An improved alphaviruses-specific RT-qPCR facilitates monitoring and prevention of alphaviruses.

Molecular surveillance is vital for monitoring arboviruses, often employing genus-specific quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Despite this, an overlooked chikungunya fever outbreak occurred in Yunnan province, China, in 2019 and false negatives are commonly encountered during alphaviruses screening practice, highlighting the need for improved detection methods. In this study, we developed an improved alphaviruses-specific RT-qPCR capable of detecting chikungunya virus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Sindbis virus, Mayaro virus, and Ross River virus with high sensitivity and specificity. The assay identified three chikungunya virus-positive cases out of 188 sera retrospectively. Later genetic characterization suggested that imported cases from neighboring countries may be responsible for the neglected chikungunya fever outbreak of 2019 in Yunnan. Our findings underscore the value of improved alphaviruses-specific RT-qPCR in bolstering alphaviruses surveillance and informing preventive strategies.

Advances in Alphavirus and Flavivirus Research.

Newly emerging viruses, primarily zoonotic or vector-borne, pose a persistent threat to public health and have led to outbreaks of global concern [...].

Pathogenic differences of cynomolgus macaques after Taï Forest virus infection depend on the viral stock propagation.

Taï Forest virus (TAFV) is a negative-sense RNA virus in the Filoviridae family. TAFV has caused only a single human infection, but several disease outbreaks in chimpanzees have been linked to this virus. Limited research has been done on this human-pathogenic virus. We sought to establish an animal model to assess TAFV disease progression and pathogenicity at our facility. We had access to two different viral stock preparations from different institutions, both originating from the single human case. Type I interferon receptor knockout mice were inoculated with TAFV stock 1 or stock 2 by the intraperitoneal route. Inoculation resulted in 100% survival with no disease regardless of viral stock preparation or infectious dose. Next, cynomolgus macaques were inoculated with TAFV stock 1 or stock 2. Inoculation with TAFV stock 1 resulted in 100% survival and robust TAFV glycoprotein-specific IgG responses including neutralizing antibodies. In contrast, macaques infected with TAFV stock 2 developed disease and were euthanized 8-11 days after infection exhibiting viremia, thrombocytopenia, and increased inflammatory mediators identified by transcriptional analysis. Histopathologic analysis of tissue samples collected at necropsy confirmed classic filovirus disease in numerous organs. Genomic differences in both stock preparations were mapped to several viral genes which may have contributed to disease severity. Taken together, we demonstrate that infection with the two TAFV stocks resulted in no disease in mice and opposing disease phenotypes in cynomolgus macaques, highlighting the impact of viral stock propagation on pathogenicity in animal models.

Co-circulation of two Alphaviruses in Burkina Faso: Chikungunya and O'nyong nyong viruses.

BACKGROUND: Chikungunya virus (CHIKV) and O'nyong nyong virus (ONNV) are phylogenetically related alphaviruses in the Semliki Forest Virus (SFV) antigenic complex of the Togaviridae family. There are limited data on the circulation of these two viruses in Burkina Faso. The aim of our study was to assess their circulation in the country by determining seroprevalence to each of the viruses in blood donor samples and by retrospective molecular and serological testing of samples collected as part of national measles and rubella surveillance. METHODOLOGY/PRINCIPAL FINDINGS: All blood donor samples were analyzed on the Luminex platform using CHIKV and ONNV E2 antigens. Patient samples collected during national measles-rubella surveillance were screened by an initial ELISA for CHIKV IgM (CHIKjj Detect IgM ELISA) at the national laboratory. The positive samples were then analyzed by a second ELISA test for CHIKV IgM (CDC MAC-ELISA) at the reference laboratory. Finally, samples that had IgM positive results for both ELISA tests and had sufficient residual volume were tested by plaque reduction neutralization testing (PRNT) for CHIKV and ONNV. These same patient samples were also analyzed by rRT-PCR for CHIKV. Among the blood donor specimens, 55.49% of the samples were positive for alphaviruses including both CHIKV and ONNV positive samples. Among patient samples collected as part of national measles and rubella surveillance, 3.09% were IgM positive for CHIKV, including 2.5% confirmed by PRNT. PRNT failed to demonstrate any ONNV infections in these samples. No samples tested by RT-qPCR. had detectable CHIKV RNA. CONCLUSIONS/SIGNIFICANCE: Our results suggest that CHIKV and ONNV have been circulating in the population of Burkina Faso and may have been confused with malaria, dengue fever or other febrile diseases such as measles or rubella. Our study underscores the necessity to enhance arbovirus surveillance systems in Burkina Faso.

