also lower than in other strokes. The aim of this study is to explore whether there are any differences in intravenous thrombolysis care times in minor strokes. PATIENTS AND METHODS: We conducted a retrospective review of strokes treated with intravenous thrombolysis at our centre and a comparative analysis of the care times in minor strokes and in the other types. RESULTS: Longer times were found in minor strokes in terms of door-to-CT scan and door-to-needle time. This was not the case, however, for the time from the onset of symptoms to arrival at the hospital. CONCLUSIONS: The presence of few symptoms in minor strokes can make them difficult to recognise and could be a reason for delaying treatment. Training among staff caring for these patients is essential to improve this aspect.

TITLE: Retraso en la administración de tratamiento trombolítico en el ictus minor.Introducción. Se considera ictus minor a aquel que se presenta con escasos síntomas; sin embargo, hasta un 40% presenta discapacidad a largo plazo. La tasa de trombólisis en estos pacientes también es inferior a la del resto de ictus. En este estudio se pretende explorar si existen diferencias en los tiempos de atención en la trombólisis intravenosa en los pacientes con ictus minor. Material y métodos. Revisión retrospectiva de los ictus tratados con trombólisis intravenosa en nuestro centro y análisis comparativo de los tiempos de asistencia entre ictus minor y el resto. Resultados. Se encontraron tiempos más alargados en los casos de ictus minor en cuanto al tiempo puerta-tomografía computarizada y puerta-aguja. No fue así, sin embargo, para el tiempo desde el inicio de los síntomas hasta la llegada al hospital. Conclusiones. La presencia de escasos síntomas en el ictus minor puede hacer difícil su reconocimiento y podría ser un motivo de retraso en el tratamiento. La formación entre el personal que atiende a estos pacientes es fundamental para mejorar este aspecto.

Retraso en la administración de tratamiento trombolítico en el ictus minor / Delayed administration of thrombolytic therapy in minor stroke

Introducción: Se considera ictus minor a aquel que se presenta con escasos síntomas; sin embargo, hasta un 40% presenta discapacidad a largo plazo. La tasa de trombólisis en estos pacientes también es inferior a la del resto de ictus. En este estudio se pretende explorar si existen diferencias en los tiempos de atención en la trombólisis intravenosa en los pacientes con ictus minor. Material y métodos. Revisión retrospectiva de los ictus tratados con trombólisis intravenosa en nuestro centro y análisis comparativo de los tiempos de asistencia entre ictus minor y el resto. Resultados: Se encontraron tiempos más alargados en los casos de ictus minor en cuanto al tiempo puerta-tomografía computarizada y puerta-aguja. No fue así, sin embargo, para el tiempo desde el inicio de los síntomas hasta la llegada al hospital. Conclusiones: La presencia de escasos síntomas en el ictus minor puede hacer difícil su reconocimiento y podría ser un motivo de retraso en el tratamiento. La formación entre el personal que atiende a estos pacientes es fundamental para mejorar este aspecto.(AU)

Introduction: Minor strokes are considered to be those that present with few symptoms, although up to 40% of them entail long-term disability. The rate of thrombolysis in these patients is also lower than in other strokes. The aim of this study is to explore whether there are any differences in intravenous thrombolysis care times in minor strokes. Patients and methods: We conducted a retrospective review of strokes treated with intravenous thrombolysis at our centre and a comparative analysis of the care times in minor strokes and in the other types. Results: Longer times were found in minor strokes in terms of door-to-CT scan and door-to-needle time. This was not the case, however, for the time from the onset of symptoms to arrival at the hospital. Conclusions: The presence of few symptoms in minor strokes can make them difficult to recognise and could be a reason for delaying treatment. Training among staff caring for these patients is essential to improve this aspect.(AU)

Species Traits and Hotspots Associated with Ross River Virus Infection in Nonhuman Vertebrates in South East Queensland.

