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Anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis has diverse patterns of injury including microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA). Necrotizing and crescentic glomerulonephritis (NCGN) occurs in all syndromes and as renal limited vasculitis (RLV). Single-dose intravenous ANCA IgG-specific for mouse myeloperoxidase (MPO) causes RLV in mice. Although multiple mouse models have elucidated ANCA-IgG induced necrotizing and crescentic glomerulonephritis (NCGN), pathogenesis of ANCA-induced granulomatosis and vasculitis outside the kidney has not been clarified. To investigate this, we used intravenous MPO-ANCA IgG in the same strain of mice to induce different patterns of lung disease mirroring patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). Repeated intravenous MPO-ANCA IgG induced GPA with NCGN, lung capillaritis, arteritis and granulomatosis. Lung leukocyte phenotypes were evaluated by immunohistochemical image analysis and by flow cytometry. ANCA lung capillaritis and microabscesses began within one day and evolved into granulomas in under seven days. Influenza plus single-dose MPO-ANCA IgG induced MPA with NCGN, lung capillaritis and arteritis, but no granulomatosis. Allergic airway disease caused by house dust mites or ovalbumin plus single-dose intravenous MPO-ANCA IgG induced EGPA with eosinophilic bronchiolitis, NCGN, capillaritis, arteritis, and granulomatosis. Thus, our study shows that the occurrence and pattern of lung lesions are determined by the same ANCA IgG accompanied by different synergistic immune factors., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Chikungunya virus (CHIKV) is an enveloped, positive-sense RNA virus that has re-emerged to cause millions of human infections worldwide. In humans, acute CHIKV infection causes fever and severe muscle and joint pain. Chronic and debilitating arthritis and joint pain can persist for months to years. To date, there are no approved antivirals against CHIKV. Recently, the ribonucleoside analog 4'-fluorouridine (4'-FlU) was reported as a highly potent orally available inhibitor of SARS-CoV-2, respiratory syncytial virus, and influenza virus replication. In this study, we assessed 4'-FlU's potency and breadth of inhibition against a panel of alphaviruses including CHIKV, and found that it broadly suppressed alphavirus production in cell culture. 4'-FlU acted on the viral RNA replication step, and the first 4 hours post-infection were the critical time for its antiviral effect. In vitro replication assays identified nsP4 as the target of inhibition. In vivo , treatment with 4'-FlU reduced disease signs, inflammatory responses, and viral tissue burden in mouse models of CHIKV and Mayaro virus infection. Treatment initiated at 2 hours post-infection was most effective; however, treatment initiated as late as 24-48 hours post-infection produced measurable antiviral effects in the CHIKV mouse model. 4'-FlU showed effective oral delivery in our mouse model and resulted in the accumulation of both 4'-FlU and its bioactive triphosphate form in tissues relevant to arthritogenic alphavirus pathogenesis. Together, our data indicate that 4'-FlU inhibits CHIKV infection in vitro and in vivo and is a promising oral therapeutic candidate against CHIKV infection.IMPORTANCEAlphaviruses including chikungunya virus (CHIKV) are mosquito-borne positive-strand RNA viruses that can cause various diseases in humans. Although compounds that inhibit CHIKV and other alphaviruses have been identified in vitro , there are no licensed antivirals against CHIKV. Here, we investigated a ribonucleoside analog, 4'-fluorouridine (4'-FlU), and demonstrated that it inhibited infectious virus production by several alphaviruses in vitro and reduced virus burden in mouse models of CHIKV and Mayaro virus infection. Our studies also indicated that 4'-FlU treatment reduced CHIKV-induced footpad swelling and reduced the production of pro-inflammatory cytokines. Inhibition in the mouse model correlated with effective oral delivery of 4'-FlU and accumulation of both 4'-FlU and its bioactive form in relevant tissues. In summary, 4'-FlU exhibits potential as a novel anti-alphavirus agent targeting the replication of viral RNA., Competing Interests: G.R.P. is a coinventor on patent WO 2019/1736002 covering the composition of matter and the use of EIDD-2749 and its analogs as an antiviral treatment. This study could affect his personal financial status. The other authors declare no conflict of interest.

We previously reported that deletion of a 44-nucleotide element in the 3' untranslated region (UTR) of the Chikungunya virus (CHIKV) genome enhances the virulence of CHIKV infection in mice. Here, we find that while this 44-nucleotide deletion enhances CHIKV fitness in murine embryonic fibroblasts in a manner independent of the type I interferon response, the same mutation decreases viral fitness in C6/36 mosquito cells. Further, the fitness advantage conferred by the UTR deletion in mammalian cells is maintained in vivo in a mouse model of CHIKV dissemination. Finally, SHAPE-MaP analysis of the CHIKV 3' UTR revealed this 44-nucleotide element forms a distinctive two-stem-loop structure that is ablated in the mutant 3' UTR without altering additional 3' UTR RNA secondary structures.

Alphaviruses are mosquito-transmitted pathogens that induce high levels of viremia, which facilitates dissemination and vector transmission. One prevailing paradigm is that, after skin inoculation, alphavirus-infected resident dendritic cells migrate to the draining lymph node (DLN), facilitating further rounds of infection and dissemination. Here, we assess the contribution of infiltrating myeloid cells to alphavirus spread. We observe two phases of virus transport to the DLN, one that occurs starting at 1 h post infection and precedes viral replication, and a second that requires replication in the skin, enabling transit to the bloodstream. Depletion of Ly6C + monocytes reduces local chikungunya (CHIKV) or Ross River virus (RRV) infection in the skin, diminishes the second phase of virus transport to the DLN, and delays spread to distal sites. Our data suggest that infiltrating monocytes facilitate alphavirus infection at the initial infection site, which promotes more rapid spread into circulation., Competing Interests: Declaration of interests M.S.D. is a consultant or member of a Scientific Advisory Board for Inbios, Vir Biotechnology, IntegerBio, Merck, GlaxoSmithKline, Akagera Medicines, and Moderna. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Emergent BioSolutions, Moderna, IntegerBio, and Vir Biotechnology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antigens from viruses or immunizations can persist or are archived in lymph node stromal cells such as lymphatic endothelial cells (LEC) and fibroblastic reticular cells (FRC). Here, we find that, during the time frame of antigen archiving, LEC apoptosis caused by a second, but unrelated, innate immune stimulus such as vaccina viral infection or CpG DNA administration resulted in cross-presentation of archived antigens and boosted memory CD8 + T cells specific to the archived antigen. In contrast to "bystander" activation associated with unrelated infections, the memory CD8 + T cells specific to the archived antigen from the immunization were significantly higher than memory CD8 + T cells of a different antigen specificity. Finally, the boosted memory CD8 + T cells resulted in increased protection against Listeria monocytogenes expressing the antigen from the immunization, but only for the duration that the antigen was archived. These findings outline an important mechanism by which lymph node stromal cell archived antigens, in addition to bystander activation, can augment memory CD8 + T cell responses during repeated inflammatory insults., (© 2024. The Author(s).)

