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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) continues its significant health and economic impact globally. Despite the success of spike-protein vaccines in preventing severe disease, long-lasting protection against emerging variants and the prevention of breakthrough infections and transmission remain elusive. We generate an intranasal live-attenuated SARS-CoV-2 vaccine, CDO-7N-1, using codon deoptimization. CDO-7N-1 shows highly attenuated replication and minimal or no lung pathology in vivo over multiple passages. It induces robust mucosal and systemic neutralizing antibody and T-cell subset responses, in mice (female K18-hACE2 and male HFH4-hACE2 mice), hamsters, and macaques triggered by a single immunization. Mice and hamsters vaccinated with CDO-7N-1 are protected from challenge with wild-type (WT) SARS-CoV-2 and other variants of concern. Serum from vaccinated animals neutralizes WT SARS-CoV-2, variants of concern (beta and delta), variants of interest (omicron XBB.1.5) and SARS-CoV-1. Antibody responses are sustained and enhanced by repeated immunization or infection with WT SARS-CoV-2. Immunity against all SARS-CoV-2 proteins by CDO-7N-1 should improve efficacy against future SARS-CoV-2 variants., (© 2024. The Author(s).)

Whole virus-based inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous coronavirus infection, the emergence of novel variants and the presence of large zoonotic reservoirs harboring novel heterologous coronaviruses provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes like vaccine-associated enhanced respiratory disease. Here, we use a female mouse model of coronavirus disease to evaluate inactivated vaccine performance against either homologous challenge with SARS-CoV-2 or heterologous challenge with a bat-derived coronavirus that represents a potential emerging disease threat. We show that inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide can cause enhanced respiratory disease during heterologous infection, while use of an alternative adjuvant does not drive disease and promotes heterologous viral clearance. In this work, we highlight the impact of adjuvant selection on inactivated vaccine safety and efficacy against heterologous coronavirus infection., (© 2024. The Author(s).)

The COVID-19 pandemic led to the rapid and worldwide development of highly effective vaccines against SARS-CoV-2. However, there is significant individual-to-individual variation in vaccine efficacy due to factors including viral variants, host age, immune status, environmental and host genetic factors. Understanding those determinants driving this variation may inform the development of more broadly protective vaccine strategies. While host genetic factors are known to impact vaccine efficacy for respiratory pathogens such as influenza and tuberculosis, the impact of host genetic variation on vaccine efficacy against COVID-19 is not well understood. To model the impact of host genetic variation on SARS-CoV-2 vaccine efficacy, while controlling for the impact of non-genetic factors, we used the Diversity Outbred (DO) mouse model. We found that DO mice immunized against SARS-CoV-2 exhibited high levels of variation in vaccine-induced neutralizing antibody responses. While the majority of the vaccinated mice were protected from virus-induced disease, similar to human populations, we observed vaccine breakthrough in a subset of mice. Importantly, we found that this variation in neutralizing antibody, virus-induced disease, and viral titer is heritable, indicating that the DO serves as a useful model system for studying the contribution of genetic variation of both vaccines and disease outcomes.

Importance: Viral encephalomyelitis outcome is dependent on host responses to neuronal infection. Interferon (IFN) is an important component of the innate response, and IFN regulatory factor (IRF) 7 is an inducible transcription factor for the synthesis of IFN-α. IRF7-deficient mice develop fatal paralysis after CNS infection with Sindbis virus, while wild-type mice recover. Irf7 -/- mice produce low levels of IFN-α but high levels of IFN-β with induction of IFN-stimulated genes, so the reason for this difference is not understood. The current study shows that Irf7 -/- mice developed inflammation earlier but failed to clear virus from motor neuron-rich regions of the brainstem and spinal cord. Levels of IFN-γ and virus-specific antibody were comparable, indicating that IRF7 deficiency does not impair expression of these known viral clearance factors. Therefore, IRF7 is either necessary for the neuronal response to currently identified mediators of clearance or enables the production of additional antiviral factor(s) needed for clearance., Competing Interests: DEG is a member of advisory boards for GSK and Takeda Pharmaceuticals.