Development and application of a colloidal-gold immunochromatographic strip for detecting Getah virus antibodies.

Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: • We established a rapid antibody detection method that can monitor GETV infection • We developed colloidal gold test strips with high sensitivity and specificity • The development of colloidal gold test strips will aid in the field serologic detection of GETV.

MAVS disruption impairs downstream signaling and results in higher virus replication levels of salmonid alphavirus subtype 3 but not infectious pancreatic necrosis virus in vitro.

The mitochondrial anti-viral signaling (MAVS) protein is an intermediary adaptor protein of retinoic acid-inducible gene-1 (RIG-I) like receptor (RLR) signaling, which activates the transcription factor interferon (IFN) regulatory factor 3 (IRF3) and NF-kB to produce type I IFNs. MAVS expression has been reported in different fish species, but few studies have shown its functional role in anti-viral responses to fish viruses. In this study, we used the transcription activator-like effector nuclease (TALEN) as a gene editing tool to disrupt the function of MAVS in Chinook salmon (Oncorhynchus tshawytscha) embryonic cells (CHSE) to understand its role in induction of interferon I responses to infections with the (+) RNA virus salmonid alphavirus subtype 3 (SAV-3), and the dsRNA virus infectious pancreatic necrosis virus (IPNV) infection. A MAVS-disrupted CHSE clone with a 7-aa polypeptide (GVFVSRV) deletion mutation at the N-terminal of the CARD domain infected with SAV-3 resulted in significantly lower expression of IRF3, IFNa, and ISGs and increased viral titer (1.5 log10) compared to wild-type. In contrast, the IPNV titer in MAVS-disrupted cells was not different from the wild-type. Furthermore, overexpression of salmon MAVS in MAVS-disrupted CHSE cells rescued the impaired type I IFN-mediated anti-viral effect against SAV-3.

LDL receptor in alphavirus entry: structural analysis and implications for antiviral therapy.

Various low-density lipoprotein receptors (LPRs) have been identified as entry factors for alphaviruses, and structures of the corresponding virion-receptor complexes have been determined. Here, we analyze the similarities and differences in the receptor binding modes of multiple alphaviruses to understand their ability to infect a wide range of hosts. We further discuss the challenges associated with the development of broad-spectrum treatment strategies against a diverse range of alphaviruses.

Research trends on alphavirus receptors: a bibliometric analysis.

Background: Alphaviruses are a diverse group of pathogens that have garnered considerable attention due to their impact on human health. By investigating alphavirus receptors, researchers can elucidate viral entry mechanisms and gain important clues for the prevention and treatment of viral diseases. This study presents an in-depth analysis of the research progress made in the field of alphavirus receptors through bibliometric analysis. Methods: This study encompasses various aspects, including historical development, annual publication trends, author and cited-author analysis, institutional affiliations, global distribution of research contributions, reference analysis with strongest citation bursts, keyword analysis, and a detailed exploration of recent discoveries in alphavirus receptor research. Results: The results of this bibliometric analysis highlight key milestones in alphavirus receptor research, demonstrating the progression of knowledge in this field over time. Additionally, the analysis reveals current research hotspots and identifies emerging frontiers, which can guide future investigations and inspire novel therapeutic strategies. Conclusion: This study provides an overview of the state of the art in alphavirus receptor research, consolidating the existing knowledge and paving the way for further advancements. By shedding light on the significant developments and emerging areas of interest, this study serves as a valuable resource for researchers, clinicians, and policymakers engaged in combating alphavirus infections and improving public health.

Evolutionary Profile of Mayaro Virus in the Americas: An Update into Genome Variability.