Ross River virus (RRV) is a mosquito-borne zoonotic arbovirus associated with high public health and economic burdens across Australia, but particularly in South East Queensland (SEQ). Despite this high burden, humans are considered incidental hosts. Transmission of RRV is maintained among mosquitoes and many nonhuman vertebrate reservoir hosts, although the relative contributions of each of these hosts are unclear. To clarify the importance of a range of vertebrates in RRV transmission in SEQ, a total of 595 serum samples from 31 species were examined for RRV exposure using a gold-standard plaque reduction neutralization test. Data were analyzed statistically using generalized linear models and a coefficient inference tree, and spatially. RRV exposure was highly variable between and within species groups. Critically, species group ("placental mammal," "marsupial," and "bird"), which has previously been used as a proxy for reservoir hosts, was a poor correlate for exposure. Instead, we found that generalized "diet" and greater "body mass" were most strongly correlated with seropositivity. We also identified significant differences in seropositivity between the two major possum species (ringtail possums and brushtail possums), which are ecologically and taxonomically different. Finally, we identified distinct hotspots and coldspots of seropositivity in nonhuman vertebrates, which correlated with human notification data. This is the largest diversity of species tested for RRV in a single study to date. The analysis methods within this study provide a framework for analyzing serological data in combination with species traits for other zoonotic disease, but more specifically for RRV highlight areas to target further public health research and surveillance effort.

Utilising a novel surveillance system to enhance field screening activities for the leishmaniases.

Over the last decade, an arbovirus surveillance system based on the preservation of nucleic acids (RNA/DNA) has been developed using Flinders Technology Associates (FTAⓇ) cards. Soaked in honey, FTAⓇ cards are applied in the field to detect arboviruses expectorated during mosquito sugar feeding. This technique has been shown to be inexpensive and efficient, and the implementation of this system for detecting parasites could be of international importance. As Leishmania parasites are highly prevalent in developing countries, FTAⓇ cards may offer an alternative inexpensive tool to enhance field surveillance activities for leishmaniasis. The simple approach of applying the cards in programs can substitute the necessary extensive training of personnel. In our hands, Leishmania macropodum DNA was shown to be stable on FTAⓇ cards during a 10-week time course, supporting their suitability for projects where direct access to laboratories is unobtainable and samples require storage prior to processing. This method may benefit programs in remote areas where accessibility to laboratory facilities are limited and samples need to be stored long-term.•This study found that FTA cards could be a valuable tool in the surveillance of leishmaniasis.•The method is based on the long-term preservation and detection of Leishmania DNA expectorated during insect sugar feeding.•The application of FTA cards can preclude the need to screen large samples and analysis of insect populations to provide evidence of disease transmission.

Integrating statistical and mechanistic approaches with biotic and environmental variables improves model predictions of the impact of climate and land-use changes on future mosquito-vector abundance, diversity and distributions in Australia.

Changes to Australia's climate and land-use patterns could result in expanded spatial and temporal distributions of endemic mosquito vectors including Aedes and Culex species that transmit medically important arboviruses. Climate and land-use changes greatly influence the suitability of habitats for mosquitoes and their behaviors such as mating, feeding and oviposition. Changes in these behaviors in turn determine future species-specific mosquito diversity, distribution and abundance. In this review, we discuss climate and land-use change factors that influence shifts in mosquito distribution ranges. We also discuss the predictive and epidemiological merits of incorporating these factors into a novel integrated statistical (SSDM) and mechanistic species distribution modelling (MSDM) framework. One potentially significant merit of integrated modelling is an improvement in the future surveillance and control of medically relevant endemic mosquito vectors such as Aedes vigilax and Culex annulirostris, implicated in the transmission of many arboviruses such as Ross River virus and Barmah Forest virus, and exotic mosquito vectors such as Aedes aegypti and Aedes albopictus. We conducted a focused literature search to explore the merits of integrating SSDMs and MSDMs with biotic and environmental variables to better predict the future range of endemic mosquito vectors. We show that an integrated framework utilising both SSDMs and MSDMs can improve future mosquito-vector species distribution projections in Australia. We recommend consideration of climate and environmental change projections in the process of developing land-use plans as this directly impacts mosquito-vector distribution and larvae abundance. We also urge laboratory, field-based researchers and modellers to combine these modelling approaches. Having many different variations of integrated (SDM) modelling frameworks could help to enhance the management of endemic mosquitoes in Australia. Enhanced mosquito management measures could in turn lead to lower arbovirus spread and disease notification rates.