The magnitude and duration of vertebrate viremia are critical determinants of arbovirus transmission, geographic spread, and disease severity-yet, mechanisms determining arbovirus viremia levels are poorly defined. Previous studies have drawn associations between in vitro virion-glycosaminoglycan (GAG) interactions and in vivo clearance kinetics of virions from blood circulation. From these observations, it is commonly hypothesized that GAG-binding virions are rapidly removed from circulation due to ubiquitous expression of GAGs by vascular endothelial cells, thereby limiting viremia. Using an in vivo model for viremia, we compared the vascular clearance of low and enhanced GAG-binding viral variants of chikungunya, eastern- (EEEV), and Venezuelan- (VEEV) equine encephalitis viruses. We find GAG-binding virions are more quickly removed from circulation than their non-GAG-binding variant; however individual clearance kinetics vary between GAG-binding viruses, from swift (VEEV) to slow removal from circulation (EEEV). Remarkably, we find phagocytes are required for efficient vascular clearance of some enhanced GAG-binding virions. Moreover, transient depletion of vascular heparan sulfate impedes vascular clearance of only some GAG-binding viral variants and in a phagocyte-dependent manner, implying phagocytes can mediate vascular GAG-virion interactions. Finally, in direct contrast to mice, we find enhanced GAG-binding EEEV is resistant to vascular clearance in avian hosts, suggesting the existence of species-specificity in virion-GAG interactions. In summary, these data support a role for GAG-mediated clearance of some viral particles from the blood circulation, illuminate the potential of blood-contacting phagocytes as a site for GAG-virion binding, and suggest a role for species-specific GAG structures in arbovirus ecology., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

Infection by chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes severe polyarthralgia and polymyalgia, which can last in some people for months to years. Chronic CHIKV disease signs and symptoms are associated with the persistence of viral nucleic acid and antigen in tissues. Like humans and nonhuman primates, CHIKV infection in mice results in the development of robust adaptive antiviral immune responses. Despite this, joint tissue fibroblasts survive CHIKV infection and can support persistent viral replication, suggesting that they escape immune surveillance. Here, using a recombinant CHIKV strain encoding the fluorescent protein VENUS with an embedded CD8+ T cell epitope, SIINFEKL, we observed a marked loss of both MHC class I (MHC-I) surface expression and antigen presentation by CHIKV-infected joint tissue fibroblasts. Both in vivo and ex vivo infected joint tissue fibroblasts displayed reduced cell surface levels of H2-Kb and H2-Db MHC-I proteins while maintaining similar levels of other cell surface proteins. Mutations within the methyl transferase-like domain of the CHIKV nonstructural protein 2 (nsP2) increased MHC-I cell surface expression and antigen presentation efficiency by CHIKV-infected cells. Moreover, expression of WT nsP2 alone, but not nsP2 with mutations in the methyltransferase-like domain, resulted in decreased MHC-I antigen presentation efficiency. MHC-I surface expression and antigen presentation was rescued by replacing VENUS-SIINFEKL with SIINFEKL tethered to β2-microglobulin in the CHIKV genome, which bypasses the requirement for peptide processing and TAP-mediated peptide transport into the endoplasmic reticulum. Collectively, this work suggests that CHIKV escapes the surveillance of antiviral CD8+ T cells, in part, by nsP2-mediated disruption of MHC-I antigen presentation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ware et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Intracellular Salmonella experiencing oxidative stress downregulates aerobic respiration. To maintain cellular energetics during periods of oxidative stress, intracellular Salmonella must utilize terminal electron acceptors of lower energetic value than molecular oxygen. We show here that intracellular Salmonella undergoes anaerobic respiration during adaptation to the respiratory burst of the phagocyte NADPH oxidase in macrophages and in mice. Reactive oxygen species generated by phagocytes oxidize methionine, generating methionine sulfoxide. Anaerobic Salmonella uses the molybdenum cofactor-containing DmsABC enzymatic complex to reduce methionine sulfoxide. The enzymatic activity of the methionine sulfoxide reductase DmsABC helps Salmonella maintain an alkaline cytoplasm that supports the synthesis of the antioxidant hydrogen sulfide via cysteine desulfuration while providing a source of methionine and fostering redox balancing by associated dehydrogenases. Our investigations demonstrate that nontyphoidal Salmonella responding to oxidative stress exploits the anaerobic metabolism associated with dmsABC gene products, a pathway that has accrued inactivating mutations in human-adapted typhoidal serovars., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we found that, during the first 24 hours of infection, CHIKV RNA accumulated in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response - including recruitment of myeloid cells to the LN - was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

Chikungunya virus (CHIKV) has recently emerged to cause millions of human infections worldwide. Infection can induce the formation of long intercellular extensions that project from infected cells and form stable non-continuous membrane bridges with neighbouring cells. The mechanistic role of these intercellular extensions in CHIKV infection was unclear. Here we developed a co-culture system and flow cytometry methods to quantitatively evaluate transmission of CHIKV from infected to uninfected cells in the presence of neutralizing antibody. Endocytosis and endosomal acidification were critical for virus cell-to-cell transmission, while the CHIKV receptor MXRA8 was not. By using distinct antibodies to block formation of extensions and by evaluation of transmission in HeLa cells that did not form extensions, we showed that intercellular extensions mediate CHIKV cell-to-cell transmission. In vivo, pre-treatment of mice with a neutralizing antibody blocked infection by direct virus inoculation, while adoptive transfer of infected cells produced antibody-resistant host infection. Together our data suggest a model in which the contact sites of intercellular extensions on target cells shield CHIKV from neutralizing antibodies and promote efficient intercellular virus transmission both in vitro and in vivo., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Endothelial dysfunction and inflammation contribute to the vascular pathology of coronavirus disease (COVID-19). However, emerging evidence does not support direct infection of endothelial or other vascular wall cells, and thus inflammation may be better explained as a secondary response to epithelial cell infection. In this study, we sought to determine whether lung endothelial or other resident vascular cells are susceptible to productive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and how local complement activation contributes to endothelial dysfunction and inflammation in response to hypoxia and SARS-CoV-2-infected lung alveolar epithelial cells. We found that ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) mRNA expression in lung vascular cells, including primary human lung microvascular endothelial cells (HLMVECs), pericytes, smooth muscle cells, and fibroblasts, was 20- to 90-fold lower compared with primary human alveolar epithelial type II cells. Consistently, we found that HLMVECs and other resident vascular cells were not susceptible to productive SARS-CoV-2 infection under either normoxic or hypoxic conditions. However, viral uptake without replication (abortive infection) was observed in HLMVECs when exposed to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells. Furthermore, we demonstrated that exposure of HLMVECs to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells and hypoxia resulted in upregulation of inflammatory factors such as ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), and IL-6 (interleukin 6) as well as complement components such as C3 (complement C3), C3AR1 (complement C3a receptor 1), C1QA (complement C1q A chain), and CFB (complement factor B). Taken together, our data support a model in which lung endothelial and vascular dysfunction during COVID-19 involves the activation of complement and inflammatory signaling and does not involve productive viral infection of endothelial cells.