The alphavirus chikungunya virus (CHIKV) represents a reemerging public health threat as mosquito vectors spread and viruses acquire advantageous mutations. Although primarily arthritogenic in nature, CHIKV can produce neurological disease with long-lasting sequelae that are difficult to study in humans. We therefore evaluated immunocompetent mouse strains/stocks for their susceptibility to intracranial infection with three different CHIKV strains, the East/Central/South African (ECSA) lineage strain SL15649 and Asian lineage strains AF15561 and SM2013. In CD-1 mice, neurovirulence was age- and CHIKV strain-specific, with SM2013 inducing less severe disease than SL15649 and AF15561. In 4-6-week-old C57BL/6J mice, SL15649 induced more severe disease and increased viral brain and spinal cord titers compared to Asian lineage strains, further indicating that neurological disease severity is CHIKV-strain-dependent. Proinflammatory cytokine gene expression and CD4+ T cell infiltration in the brain were also increased with SL15649 infection, suggesting that like other encephalitic alphaviruses and with CHIKV-induced arthritis, the immune response contributes to CHIKV-induced neurological disease. Finally, this study helps overcome a current barrier in the alphavirus field by identifying both 4-6-week-old CD-1 and C57BL/6J mice as immunocompetent, neurodevelopmentally appropriate mouse models that can be used to examine CHIKV neuropathogenesis and immunopathogenesis following direct brain infection.

Group 2B β-coronaviruses (sarbecoviruses) have caused regional and global epidemics in modern history. Here, we evaluate the mechanisms of cross-sarbecovirus protective immunity, currently less clear yet important for pan-sarbecovirus vaccine development, using a panel of alphavirus-vectored vaccines covering bat to human strains. While vaccination does not prevent virus replication, it protects against lethal heterologous disease outcomes in both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and clade 2 bat sarbecovirus challenge models. The spike vaccines tested primarily elicit a highly S1-specific homologous neutralizing antibody response with no detectable cross-virus neutralization. Rather, non-neutralizing antibody functions, mechanistically linked to FcgR4 and spike S2, mediate cross-protection in wild-type mice. Protection is lost in FcR knockout mice, further supporting a model for non-neutralizing, protective antibodies. These data highlight the importance of FcR-mediated cross-protective immune responses in universal pan-sarbecovirus vaccine designs., Competing Interests: Declaration of interests G.A. is a founder/equity holder in Seroymx Systems and Leyden Labs. G.A. has served as a scientific advisor for Sanofi Vaccines. G.A. has collaborative agreements with GSK, Merck, Abbvie, Sanofi, Medicago, BioNtech, Moderna, BMS, Novavax, SK Biosciences, Gilead, and Sanaria. R.S.B. has served as a consultant for Takeda and Sanofi Pasteur vaccines and is a member of the Scientific Advisory Board of VaxArt and Invivyd. R.S.B. has unrelated collaborations with J&J, Gilead, Ridgeback Biosciences, and Moderna., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Angiotensin-converting enzyme 2 (ACE2), part of the renin-angiotensin system (RAS), serves as an entry point for SARS-CoV-2, leading to viral proliferation in permissive cell types. Using mouse lines in which the Ace2 locus has been humanized by syntenic replacement, we show that regulation of basal and interferon induced ACE2 expression, relative expression levels of different ACE2 transcripts, and sexual dimorphism in ACE2 expression are unique to each species, differ between tissues, and are determined by both intragenic and upstream promoter elements. Our results indicate that the higher levels of expression of ACE2 observed in the lungs of mice relative to humans may reflect the fact that the mouse promoter drives expression of ACE2 in populous airway club cells while the human promoter drives expression in alveolar type 2 (AT2) cells. In contrast to transgenic mice in which human ACE2 is expressed in ciliated cells under the control of the human FOXJ1 promoter, mice expressing ACE2 in club cells under the control of the endogenous Ace2 promoter show a robust immune response after infection with SARS-CoV-2, leading to rapid clearance of the virus. This supports a model in which differential expression of ACE2 determines which cell types in the lung are infected, and this in turn modulates the host response and outcome of COVID-19., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Snouwaert 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.)