Utilising a novel surveillance system to investigate species of Forcipomyia (Lasiohelea) (Diptera: Ceratopogonidae) as the suspected vectors of Leishmania macropodum (Kinetoplastida: Trypanosomatidae) in the Darwin region of Australia.

Up until recently, Australia was considered free of Leishmania due to the absence of phlebotomine sandfly species (Diptera: Phlebotominae) known to transmit Leishmania parasites in other parts of the world. The discovery of Leishmania (Mundinia) macropodum (Kinetoplastida: Trypanosomatidae) in Northern Australia sparked questions as to the existence of alternative vectors of Leishmania. This has added to the complexity of fully understanding the parasite's interaction with its vector, which is known to be very specific. Previous findings demonstrated L. macropodum infection beyond the blood meal stage in the day-biting midges Forcipomyia (Lasiohelea) Kieffer (Diptera: Ceratopogonidae) implicating them in the parasite's life cycle. Currently, there is no conclusive evidence demonstrating this suspected vector to transmit L. macropodum to a naïve host. Therefore, this research aimed to investigate the vector competency of day-biting midge F. (Lasiohelea) to transmit L. macropodum utilising a novel technology that preserves nucleic acids. Honey-soaked Flinders Technology Associates (FTA®) filter-paper cards were used to obtain saliva expectorated from biting midges while sugar-feeding. F. (Lasiohelea) were aspirated directly off macropods from a known Leishmania-transmission site and were kept in a waxed-paper container holding a honey-coated FTA® card for feeding. Insect identification and Taqman quantitative real-time PCR (qPCR) screening assays revealed L. macropodum DNA in F. (Lasiohelea) up to 7 days post field-collection, and in an unidentified biting midge, previously known as F. (Lasiohelea) sp.1. Moreover, 7/145 (4.83%) of FTA® cards were confirmed positive with L. macropodum DNA after exposure to field-collected F. (Lasiohelea). Additionally, FTA® cards were found to be a valuable surveillance tool, given the ease of use in the field and laboratory. Overall, our findings support previous reports on L. macropodum transmission by an alternative vector to phlebotomine sandflies. Further studies identifying and isolating infective L. macropodum promastigotes is necessary to resolve questions on the L. macropodum vector.

Modulation of Monocyte-Driven Myositis in Alphavirus Infection Reveals a Role for CX3CR1+ Macrophages in Tissue Repair.

Arthritogenic alphaviruses such as Ross River and Chikungunya viruses cause debilitating muscle and joint pain and pose significant challenges in the light of recent outbreaks. How host immune responses are orchestrated after alphaviral infections and lead to musculoskeletal inflammation remains poorly understood. Here, we show that myositis induced by Ross River virus (RRV) infection is driven by CD11bhi Ly6Chi inflammatory monocytes and followed by the establishment of a CD11bhi Ly6Clo CX3CR1+ macrophage population in the muscle upon recovery. Selective modulation of CD11bhi Ly6Chi monocyte migration to infected muscle using immune-modifying microparticles (IMP) reduced disease score, tissue damage, and inflammation and promoted the accumulation of CX3CR1+ macrophages, enhancing recovery and resolution. Here, we detail the role of immune pathology, describing a poorly characterized muscle macrophage subset as part of the dynamics of alphavirus-induced myositis and tissue recovery and identify IMP as an effective immunomodulatory approach. Given the lack of specific treatments available for alphavirus-induced pathologies, this study highlights a therapeutic potential for simple immune modulation by IMP in infected individuals in the event of large alphavirus outbreaks.IMPORTANCE Arthritogenic alphaviruses cause debilitating inflammatory disease, and current therapies are restricted to palliative approaches. Here, we show that following monocyte-driven muscle inflammation, tissue recovery is associated with the accumulation of CX3CR1+ macrophages in the muscle. Modulating inflammatory monocyte infiltration using immune-modifying microparticles (IMP) reduced tissue damage and inflammation and enhanced the formation of tissue repair-associated CX3CR1+ macrophages in the muscle. This shows that modulating key effectors of viral inflammation using microparticles can alter the outcome of disease by facilitating the accumulation of macrophage subsets associated with tissue repair.