Arboviruses are public health threats that cause explosive outbreaks. Major determinants of arbovirus transmission, geographic spread, and pathogenesis are the magnitude and duration of viremia in vertebrate hosts. Previously, we determined that multiple alphaviruses are cleared efficiently from murine circulation by the scavenger receptor MARCO (Macrophage receptor with collagenous structure). Here, we define biochemical features on chikungunya (CHIKV), o'nyong 'nyong (ONNV), and Ross River (RRV) viruses required for MARCO-dependent clearance in vivo. In vitro, MARCO expression promotes binding and internalization of CHIKV, ONNV, and RRV via the scavenger receptor cysteine-rich (SRCR) domain. Furthermore, we observe species-specific effects of the MARCO SRCR domain on CHIKV internalization, where those from known amplification hosts fail to promote CHIKV internalization. Consistent with this observation, CHIKV is inefficiently cleared from the circulation of rhesus macaques in contrast with mice. These findings suggest a role for MARCO in determining whether a vertebrate serves as an amplification or dead-end host following CHIKV infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Infectious particles can be shared through aerosols and droplets formed as the result of normal respiration. Whether Abs within the nasal/oral fluids can similarly be shared between hosts has not been investigated. The circumstances of the SARS-CoV-2 pandemic facilitated a unique opportunity to fully examine this provocative idea. The data we show from human nasal swabs provides evidence for the aerosol transfer of Abs between immune and nonimmune hosts., (Copyright © 2023 The Authors.)

Purpose of Review: We discuss current research on the mental health effects of COVID-19 sports restrictions on youth athletes, highlighting the largest problems, as well as how organizations can help youth athletes by preparing for and responding to these problems., Recent Findings: Millions of children and adolescents worldwide participate in organized sports, which has significant physical and mental health benefits. In 2020, the COVID-19 pandemic triggered large-scale, public restrictions that forced the closure and cancelation of organized youth sports across the world. Sports cancelations not only removed these protective benefits, but also worsened the mental health of youth athletes who were no longer able to participate in their sports. Youth athletes are even more vulnerable than adults to the negative mental health effects of sports restrictions. The unexpected loss of sports from COVID-19 restrictions disrupted these youths' athletic identities and worsened youth athlete depression, anxiety, anger, sleep, and quality of life. Restrictions particularly affected certain high-risk subpopulations of youth athletes including females, high school upperclassmen, those from low socioeconomic backgrounds, and those from team sports. Sports organizations could limit the negative mental health impacts of future sports cancelations by implementing at-home training opportunities, remote check-ins with teammates, discussions about athletic identity with coaches and sports psychology professionals, and mindfulness skill-building., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The type III secretion system encoded in the Salmonella pathogenicity island-2 (SPI-2) gene cluster facilitates intracellular growth of nontyphoidal Salmonella by interfering with the maturation of Salmonella-containing vacuoles along the degradative pathway. SPI-2 gene products also protect Salmonella against the antimicrobial activity of reactive oxygen species (ROS) synthesized by the phagocyte NADPH oxidase 2 (NOX2). However, a potential relationship between inflammatory ROS and the activation of transcription of SPI-2 genes by intracellular Salmonella is unclear. Here, we show that ROS engendered in the innate host response stimulate SPI-2 gene transcription. We found that the expression of SPI-2 genes in Salmonella-sustaining oxidative stress conditions involves DksA, a protein otherwise known to regulate the stringent response of bacteria to nutritional stress. We also demonstrate that the J and zinc-2-oxidoreductase domains of DnaJ as well as the ATPase activity of the DnaK chaperone facilitate loading of DksA onto RNA polymerase complexed with SPI-2 promoters. Furthermore, the DksA-driven transcription of SPI-2 genes in Salmonella experiencing oxidative stress is contingent on upstream OmpR, PhoP, and SsrB signaling events that participate in the removal of nucleoid proteins while simultaneously recruiting RNA polymerase to SPI-2 promoter regions. Taken together, our results suggest the activation of SPI-2 gene transcription in Salmonella subjected to ROS produced by the respiratory burst of macrophages protects this intracellular pathogen against NOX2-mediated killing. We propose that Salmonella have co-opted inflammatory ROS to induce SPI-2-mediated protective responses against NOX2 host defenses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Background: There is an urgent need for harmonization between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology platforms and assays prior to defining appropriate correlates of protection and as well inform the development of new rapid diagnostic tests that can be used for serosurveillance as new variants of concern (VOC) emerge. We compared multiple SARS-CoV-2 serology reference materials to the WHO International Standard (WHO IS) to determine their utility as secondary standards, using an international network of laboratories with high-throughput quantitative serology assays. This enabled the comparison of quantitative results between multiple serology platforms., Methods: Between April and December 2020, 13 well-characterized and validated SARS-CoV-2 serology reference materials were recruited from six different providers to qualify as secondary standards to the WHO IS. All the samples were tested in parallel with the National Institute for Biological Standards and Control (NIBSC) 20/136 and parallel-line assays were used to calculate the relevant potency and binding antibody units., Results: All the samples saw varying levels of concordance between diagnostic methods at specific antigen-antibody combinations. Seven of the 12 candidate materials had high concordance for the spike-immunoglobulin G (IgG) analyte [percent coefficient of variation (%CV) between 5 and 44%]., Conclusion: Despite some concordance between laboratories, qualification of secondary materials to the WHO IS using arbitrary international units or binding antibody units per milliliter (BAU/ml) does not provide any benefit to the reference materials overall, due to the lack of consistent agreeable international unit (IU) or BAU/ml conversions between laboratories. Secondary standards should be qualified to well-characterized reference materials, such as the WHO IS, using serology assays that are similar to the ones used for the original characterization of the WHO IS., Competing Interests: GP and LP was employed by Biodesix, Inc. HZ and MK was employed by INSTAND e.V., IQVD GmbH, and GBD Gesellschaft fuer Biotechnologische Diagnostik mbH. HW was employed by Thermo Fisher Scientific. AD and DT were employed by company Oneworld Accuracy. The authors declare that this study received funding from the Bill & Melinda Gates Foundation. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Windsor, Roell, Tucker, Cheng, Suliman, Peek, Pestano, Lee, Zeichhardt, Lamb, Kammel, Wang, Kedl, Rester, Morrison, Davenport, Carson, Yates, Howard, Kulas, Walt, Dafni, Taylor and Chu.)