Emergence of the betacoronavirus SARS-CoV-2 has resulted in a historic pandemic, with millions of deaths worldwide. An unprecedented effort has been made by the medical, scientific, and public health communities to rapidly develop and implement vaccines and therapeutics to prevent and reduce hospitalizations and deaths. Although SARS-CoV-2 infection can lead to disease in many organ systems, the respiratory system is its main target, with pneumonia and acute respiratory distress syndrome as the hallmark features of severe disease. The large number of patients who have contracted COVID-19 infections since 2019 has permitted a detailed characterization of the clinical and pathologic features of the disease in humans. However, continued progress in the development of effective preventatives and therapies requires a deeper understanding of the pathogenesis of infection. Studies using animal models are necessary to complement in vitro findings and human clinical data. Multiple animal species have been evaluated as potential models for studying the respiratory disease caused by SARSCoV-2 infection. Knowing the similarities and differences between animal and human responses to infection is critical for effective translation of animal data into human medicine. This review provides a detailed summary of the respiratory disease and associated pathology induced by SARS-CoV-2 infection in humans and compares them with the disease that develops in 3 commonly used models: NHP, hamsters, and mice. The effective use of animals to study SARS-CoV-2-induced respiratory disease will enhance our understanding of SARS-CoV-2 pathogenesis, allow the development of novel preventatives and therapeutics, and aid in the preparation for the next emerging virus with pandemic potential.

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to severe acute respiratory syndrome coronavirus (SARS-CoV) disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6 , that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2, and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species. IMPORTANCE Host genetic variation is an important determinant that predicts disease outcomes following infection. In the setting of highly pathogenic coronavirus infections genetic determinants underlying host susceptibility and mortality remain unclear. To elucidate the role of host genetic variation on sarbecovirus pathogenesis and disease outcomes, we utilized the Collaborative Cross (CC) mouse genetic reference population as a model to identify susceptibility alleles to SARS-CoV and SARS-CoV-2 infections. Our findings reveal that a multitrait loci found in chromosome 9 is an important regulator of sarbecovirus pathogenesis in mice. Within this locus, we identified and validated CCR9 and CXCR6 as important regulators of host disease outcomes. Specifically, both CCR9 and CXCR6 are protective against severe SARS-CoV, SARS-CoV-2, and SARS-related HKU3 virus disease in mice. This chromosome 9 multitrait locus may be important to help identify genes that regulate coronavirus disease outcomes in humans.

The COVID-19 pandemic has highlighted the critical role that animal models play in elucidating the pathogenesis of emerging diseases and rapidly analyzing potential medical countermeasures. Relevant pathologic outcomes are paramount in evaluating preclinical models and therapeutic outcomes and require careful advance planning. While there are numerous guidelines for attaining high-quality pathology specimens in routine animal studies, preclinical studies using coronaviruses are often conducted under biosafety level-3 (BSL3) conditions, which pose unique challenges and technical limitations. In such settings, rather than foregoing pathologic outcomes because of the inherent constraints of high-containment laboratory protocols, modifications can be made to conventional best practices of specimen collection. Particularly for those unfamiliar with working in a high-containment laboratory, the authors describe the logistics of conducting such work, focusing on animal experiments in BSL3 conditions. To promote scientific rigor and reproducibility and maximize the value of animal use, the authors provide specific points to be considered before, during, and following a high-containment animal study. The authors provide procedural modifications for attaining good quality pathologic assessment of the mouse lung, central nervous system, and blood specimens under high-containment conditions while being conscientious to maximize animal use for other concurrent assays.

Mice are an invaluable resource for studying virus-induced disease. They are a small, genetically modifiable animal for which a large arsenal of genetic and immunologic tools is available for evaluation of pathogenesis and potential vaccines and therapeutics. SARS-CoV-2, the betacoronavirus responsible for the COVID-19 pandemic, does not naturally replicate in wild-type mice, due to structural differences between human and mouse ACE2, the primary receptor for SARS-CoV-2 entry into cells. However, several mouse strains have been developed that allow for SARS-CoV-2 replication and clinical disease. Two broad strategies have primarily been deployed for developing mouse strains susceptible to COVID-19-like disease: adding in the human ACE2 gene and adapting the virus to the mouse ACE2 receptor. Both approaches result in mice that develop several of the clinical and pathologic hallmarks of COVID-19, including acute respiratory distress syndrome and acute lung injury. In this review, we describe key acute pulmonary and extrapulmonary pathologic changes seen in COVID-19 patients that mouse models of SARS-CoV-2 infection ideally replicate, the essential development of mouse models for the study of Severe Acute Respiratory Syndrome and Middle Eastern Respiratory Syndrome and the basis of many of the models of COVID-19, and key clinical and pathologic features of currently available mouse models of SARS-CoV-2 infection.