The Mayaro virus (MAYV) is an arbovirus with emerging potential, though with a limited understanding of its epidemiology and evolution due to the lack of studies and surveillance. Here, we investigated 71 MAYV genome sequences from the Americas available at GenBank and characterized the phylogenetic relationship among virus strains. A phylogenetic analysis showed that sequences were grouped according to the genotypes L, D, and N. Genotype D sequences were closely related to sequences collected in adjacent years and from their respective countries, suggesting that isolates may have originated from circulating lineages. The coalescent analysis demonstrated similar results, indicating the continuous circulation of the virus between countries as well. An unidentified sequence from the USA was grouped with genotype D, suggesting the insertion of this genotype in the country. Furthermore, the recombination analysis detected homologous and three heterologous hybrids which presented an insertion into the nsP3 protein. Amino acid substitutions among sequences indicated selective pressure sites, suggesting viral adaptability. This also impacted the binding affinity between the E1-E2 protein complex and the Mxra8 receptor, associated with MAYV entry into human cells. These results provide information for a better understanding of genotypes circulating in the Americas.

Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

Mayaro virus, a potential threat for Europe: vector competence of autochthonous vector species.

BACKGROUND: Mayaro virus (MAYV) is an emerging alphavirus, primarily transmitted by the mosquito Haemagogus janthinomys in Central and South America. However, recent studies have shown that Aedes aegypti, Aedes albopictus and various Anopheles mosquitoes can also transmit the virus under laboratory conditions. MAYV causes sporadic outbreaks across the South American region, particularly in areas near forests. Recently, cases have been reported in European and North American travelers returning from endemic areas, raising concerns about potential introductions into new regions. This study aims to assess the vector competence of three potential vectors for MAYV present in Europe. METHODS: Aedes albopictus from Italy, Anopheles atroparvus from Spain and Culex pipiens biotype molestus from Belgium were exposed to MAYV and maintained under controlled environmental conditions. Saliva was collected through a salivation assay at 7 and 14 days post-infection (dpi), followed by vector dissection. Viral titers were determined using focus forming assays, and infection rates, dissemination rates, and transmission efficiency were calculated. RESULTS: Results indicate that Ae. albopictus and An. atroparvus from Italy and Spain, respectively, are competent vectors for MAYV, with transmission possible starting from 7 dpi under laboratory conditions. In contrast, Cx. pipiens bioform molestus was unable to support MAYV infection, indicating its inability to contribute to the transmission cycle. CONCLUSIONS: In the event of accidental MAYV introduction in European territories, autochthonous outbreaks could potentially be sustained by two European species: Ae. albopictus and An. atroparvus. Entomological surveillance should also consider certain Anopheles species when monitoring MAYV transmission.

Phenotypic and transcriptional changes in peripheral blood mononuclear cells during alphavirus encephalitis in mice.

Sindbis virus (SINV) infection of mice provides a model system for studying the pathogenesis of alphaviruses that infect the central nervous system (CNS) to cause encephalomyelitis. While studies of human viral infections typically focus on accessible cells from the blood, this compartment is rarely evaluated in mice. To bridge this gap, single-cell RNA sequencing (scRNAseq) was combined with flow cytometry to characterize the transcriptional and phenotypic changes of peripheral blood mononuclear cells (PBMCs) from SINV-infected mice. Twenty-one clusters were identified by scRNAseq at 7 days after infection, with a unique cluster and overall increase in naive B cells for infected mice. Uninfected mice had fewer immature T cells and CCR9+ CD4 T cells and a unique immature T cell cluster. Gene expression was most altered in the Ki67+ CD8 T cell cluster, with chemotaxis and proliferation-related genes upregulated. Global analysis indicated metabolic changes in myeloid cells and increased expression of Ccl5 by NK cells. Phenotypes of PBMCs and cells infiltrating the CNS were analyzed by flow cytometry over 14 days after infection. In PBMCs, CD8 and Th1 CD4 T cells increased in representation, while B cells showed a transient decrease at day 5 in total, Ly6a+, and naive cells, and an increase in activated B cells. In the brain, CD8 T cells increased for the first 7 days, while Th1 CD4 T cells and naive and Ly6a+ B cells continued to accumulate for 14 days. Therefore, dynamic immune cell changes can be identified in the blood as well as the CNS during viral encephalomyelitis. IMPORTANCE: The outcome of viral encephalomyelitis is dependent on the host immune response, with clearance and resolution of infection mediated by the adaptive immune response. These processes are frequently studied in mouse models of infection, where infected tissues are examined to understand the mechanisms of clearance and recovery. However, studies of human infection typically focus on the analysis of cells from the blood, a compartment rarely examined in mice, rather than inaccessible tissue. To close this gap, we used single-cell RNA sequencing and flow cytometry to profile the transcriptomic and phenotypic changes of peripheral blood mononuclear cells (PBMCs) before and after central nervous system (CNS) infection in mice. Changes to T and B cell gene expression and cell composition occurred in PBMC and during entry into the CNS, with CCL5 being a differentially expressed chemokine. Therefore, dynamic changes occur in the blood as well as the CNS during the response of mice to virus infection, which will inform the analysis of human studies.