The response by vaccine developers to the COVID-19 pandemic has been extraordinary with effective vaccines authorized for emergency use in the United States within 1 year of the appearance of the first COVID-19 cases. However, the emergence of SARS-CoV-2 variants and obstacles with the global rollout of new vaccines highlight the need for platforms that are amenable to rapid tuning and stable formulation to facilitate the logistics of vaccine delivery worldwide. We developed a "designer nanoparticle" platform using phage-like particles (PLPs) derived from bacteriophage lambda for a multivalent display of antigens in rigorously defined ratios. Here, we engineered PLPs that display the receptor-binding domain (RBD) protein from SARS-CoV-2 and MERS-CoV, alone (RBD SARS -PLPs and RBD MERS -PLPs) and in combination (hCoV-RBD PLPs). Functionalized particles possess physiochemical properties compatible with pharmaceutical standards and retain antigenicity. Following primary immunization, BALB/c mice immunized with RBD SARS - or RBD MERS -PLPs display serum RBD-specific IgG endpoint and live virus neutralization titers that, in the case of SARS-CoV-2, were comparable to those detected in convalescent plasma from infected patients. Further, these antibody levels remain elevated up to 6 months post-prime. In dose-response studies, immunization with as little as one microgram of RBD SARS -PLPs elicited robust neutralizing antibody responses. Finally, animals immunized with RBD SARS -PLPs, RBD MERS -PLPs, and hCoV-RBD PLPs were protected against SARS-CoV-2 and/or MERS-CoV lung infection and disease. Collectively, these data suggest that the designer PLP system provides a platform for facile and rapid generation of single and multi-target vaccines., (© 2022. The Author(s).)

Alphaviruses infect cells by a low pH-dependent fusion reaction between viral and host cell membranes that is mediated by the viral E1 glycoprotein. Most reported alphavirus E1 sequences include two phenylalanines (F87 and F95) in the fusion loop, yet the role of these residues in viral infectivity remains to be defined. Following introduction of wild type (WT), E1-F87A, and E1-F95A chikungunya virus (CHIKV) RNA genomes into cells, viral particle production was similar in magnitude. However, CHIKV E1-F87A and E1-F95A virions displayed impaired infectivity compared with WT CHIKV particles. Although WT, E1-F87A, and E1-F95A particles bound cells with similar efficiencies, E1-F87A and E1-F95A particles were unable to undergo fusion and entry into cells. Introduction of an F95A mutation in the E1 fusion loop of Mayaro virus or Venezuelan equine encephalitis virus also resulted in poorly infectious virions. We further tested whether an E1-F87A or E1-F95A mutation could be incorporated into a live-attenuated vaccine strain, CHIKV 181/25, to enhance vaccine safety. Infection of immunocompromised Ifnar1 -/- and Irf3 -/- Irf5 -/- Irf7 -/- mice with 181/25 E1-F87A or 181/25 E1-F95A resulted in 0% mortality, compared with 100% mortality following 181/25 infection. Despite this enhanced attenuation, surviving Ifnar1 -/- and Irf3 -/- Irf5 -/- Irf7 -/- mice were protected against virulent virus re-challenge. Moreover, single-dose immunization of WT mice with either 181/25, 181/25 E1-F87A , or 181/25 E1-F95A elicited CHIKV-specific antibody responses and protected against pathogenic CHIKV challenge. These studies define a critical function for residues E1-F87 and E1-F95 in alphavirus fusion and entry into target cells and suggest that incorporation of these mutations could enhance the safety of live-attenuated alphavirus vaccine candidates. IMPORTANCE Alphaviruses are human pathogens that cause both debilitating acute and chronic musculoskeletal disease and potentially fatal encephalitis. In this study, we determined that two highly conserved phenylalanine residues in the alphavirus E1 glycoprotein are required for fusion of viral and host cell membranes and viral entry into target cells. We further demonstrated that mutation of these phenylalanines results in a substantial loss of viral virulence but not immunogenicity. These data enhance an understanding of the viral determinants of alphavirus entry into host cells and could contribute to the development of new antivirals targeting these conserved phenylalanines or new live-attenuated alphavirus vaccines.

Many viruses utilize the lymphohematogenous route for dissemination; however, they may not freely use this highway unchecked. The reticuloendothelial system (RES) is an innate defense system that surveys circulating blood, recognizing and capturing viral particles. Examination of the literature shows that the bulk of viral clearance is mediated by the liver; however, the precise mechanism(s) mediating viral vascular clearance vary between viruses and, in many cases, remains poorly defined. Herein, we summarize what is known regarding the recognition and capture of virions from the circulation prior to the generation of a specific antibody response. We also discuss the consequences of viral capture on viral pathogenesis and the fate of the captor cell. Finally, this understudied topic has implications beyond viral pathogenesis, including effects on arbovirus ecology and the application of virus-vectored gene therapies., Competing Interests: The authors have declared that no competing interests exist.

Co-occurring posttraumatic stress disorder (PTSD) and alcohol use disorder (AUD) is common and particularly associated with elevation of hyperarousal compared to PTSD alone. Treatment options are limited. Oxytocin regulates physiological stress response. Intranasal oxytocin administration has demonstrated potential in reducing symptoms of both PTSD and AUD. This study addresses a gap in the literature by investigating effects of intranasal oxytocin on startle reactivity, an important potential marker of both PTSD and AUD symptomatology. This is a randomized, double-blind, placebo-controlled, within- and between-participant, crossover, dose-ranging study examining the effects of a single administration of oxytocin 20 IU versus 40 IU versus placebo on psychophysiological responses to a common laboratory fear-potentiated acoustic startle paradigm in participants with PTSD-AUD (n = 47) and controls (n = 37) under three different levels of threat. Contrary to our hypothesis, for the PTSD-AUD group, oxytocin 20 IU had no effect on startle reactivity, while oxytocin 40 IU increased measures of startle reactivity. Additionally, for PTSD-AUD only, ambiguous versus low threat was associated with an elevated skin conductance response. For controls only, oxytocin 20 IU versus placebo was associated with reduced startle reactivity., (Copyright © 2021. Published by Elsevier B.V.)