Identification of Natural Molecular Determinants of Ross River Virus Type I Interferon Modulation.

Ross River virus (RRV) belongs to the genus Alphavirus and is prevalent in Australia. RRV infection can cause arthritic symptoms in patients and may include rash, fever, arthralgia, and myalgia. Type I interferons (IFN) are the primary antiviral cytokines and trigger activation of the host innate immune system to suppress the replication of invading viruses. Alphaviruses are able to subvert the type I IFN system, but the mechanisms used are ill defined. In this study, seven RRV field strains were analyzed for induction of and sensitivity to type I IFN. The sensitivities of these strains to human IFN-ß varied significantly and were highest for the RRV 2548 strain. Compared to prototype laboratory strain RRV-T48, RRV 2548 also induced higher type I IFN levels both in vitro and in vivo and caused milder disease. To identify the determinants involved in type I IFN modulation, the region encoding the nonstructural proteins (nsPs) of RRV 2548 was sequenced, and 42 amino acid differences from RRV-T48 were identified. Using fragment swapping and site-directed mutagenesis, we discovered that substitutions E402A and R522Q in nsP1 as well as Q619R in nsP2 were responsible for increased sensitivity of RRV 2548 to type I IFN. In contrast, substitutions A31T, N219T, S580L, and Q619R in nsP2 led to induction of higher levels of type I IFN. With exception of E402A, all these variations are common for naturally occurring RRV strains. However, they are different from all known determinants of type I IFN modulation reported previously in nsPs of alphaviruses.IMPORTANCE By identifying natural Ross River virus (RRV) amino acid determinants for type I interferon (IFN) modulation, this study gives further insight into the mechanism of type I IFN modulation by alphaviruses. Here, the crucial role of type I IFN in the early stages of RRV disease pathogenesis is further demonstrated. This study also provides a comparison of the roles of different parts of the RRV nonstructural region in type I IFN modulation, highlighting the importance of nonstructural protein 1 (nsP1) and nsP2 in this process. Three substitutions in nsP1 and nsP2 were found to be independently associated with enhanced type I IFN sensitivity, and four independent substitutions in nsP2 were important in elevated type I IFN induction. Such evidence has clear implications for RRV immunobiology, persistence, and pathology. The identification of viral proteins that modulate type I IFN may also have importance for the pathogenesis of other alphaviruses.

PG545 treatment reduces RRV-induced elevations of AST, ALT with secondary lymphoid organ alterations in C57BL/6 mice.

Recently the anti-viral effects of prophylactic treatment with the low-molecular-weight heparan sulfate mimetic PG545 in Ross River virus (RRV) infected mice were reported. We further investigated the related, transient pathophysiology of PG545 drug treatment in RRV-infected and mock-infected PG545-treated mice. PG545 treatment resulted in mild lethargy and piloerection, on days after the drug administration. Mice were treated with two or three doses of PG545 within a ten-day period and were subsequently culled at peak disease or at disease resolution. The treatment responses of the spleen and liver were assessed through histology, flow cytometry, gene arrays and serum biochemistry. Microscopy showed an expanded red pulp in the spleen following either two or three treatments with PG545. The red pulp expansion was further demonstrated by the proliferation of megakaryocytes and erythrocyte precursors within the spleen. In addition, flow cytometry and gene array analyses revealed a reduction of lymphocytes within the spleens of PG545-treated mice. Previously unreported, RRV-induced elevations of aspartate aminotransferase (AST) and alanine transaminase (ALT) enzymes and creatinine were also noted in the RRV-infected mice. However, PG545 only reduced AST and ALT levels but not the creatinine levels in infected mice during treatment. Mice treated with three doses of PG545 also showed hepatosplenomegaly and anaemia, which were reversed upon discontinuation of the treatment. In summary, this study demonstrates that dose and frequency related haemopoietic pathophysiology such as hepatosplenomegaly and anaemia, occurred in C57BL/6 mice treated with PG545. However, this effect was reversible once drug administration is terminated.