Current challenges in the discovery of treatments against Mayaro fever.

INTRODUCTION: Mayaro fever is an emerging viral disease that manifests as an acute febrile illness. The disease is self-limiting, however joint pain can persist for months leading to chronic arthralgia. There is no specific treatment available, which ultimately leads to socioeconomic losses in populations at risk as well as strains to the public health systems. AREAS COVERED: We reviewed the candidate treatments proposed for Mayaro virus (MAYV) infection and disease, including antiviral compounds targeting viral or host mechanisms, and pathways involved in disease development and pathogenicity. We assessed compound screening technologies and experimental infection models used in these studies and indicated the advantages and limitations of available technologies and intended therapeutic strategies. EXPERT OPINION: Although several compounds have been suggested as candidate treatments against MAYV infection, notably those with antiviral activity, most compounds were assessed only in vitro. Compounds rarely progress toin vivo or preclinical studies, and such difficulty may be associated with limited experimental models. MAYV biology is largely inferred from related alphaviruses and reflected by few studies focusing on target proteins or mechanisms of action for MAYV. Therapeutic strategies targeting pathogenic inflammatory responses have shown potential against MAYV-induced disease in vivo, which might reduce long-term sequelae.

Pathogenicity and virulence of O'nyong-nyong virus: A less studied Togaviridae with pandemic potential.

O'nyong-nyong virus (ONNV) is a neglected mosquito-borne alphavirus belonging to the Togaviridae family. ONNV is known to be responsible for sporadic outbreaks of acute febrile disease and polyarthralgia in Africa. As climate change increases the geographical range of known and potential new vectors, recent data indicate a possibility for ONNV to spread outside of the African continent and grow into a greater public health concern. In this review, we summarise the current knowledge on ONNV epidemiology, host-pathogen interactions, vector-virus responses, and insights into possible avenues to control risk of further epidemics. In this review, the limited ONNV literature is compared and correlated to other findings on mainly Old World alphaviruses. We highlight and discuss studies that investigate viral and host factors that determine viral-vector specificity, along with important mechanisms that determine severity and disease outcome of ONNV infection.

Trans-Amplifying RNA: A Journey from Alphavirus Research to Future Vaccines.

Replicating RNA, including self-amplifying RNA (saRNA) and trans-amplifying RNA (taRNA), holds great potential for advancing the next generation of RNA-based vaccines. Unlike in vitro transcribed mRNA found in most current RNA vaccines, saRNA or taRNA can be massively replicated within cells in the presence of RNA-amplifying enzymes known as replicases. We recently demonstrated that this property could enhance immune responses with minimal injected RNA amounts. In saRNA-based vaccines, replicase and antigens are encoded on the same mRNA molecule, resulting in very long RNA sequences, which poses significant challenges in production, delivery, and stability. In taRNA-based vaccines, these challenges can be overcome by splitting the replication system into two parts: one that encodes replicase and the other that encodes a short antigen-encoding RNA called transreplicon. Here, we review the identification and use of transreplicon RNA in alphavirus research, with a focus on the development of novel taRNA technology as a state-of-the art vaccine platform. Additionally, we discuss remaining challenges essential to the clinical application and highlight the potential benefits related to the unique properties of this future vaccine platform.