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020. The immunoglobulin subtypes (total Ig, IgM, and IgG) and their respective reactivity to severe acute respiratory syndrome coronavirus 2 nucleocapsid, spike, and receptor-binding domain antigens of the samples were delineated and compared with the WHO International Standard to elucidate the exact binding antibody units of each CSCP sample and ensure the CSCP provides adequate reactivity for different types of serological test platforms. We distribute the CSCP as a kit with five coded tubes to laboratories around the world to be used to compare test kits for external quality assurance, for harmonizing laboratory testing, and for use as training materials for laboratory workers.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that has caused both small- and large-scale epidemics of incapacitating musculoskeletal disease across the globe. A substantial proportion of infected individuals experience debilitating arthralgia and/or arthritis that can persist in relapsing or continuous forms for months to years, an occurrence that appears independent of viral strain and outbreak location. Due to the lack of CHIKV-specific vaccine or therapeutics, treatment of chronic CHIKV disease is limited to supportive care. Although the epidemiologic and molecular mechanisms that dictate resolution or chronicity of CHIKV disease remain unclear, several risk factors and immunological responses have been implicated in the development of chronic CHIKV disease. Mounting evidence from animal models and limited case studies indicates that chronic disease is likely a result of induced autoimmunity and/or viral persistence in joint-associated tissue. Due to the global spread and explosive, often unpredictable nature of CHIKV epidemics, concerted efforts to obtain a more precise understanding of the development and maintenance of chronic CHIKV disease must be at the forefront of investigative endeavors., (© 2018. Springer Nature Switzerland AG.)

Alphaviruses are a large group (>30 species) of enveloped, positive-strand RNA viruses. The re-emergence of mosquito-transmitted alphaviruses associated with human diseases ranging from severe and potentially fatal neurological disease to chronic arthritic disease highlights the need to understand the biology and pathogenesis of alphaviruses. Here, we review the development and use of animal models of alphavirus transmission and human disease, and discuss areas for continued refinement of these models including possible avenues for future investigation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO -/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection., (© 2021 The Authors.)

Mitigation of the COVID-19 pandemic requires an understanding of the antibody response to SARS-CoV-2. However, throughout the development of SARS-CoV-2 IgG antibody assays during the past year, cross-reactivity to other coronaviruses remained a question. To address these issues, we evaluated IgG in COVID-19 convalescent plasma samples for reactivity against three SARS-CoV-2 antigens including full-length spike, receptor binding domain, and the proximal extracellular fusion domain, and spike antigens from other coronaviruses (SARS-CoV, MERS-CoV, hCoV-HKU1, hCoV-OC43, hCoV-229E and hCoV-NL63) using the VaxArray Coronavirus SeroAssay which is a multiplexed antigen assay developed by InDevR Inc. These results were compared to two commercial SARS-CoV-2 IgG ELISAs targeting either the SARS-CoV-2 nucleocapsid or spike antigens and a live virus focus reduction neutralizing antibody test (FRNT). The VaxArray platform showed high specificity for detection of SARS-CoV-2 IgG, evident from lack of reactivity to SARS-CoV-2 antigens despite significant reactivity to endemic coronavirus antigens in pre-pandemic samples. SARS-CoV-2 IgG positive samples reacted weakly to SARS-CoV spike but not to MERS-CoV. While the VaxArray platform had overall comparable results to the spike and nucleocapsid IgG ELISAs, results were more similar to the spike antigen ELISA and the platform displayed a higher sensitivity and specificity than both ELISAs. Samples with FRNT titers below 1/23 reported negative on VaxArray, while positive samples on VaxArray had significantly higher neutralizing antibody titers. These results suggest that the VaxArray Coronavirus SeroAssay performs with high sensitivity and specificity for the detection of SARS-CoV-2 IgG, and positive results on the platform indicate SARS-CoV-2 neutralizing activity., (Copyright © 2021. Published by Elsevier B.V.)

Venezuelan equine encephalitis virus (VEEV) is a reemerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses, such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time-of-addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that conferred VEEV resistance on this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of chikungunya virus (CHIKV) to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.

The prior existence of human ACE2 protein-expressing mice used to study SARS-CoV and the rapid development of mouse-adapted virus strains have allowed the study of SARS-CoV-2 in mice, even as we are still learning about its natural pathology in humans. With myriad genetically altered strains on the C57BL/6 background and the abundance of immunological reagents available to interrogate its immune responses, the C57BL/6 mice may provide useful insight into the immunology of SARS-CoV-2 infection and vaccination. To conduct more detailed studies on their T cell responses to vaccines and infection, the epitopes eliciting those responses must be characterized in further detail. In this study, we mapped CD8 T cell epitopes within the receptor binding domain of the SARS-CoV-2 spike protein in C57BL/6 mice. Our study identified five major CD8 T cell epitopes in immunized C57BL/6 mice, including one, VVLSFELL, presented by H-2K b and common between SARS-CoV and SARS-CoV-2., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Serological testing of large representative populations for antibodies to SARS-CoV-2 is needed to estimate seroprevalence, transmission dynamics, and the duration of antibody responses from natural infection and vaccination. In this study, a high-throughput SARS-CoV-2 multiplex microsphere immunoassay (MMIA) was developed for the receptor binding domain (RBD) and nucleocapsid (N) that was more sensitive than enzyme-linked immunosorbent assay (ELISA) (98% versus 87%). The MMIA was then applied and validated in 264 first responders in Colorado using serum and dried blood spot (DBS) eluates, compared to ELISA, and evaluated for neutralizing antibodies. Four percent (11/264) of first responders were seropositive in July to August 2020. Serum and DBS were highly correlated for anti-RBD and anti-N antibodies ( R  = 0.83, P  < 0.0001 and R  = 0.87, P  < 0.0001, respectively) by MMIA. The MMIA accurately predicted SARS-CoV-2 neutralizing antibodies using DBS ( R  = 0.76, P  = 0.037). On repeat antibody testing 3 months later, anti-RBD IgG decreased less rapidly than anti-N IgG measured by MMIA, with a median change in geometric median fluorescence intensity of 62% versus 79% ( P <  0.01) for anti-RBD and anti-N IgG, respectively. This novel MMIA using DBS could be scalable for rapid and affordable SARS-CoV-2 serosurveillance in the United States and globally., (Copyright © 2021 American Society for Microbiology.)