Mosquito antiviral defense mechanisms: a delicate balance between innate immunity and persistent viral infection.

Mosquito-borne diseases are associated with major global health burdens. Aedes spp. and Culex spp. are primarily responsible for the transmission of the most medically important mosquito-borne viruses, including dengue virus, West Nile virus and Zika virus. Despite the burden of these pathogens on human populations, the interactions between viruses and their mosquito hosts remain enigmatic. Viruses enter the midgut of a mosquito following the mosquito's ingestion of a viremic blood meal. During infection, virus recognition by the mosquito host triggers their antiviral defense mechanism. Of these host defenses, activation of the RNAi pathway is the main antiviral mechanism, leading to the degradation of viral RNA, thereby inhibiting viral replication and promoting viral clearance. However, whilst antiviral host defense mechanisms limit viral replication, the mosquito immune system is unable to effectively clear the virus. As such, these viruses can establish persistent infection with little or no fitness cost to the mosquito vector, ensuring life-long transmission to humans. Understanding of the mosquito innate immune response enables the discovery of novel antivectorial strategies to block human transmission. This review provides an updated and concise summary of recent studies on mosquito antiviral immune responses, which is a key determinant for successful virus transmission. In addition, we will also discuss the factors that may contribute to persistent infection in mosquito hosts. Finally, we will discuss current mosquito transmission-blocking strategies that utilize genetically modified mosquitoes and Wolbachia-infected mosquitoes for resistance to pathogens.

Inhibition of Interleukin-1ß Signaling by Anakinra Demonstrates a Critical Role of Bone Loss in Experimental Arthritogenic Alphavirus Infections.

OBJECTIVE: Arthritogenic alphaviruses, such as Ross River virus (RRV) and chikungunya virus (CHIKV), particularly affect joints of the extremities and can lead to debilitating and potentially chronic polyarthritis/polyarthralgia. The innate immune response of the host plays a crucial role in inducing proinflammatory host factors, leading to tissue destruction and bone loss in the joints. This study was performed to assess how the inhibition of interleukin-1ß (IL-1ß) signaling using the clinical rheumatoid arthritis drug anakinra influences bone loss in mice with arthritogenic alphavirus infections. METHODS: Mice (n = 5 per group) were infected with RRV or CHIKV and then treated with anakinra. Weight gain and disease severity were measured, tissue viral titers were determined, and histologic changes in joint tissues were assessed. RESULTS: Anakinra therapy reduced RRV- and CHIKV-induced bone loss in this murine model (P < 0.001 and P < 0.05, respectively). Histologic analysis of the knee joint showed that treatment with anakinra decreased epiphyseal growth plate thinning, loss of epiphyseal bone volume, and osteoclastogenesis in the tibia. Importantly, pharmacologic IL-1 receptor (IL-1R) blockade did not improve other clinical features, including disease score, weight loss, or viremia. CONCLUSION: The present findings suggest that anakinra therapy may reduce bone loss in experimental murine models of RRV and CHIKV. Further investigations are needed to assess the potential therapeutic benefits of anakinra in patients with arthritogenic alphavirus disease.