Exposure dynamics of Ross River virus in horses - Horses as potential sentinels (a One Health approach).

Ross River virus (RRV), the most medically and economically important arbovirus in Australia, has been the most prevalent arbovirus infections in humans for many years. Infected humans and horses often suffer similar clinical symptoms. We conducted a prospective longitudinal study over a 3.5-year period to investigate the exposure dynamics of RRV in three foal cohorts (n = 32) born in a subtropical region of South East Queensland, Australia, between 2020 and 2022. RRV-specific seroconversion was detected in 56% (n = 18) of foals with a median time to seroconversion, after waning of maternal antibodies, of 429 days (95% CI: 294-582). The median age at seroconversion was 69 weeks (95% CI: 53-57). Seroconversion events were only detected between December and March (Southern Hemisphere summer) over the entire study period. Cox proportion hazards regression analyses revealed that seroconversions were significantly (p < 0.05) associated with air temperature in the month of seroconversion. Time-lags in meteorological variables were not significantly (p > 0.05) associated with seroconversion, except for relative humidity (p = 0.036 at 2-month time-lag). This is in contrast to research results of RRV infection in humans, which peaked between March and May (Autumn) and with a 0-3 month time-lag for various meteorological risk factors. Therefore, horses may be suitable sentinels for monitoring active arbovirus circulation and could be used for early arbovirus outbreak detection in human populations.

Molecular Epidemiology of Mayaro Virus among Febrile Patients, Roraima State, Brazil, 2018-2021.

We detected Mayaro virus (MAYV) in 3.4% (28/822) of febrile patients tested during 2018-2021 from Roraima State, Brazil. We also isolated MAYV strains and confirmed that these cases were caused by genotype D. Improved surveillance is needed to better determine the burden of MAYV in the Amazon Region.

DEAD box RNA helicase 5 is a new pro-viral host factor for Sindbis virus infection.

BACKGROUND: RNA helicases are emerging as key factors regulating host-virus interactions. The DEAD-box ATP-dependent RNA helicase DDX5, which plays an important role in many aspects of cellular RNA biology, was also found to either promote or inhibit viral replication upon infection with several RNA viruses. Here, our aim is to examine the impact of DDX5 on Sindbis virus (SINV) infection. METHODS: We analysed the interaction between DDX5 and the viral RNA using imaging and RNA-immunoprecipitation approaches. The interactome of DDX5 in mock- and SINV-infected cells was determined by mass spectrometry. We validated the interaction between DDX17 and the viral capsid by co- immunoprecipitation in the presence or absence of an RNase treatment. We determined the subcellular localization of DDX5, its cofactor DDX17 and the viral capsid protein by co-immunofluorescence. Finally, we investigated the impact of DDX5 depletion and overexpression on SINV infection at the viral protein, RNA and infectious particle accumulation level. The contribution of DDX17 was also tested by knockdown experiments. RESULTS: In this study we demonstrate that DDX5 interacts with the SINV RNA during infection. Furthermore, the proteomic analysis of the DDX5 interactome in mock and SINV-infected HCT116 cells identified new cellular and viral partners and confirmed the interaction between DDX5 and DDX17. Both DDX5 and DDX17 re-localize from the nucleus to the cytoplasm upon SINV infection and interact with the viral capsid protein. We also show that DDX5 depletion negatively impacts the viral replication cycle, while its overexpression has a pro-viral effect. Finally, we observed that DDX17 depletion reduces SINV infection, an effect which is even more pronounced in a DDX5-depleted background, suggesting a synergistic pro-viral effect of the DDX5 and DDX17 proteins on SINV. CONCLUSIONS: These results not only shed light on DDX5 as a novel and important host factor to the SINV life cycle, but also expand our understanding of the roles played by DDX5 and DDX17 as regulators of viral infections.