The outbreak of COVID-19 disease, caused by SARS-CoV-2 beta-coronovirus, urges a focused search for the underlying mechanisms and treatment options. The lung is the major target organ of COVID-19, wherein the primary cause of mortality is hypoxic respiratory failure, resulting from acute respiratory distress syndrome, with severe hypoxemia, often requiring assisted ventilation. While similar in some ways to acute respiratory distress syndrome secondary to other causes, lungs of some patients dying with COVID-19 exhibit distinct features of vascular involvement, including severe endothelial injury and cell death via apoptosis and/or pyroptosis, widespread capillary inflammation, and thrombosis. Furthermore, the pulmonary pathology of COVID-19 is characterized by focal inflammatory cell infiltration, impeding alveolar gas exchange resulting in areas of local tissue hypoxia, consistent with potential amplification of COVID-19 pathogenicity by hypoxia. Vascular endothelial cells play essential roles in both innate and adaptive immune responses, and are considered to be "conditional innate immune cells" centrally participating in various inflammatory, immune pathologies. Activated endothelial cells produce cytokines/chemokines, dynamically recruit and activate inflammatory cells and platelets, and centrally participate in pro-thrombotic processes (thrombotic microangiopathies). Initial reports presented pathological findings of localized direct infection of vascular endothelial cells with SARS-CoV-2, yet emerging evidence does not support direct infection of endothelial or other vascular wall cell and thus widespread endothelial cell dysfunction and inflammation may be better explained as secondary responses to epithelial cell infection and inflammation. Endothelial cells are also actively engaged in a cross-talk with the complement system, the essential arm of innate immunity. Recent reports present evidence for complement deposition in SARS-CoV-2-damaged lung microcirculation, further strengthening the idea that, in severe cases of COVID-19, complement activation is an essential player, generating destructive hemorrhagic, capillaritis-like tissue damage, clotting, and hyperinflammation. Thus, complement-targeted therapies are actively in development. This review is intended to explore in detail these ideas., (© The Author(s) 2021.)

A benzo[6]annulene, 4-( tert -butyl)- N -(3-methoxy-5,6,7,8-tetrahydronaphthalen-2-yl) benzamide ( 1a ), was identified as an inhibitor against Chikungunya virus (CHIKV) with antiviral activity EC 90 = 1.45 μM and viral titer reduction (VTR) of 2.5 log at 10 μM with no observed cytotoxicity (CC 50 = 169 μM) in normal human dermal fibroblast cells. Chemistry efforts to improve potency, efficacy, and drug-like properties of 1a resulted in a novel lead compound 8q , which possessed excellent cellular antiviral activity (EC 90 = 270 nM and VTR of 4.5 log at 10 μM) and improved liver microsomal stability. CHIKV resistance to an analog of 1a , compound 1c , tracked to a mutation in the nsP3 macrodomain. Further mechanism of action studies showed compounds working through inhibition of human dihydroorotate dehydrogenase in addition to CHIKV nsP3 macrodomain. Moderate efficacy was observed in an in vivo CHIKV challenge mouse model for compound 8q as viral replication was rescued from the pyrimidine salvage pathway.

As exemplified by the COVID-19 pandemic, highly infective respiratory viruses can spread rapidly in the population because of lack of effective approaches to control viral replication and spread. Niclosamide (NCM) is an old anthelminthic drug (World Health Organization essential medicine list) with pleiotropic pharmacological activities. Several recent publications demonstrated that NCM has broad antiviral activities and potently inhibits viral replication, including replication of SARS-CoV-2, SARS-CoV, and dengue viruses. Unfortunately, NCM is almost completely insoluble in water, which limits its clinical use. We developed a cost-effective lipid nanoparticle formulation of NCM (nano NCM) using only FDA-approved excipient and demonstrated potency against SARS-CoV-2 infection in cells (Vero E6 and ACE2-expressing lung epithelium cells).

Background: In March 2020, the Food and Drug Administration (FDA) approved use of COVID-19 convalescent plasma (CCP) as an investigational new drug for treatment of COVID-19. Since then, collection of CCP from COVID-19-recovered patients has been implemented in donor centers nationwide. Children's Hospital Colorado rapidly put into practice a CCP collection protocol, necessitating development and implementation of assays to evaluate SARS-CoV-2 antibodies in CCP units., Study Design and Methods: We evaluated 87 units of CCP collected from 36 donors over two to four sequential donations using both antigen-binding assays for SARS-CoV-2 nucleoprotein and spike antigens and a live virus focus reduction neutralization test (FRNT 50 )., Results: Our data show that the majority of donors (83%) had a FRNT 50 titer of at least 80, and 61% had a titer of at least 160, which met the FDA's criteria for acceptable CCP units. Additionally, our data indicate that analysis of antibodies to a single SARS-CoV-2 antigen is likely to miss a percentage of seroconverters; however, these individuals tend to have neutralizing antibody titers of less than 80. There was considerable variability in the short-term, sustained antibody response, measured by neutralizing antibody titers, among our donor population., Conclusion: The correlation of neutralizing activity and antigen-binding assays is necessary to qualify CCP for therapeutic use. Since SARS-CoV-2 antibody levels decline in a percentage of donors, and such a decline is not detectable by current qualitative assays implemented in many laboratories, robust, quantitative assays are necessary to evaluate CCP units best suited for therapeutic infusion in COVID-19 patients., (© 2021 AABB.)

Mayaro virus (MAYV) is an alphavirus endemic to South and Central America associated with sporadic outbreaks in humans. MAYV infection causes severe joint and muscle pain that can persist for weeks to months. Currently, there are no approved vaccines or therapeutics to prevent MAYV infection or treat the debilitating musculoskeletal inflammatory disease. In the current study, a prophylactic MAYV vaccine expressing the complete viral structural polyprotein was developed based on a non-replicating human adenovirus V (AdV) platform. Vaccination with AdV-MAYV elicited potent neutralizing antibodies that protected WT mice against MAYV challenge by preventing viremia, reducing viral dissemination to tissues and mitigating viral disease. The vaccine also prevented viral-mediated demise in IFN⍺R1-/- mice. Passive transfer of immune serum from vaccinated animals similarly prevented infection and disease in WT mice as well as virus-induced demise of IFN⍺R1-/- mice, indicating that antiviral antibodies are protective. Immunization with AdV-MAYV also generated cross-neutralizing antibodies against two related arthritogenic alphaviruses-chikungunya and Una viruses. These cross-neutralizing antibodies were protective against lethal infection in IFN⍺R1-/- mice following challenge with these heterotypic alphaviruses. These results indicate AdV-MAYV elicits protective immune responses with substantial cross-reactivity and protective efficacy against other arthritogenic alphaviruses. Our findings also highlight the potential for development of a multi-virus targeting vaccine against alphaviruses with endemic and epidemic potential in the Americas., Competing Interests: The authors have declared that no competing interests exist.