Decreased Virulence of Ross River Virus Harboring a Mutation in the First Cleavage Site of Nonstructural Polyprotein Is Caused by a Novel Mechanism Leading to Increased Production of Interferon-Inducing RNAs.

Infection with Ross River virus (RRV) causes debilitating polyarthritis and arthralgia in individuals. Alphaviruses are highly sensitive to type I interferon (IFN). Mutations at the conserved P3 position of the cleavage site between nonstructural protein 1 (nsP1) and nsP2 (1/2 site) modulate type I IFN induction for both RRV and Sindbis virus (SINV). We constructed and characterized RRV-T48A534V, a mutant harboring an A534V substitution in the P1 position of the 1/2 site, and compared it to parental RRV-T48 and to RRV-T48A532V, SINVI538 and SINVT538 harboring different substitutions in the same region. A534V substitution resulted in impaired processing of RRV nonstructural polyprotein and in elevated production of replicase-generated pathogen-associated molecular pattern (PAMP) RNAs that induce expression of type I IFN. Both A532V and A534V substitutions affected synthesis of viral RNAs, though the effects of these closely located mutations were drastically different affecting mostly either the viral negative-strand RNA or genomic and subgenomic RNA levels, respectively. Synthesis of PAMP RNAs was also observed for SINV replicase, and it was increased by I538T substitution. In comparison to RRV-T48, RRV-T48A534V was attenuated in vitro and in vivo Interestingly, when type I IFN-deficient cells and type I IFN receptor-deficient mice were infected with RRV-T48 or RRV-T48A534V, differences between these viruses were no longer apparent. Compared to RRV-T48, RRV-T48A534V infection was associated with increased upregulation of type I IFN signaling proteins. We demonstrate novel mechanisms by which the A534V mutation affect viral nonstructural polyprotein processing that can impact PAMP RNA production, type I IFN induction/sensitivity, and disease.IMPORTANCE This study gives further insight into mechanisms of type I IFN modulation by the medically important alphaviruses Ross River virus (RRV) and Sindbis virus (SINV). By characterizing attenuated RRV mutants, the crucial role of amino acid residues in P1 and P3 positions (the first and third amino acid residues preceding the scissile bond) of the cleavage site between nsP1 and nsP2 regions was highlighted. The study uncovers a unique relationship between alphavirus nonstructural polyprotein processing, RNA replication, production of different types of pathogen-associated molecular pattern (PAMP) RNAs, type I IFN induction, and disease pathogenesis. This study also highlights the importance of the host innate immune response in RRV infections. The viral determinants of type I IFN modulation provide potential drug targets for clinical treatment of alphaviral disease and offer new approaches for rational attenuation of alphaviruses for construction of vaccine candidates.

How myeloid cells contribute to the pathogenesis of prominent emerging zoonotic diseases.

Up to 75 % of emerging human diseases are zoonoses, spread from animals to humans. Although bacteria, fungi and parasites can be causative agents, the majority of zoonotic infections are caused by viral pathogens. During the past 20 years many factors have converged to cause a dramatic resurgence or emergence of zoonotic diseases. Some of these factors include demographics, social changes, urban sprawl, changes in agricultural practices and global climate changes. In the period between 2014-2017 zoonotic viruses including ebola virus (EBOV), chikungunya virus (CHIKV), dengue virus (DENV) and zika virus (ZIKV), caused prominent outbreaks resulting in significant public health and economic burdens, especially in developing areas where these diseases are most prevalent. When a viral pathogen invades a new human host, it is the innate immune system that serves as the first line of defence. Myeloid cells are especially important to help fight viral infections, including those of zoonotic origins. However, viruses such as EBOV, CHIKV, DENV and ZIKV have evolved mechanisms that allow circumvention of the host's innate immune response, avoiding eradication and leading to severe clinical disease. Herein, the importance of myeloid cells in host defence is discussed and the mechanisms by which these viruses exploit myeloid cells are highlighted. The insights provided in this review will be invaluable for future studies looking to identify potential therapeutic targets towards the treatment of these emerging diseases.