Many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology tests have proven to be less accurate than expected and do not assess antibody function as neutralizing, correlating with protection from reinfection. A new assay technology measuring the interaction of the purified SARS-CoV-2 spike protein receptor binding domain (RBD) with the extracellular domain of the human angiotensin-converting enzyme 2 (hACE2) receptor detects these important antibodies. The cPass surrogate virus neutralization test (sVNT), compared directly with eight SARS-CoV-2 IgG serology and two live-cell neutralization tests, gives similar or improved accuracy for qualitative delineation between positive and negative individuals in a fast, scalable, and high-throughput assay. The combined data support the cPass sVNT as a tool for highly accurate SARS-CoV-2 immunity surveillance of infected/recovered and/or vaccinated individuals as well as drug and convalescent-phase donor screening. The data also preview a novel application for the cPass sVNT in calibrating the stringency of live-cell neutralization tests and its use in longitudinal testing of recovered and/or vaccinated patients., (Copyright © 2021 Taylor et al.)

Objectives: Define the seroprevalence and risk factors for SARS-CoV-2 antibodies in Arapahoe County, Colorado first responders (eg, law enforcement, human services, fire departments)., Methods: Two hundred sixty four first responders were enrolled June to July 2020. SARS-CoV-2 seropositivity was defined as detection of immunoglobulin G (IgG) antibodies to both spike receptor binding domain and nucleocapsid in venous blood by validated enzyme-linked immunosorbent assay. We compared risk factors for being seropositive versus seronegative., Results: 4% (11/264) were SARS-CoV-2 seropositive. Seropositive participants were significantly more likely to have lung disease (% seropositive, % seronegative; P-value) (36%, 8%; P = 0.01), prior SARS-CoV-2/COVID-19 testing (36%, 8%; P ≤ 0.01), a prior positive result (18%, less than 1%), and to believe they previously had COVID-19 (64%, 15%; P < 0.01). Only 15% of those believing they had COVID-19 had anti-SARS-CoV-2 antibodies., Conclusions: Human services employees and individuals with lung disease are at SARS-CoV-2 exposure risk. Few individuals believed they had COVID-19 had prior exposure., Competing Interests: Conflict of Interest: None declared., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American College of Occupational and Environmental Medicine.)

Ross River virus (RRV) is a mosquito-borne alphavirus that causes epidemics of debilitating musculoskeletal disease. To define the innate immune mechanisms that mediate control of RRV infection, we studied a RRV strain encoding 6 nonsynonymous mutations in nsP1 (RRV-T48-nsP1 6M ) that is attenuated in wild-type (WT) mice and Rag1 -/- mice, which are unable to mount adaptive immune responses, but not in mice that lack the capacity to respond to type I interferon (IFN) ( Ifnar1 -/- mice). Utilizing this attenuated strain, our prior studies revealed that mitochondrial antiviral signaling (MAVS)-dependent production of type I IFN by Ly6C hi monocytes is critical for control of acute RRV infection. Here, we infected Mavs -/- mice with either WT RRV or RRV-T48-nsP1 6M to elucidate MAVS-independent protective mechanisms. Mavs -/- mice infected with WT RRV developed severe disease and succumbed to infection, whereas those infected with RRV-T48-nsP1 6M exhibited minimal disease signs. Mavs -/- mice infected with RRV-T48-nsP1 6M had higher levels of systemic type I IFN than Mavs -/- mice infected with WT virus, and treatment of Mavs -/- mice infected with the attenuated nsP1 mutant virus with an IFNAR1-blocking antibody resulted in a lethal infection. In vitro , type I IFN expression was induced in plasmacytoid dendritic cells (pDCs) cocultured with RRV-infected cells in a MAVS-independent manner, and depletion of pDCs in Mavs -/- mice resulted in increased viral burdens in joint and muscle tissues, suggesting that pDCs are a source of the protective IFN in Mavs -/- mice. These data suggest that pDC production of type I IFN through a MAVS-independent pathway contributes to control of RRV infection. IMPORTANCE Arthritogenic alphaviruses, including Ross River virus (RRV), are human pathogens that cause debilitating acute and chronic musculoskeletal disease and are a significant public health burden. Using an attenuated RRV with enhanced susceptibility to host innate immune responses has revealed key cellular and molecular mechanisms that can mediate control of attenuated RRV infection and that are evaded by more virulent RRV strains. In this study, we found that pDCs contribute to the protective type I interferon response during RRV infection through a mechanism that is independent of the mitochondrial antiviral signaling (MAVS) adaptor protein. These findings highlight a key innate immune mechanism that contributes to control of alphavirus infections., (Copyright © 2021 American Society for Microbiology.)

Introduction: Mimicking movements of others makes both the imitating and imitated partners feel closer. Oxytocin may increase focus on others and has been shown to increase automatic imitation in healthy controls (HC). However, this has not been replicated, and oxytocin's effects on automatic imitation have not been demonstrated in clinical populations. This study attempts to replicate effects on HC and examine effects on people with comorbid posttraumatic stress disorder and alcohol use disorder (PTSD-AUD)., Methods: Fifty-four males with PTSD-AUD and 43 male HC received three intranasal treatment conditions (placebo, oxytocin 20 International Units (IU), and oxytocin 40 IU) in a randomized order, across three separate testing days, as part of a double-blind, crossover parent study. At 135 min post-administration, each performed the imitation-inhibition task, which quantifies automatic imitation as the congruency effect (CE). After exclusions, the final analyzed data set included 49 participants with PTSD-AUD and 38 HC., Results: In HC, oxytocin 20 IU demonstrated a statistically significant increase in CE, and 40 IU showed a trend-level increase. In PTSD-AUD, oxytocin did not significantly increase CE. Post-hoc analysis showed the PTSD-AUD group had higher CE than HC on placebo visits., Discussion: Our data suggest PTSD-AUD is associated with higher automatic imitation than HC in the absence of oxytocin administration. We successfully replicated findings that oxytocin increases automatic imitation in HC. This demonstrates an unconscious motor effect induced by oxytocin, likely relevant to more complex forms of imitative movements, which have the potential to improve social connection. We did not find a significant effect of oxytocin on automatic imitation in PTSD-AUD. Future research should examine imitation in both sexes, at peak oxytocin levels, and on increasingly complex forms of imitation., (Published by Elsevier Ltd.)

The Sementis Copenhagen Vector (SCV) is a new vaccinia virus-derived, multiplication-defective, vaccine technology assessed herein in non-human primates. Indian rhesus macaques ( Macaca mulatta ) were vaccinated with a multi-pathogen recombinant SCV vaccine encoding the structural polyproteins of both Zika virus (ZIKV) and chikungunya virus (CHIKV). After one vaccination, neutralising antibody responses to ZIKV and four strains of CHIKV, representative of distinct viral genotypes, were generated. A second vaccination resulted in significant boosting of neutralising antibody responses to ZIKV and CHIKV. Following challenge with ZIKV, SCV-ZIKA/CHIK-vaccinated animals showed significant reductions in viremias compared with animals that had received a control SCV vaccine. Two SCV vaccinations also generated neutralising and IgG ELISA antibody responses to vaccinia virus. These results demonstrate effective induction of immunity in non-human primates by a recombinant SCV vaccine and illustrates the utility of SCV as a multi-disease vaccine platform capable of delivering multiple large immunogens., Competing Interests: Competing interestsA.S., N.A.P., P.E., L.L., T.H.C. and J.D.H. own Sementis shares. J.D.H.’s research group conducts collaborative research with Sementis financially supported, whole or in part, by Sementis. A.S. was an unpaid Scientific Advisory Board member of Sementis (2011–2019) and was an occasional paid consultant for Sementis. P.M.H. was the CEO/CSO of Sementis. L.L., T.H.C., K.R.D., P.E. and N.A.P. have had, and/or currently have, salary and/or project support from funds provided, whole or in part, by Sementis. The NHP study was undertaken by Southern Research and was contracted and overseen by the NIAID (NIH, USA) and constitutes an independent assessment of the SCV vaccine vector technology. K.W., R.K., J.G. and F.K. are employees of Southern Research. VACV antibody analyses on the stored serum samples were undertaken at Southern Research under a separate R&D contract paid for by Sementis. CHIKV serology was undertaken at the University of Colorado School of Medicine and was conducted independently of Sementis using serum samples provided by Southern Research. Sementis had no influence on the analysis and interpretation of the data., (© The Author(s) 2020.)

Ross River fever is a mosquito-transmitted viral disease that is endemic to Australia and the surrounding Pacific Islands. Ross River virus (RRV) belongs to the arthritogenic group of alphaviruses, which largely cause disease characterized by debilitating polyarthritis, rash, and fever. There is no specific treatment or licensed vaccine available, and the mechanisms of protective humoral immunity in humans are poorly understood. Here, we describe naturally occurring human mAbs specific to RRV, isolated from subjects with a prior natural infection. These mAbs potently neutralize RRV infectivity in cell culture and block infection through multiple mechanisms, including prevention of viral attachment, entry, and fusion. Some of the most potently neutralizing mAbs inhibited binding of RRV to Mxra8, a recently discovered alpahvirus receptor. Epitope mapping studies identified the A and B domains of the RRV E2 protein as the major antigenic sites for the human neutralizing antibody response. In experiments in mice, these mAbs were protective against cinical disease and reduced viral burden in multiple tissues, suggesting a potential therapeutic use for humans., Competing Interests: M.S.D. is a consultant for Inbios and is on the Scientific Advisory Board of Moderna. J.E.C. has served as a consultant for Takeda Vaccines, Sanofi Pasteur, Pfizer, and Novavax, is on the Scientific Advisory Boards of CompuVax and Meissa Vaccines and is Founder of IDBiologics.

Host genetic factors play a fundamental role in regulating humoral immunity to viral infection, including influenza A virus (IAV). Here, we utilize the Collaborative Cross (CC), a mouse genetic reference population, to study genetic regulation of variation in antibody response following IAV infection. CC mice show significant heritable variation in the magnitude, kinetics, and composition of IAV-specific antibody response. We map 23 genetic loci associated with this variation. Analysis of a subset of these loci finds that they broadly affect the antibody response to IAV as well as other viruses. Candidate genes are identified based on predicted variant consequences and haplotype-specific expression patterns, and several show overlap with genes identified in human mapping studies. These findings demonstrate that the host antibody response to IAV infection is under complex genetic control and highlight the utility of the CC in modeling and identifying genetic factors with translational relevance to human health and disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes explosive epidemics of a febrile illness characterized by debilitating arthralgia and arthritis that can endure for months to years following infection. In mouse models, CHIKV persists in joint tissues for weeks to months and is associated with chronic synovitis. Using a recombinant CHIKV strain encoding a CD8 + T cell receptor epitope from ovalbumin, as well as a viral peptide-specific major histocompatibility complex class I tetramer, we interrogated CD8 + T cell responses during CHIKV infection. Epitope-specific CD8 + T cells, which were reduced in Batf3 -/- and Wdfy4 -/- mice with known defects in antigen cross-presentation, accumulated in joint tissue and the spleen. Antigen-specific ex vivo restimulation assays and in vivo killing assays demonstrated that CD8 + T cells produce cytokine and have cytolytic activity. Despite the induction of a virus-specific CD8 + T cell response, the CHIKV burden in joint-associated tissues and the spleen were equivalent in wild-type (WT) and CD8α -/- mice during both the acute and the chronic phases of infection. In comparison, CD8 + T cells were essential for the control of acute and chronic lymphocytic choriomeningitis virus infection in the joint and spleen. Moreover, adoptive transfer of virus-specific effector CD8 + T cells or immunization with a vaccine that induces virus-specific effector CD8 + T cells prior to infection enhanced the clearance of CHIKV infection in the spleen but had a minimal impact on CHIKV infection in the joint. Collectively, these data suggest that CHIKV establishes and maintains a persistent infection in joint-associated tissue in part by evading CD8 + T cell immunity. IMPORTANCE CHIKV is a reemerging mosquito-transmitted virus that in the last decade has spread into Europe, Asia, the Pacific Region, and the Americas. Joint pain, swelling, and stiffness can endure for months to years after CHIKV infection, and epidemics have a severe economic impact. Elucidating the mechanisms by which CHIKV subverts antiviral immunity to establish and maintain a persistent infection may lead to the development of new therapeutic strategies against chronic CHIKV disease. In this study, we found that CHIKV establishes and maintains a persistent infection in joint-associated tissue in part by evading antiviral CD8 + T cell immunity. Thus, immunomodulatory therapies that improve CD8 + T cell immune surveillance and clearance of CHIKV infection could be a strategy for mitigating chronic CHIKV disease., (Copyright © 2020 American Society for Microbiology.)