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: The University of Minnesota has filed a patent on the lumazine synthase nanoparticle-based SARS-CoV-2 RBD vaccine with F.L, Q.G., Y.W., J.S., M.J., S.H., L.D. and T.W. as inventors. Other authors have declared that no competing interests exist.

Recovery from measles results in life-long protective immunity. To understand induction of long-term immunity, rhesus macaques were studied for 6 months after infection with wild-type measles virus (MeV). Infection caused viremia and rash, with clearance of infectious virus by day 14. MeV RNA persisted in PBMCs for 30-90 days and in lymphoid tissue for 6 months most often in B cells but was rarely detected in BM. Antibody with neutralizing activity and binding specificity for MeV nucleocapsid (N), hemagglutinin (H), and fusion proteins appeared with the rash and avidity matured over 3-4 months. Lymph nodes had increasing numbers of MeV-specific antibody-secreting cells (ASCs) and germinal centers with late hyalinization. ASCs appeared in circulation with the rash and continued to appear along with peripheral T follicular helper cells for the study duration. ASCs in lymph nodes and PBMCs produced antibody against both H and N, with more H-specific ASCs in BM. During days 14-21, 20- to 100-fold more total ASCs than MeV-specific ASCs appeared in circulation, suggesting mobilization of preexisting ASCs. Therefore, persistence of MeV RNA in lymphoid tissue was accompanied by continued germinal center formation, ASC production, avidity maturation, and accumulation of H-specific ASCs in BM to sustain neutralizing antibody and protective immunity.

Infection of mice with Sindbis virus (SINV) provides a model for examining the role of the immune response to alphavirus infection of the central nervous system (CNS). Interferon-gamma (IFN-γ) is an important component of this response, and we show that SINV-infected differentiated neurons respond to IFN-γ in vitro by induction of antiviral genes and suppression of virus replication. To determine the in vivo effects of IFN-γ on SINV clearance and T cell responses, C57BL/6 mice lacking IFN-γ or IFN-γ receptor-1 were compared to wild-type (WT) mice after intracranial SINV infection. In WT mice, IFN-γ was first produced in the CNS by natural killer cells and then by CD4 + and CD8 + T cells. Mice with impaired IFN-γ signaling initiated clearance of viral RNA earlier than WT mice associated with CNS entry of more granzyme B-producing CD8 + T cells. However, these mice established fewer CD8 + tissue-resident memory T (T RM ) cells and were more likely to experience reactivation of viral RNA synthesis late after infection. Therefore, IFN-γ suppresses the local development of granzyme B-expressing CD8 + T cells and slows viral RNA clearance but promotes CD8 + T RM cell establishment., Competing Interests: The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. The authors declare no conflicts of interest.

Floor contamination control practices in rodent housing facilities commonly include disposable shoe covers despite the lack of evidence for their usefulness in bioexclusion. Contamination control flooring mats are advertised as an economical and environmentally-responsible alternative to shoe covers, yet little is published regarding their efficacy in preventing the transfer of organic material and the introduction of infectious agents into facilities. We evaluated 4 floor contamination control strategies-shoe covers (ShCv), contamination control flooring (CCF), using both products concurrently (ShCv+CCF) compared with using neither-in preventing bacterial transfer and reducing organic load on facility floors and maintaining murine colony health status. According to PCR assay and culture analysis, ShCv provided the greatest reduction in bacte- rial numbers. Either ShCv, CCF, or ShCv+CCF significantly decreased ATP levels within the facility compared with those at facility entrances, with ShCv+CCF yielding the greatest reduction; however, even when neither ShCv nor CCF was used, intrafacility floor ATP levels were about half those at entrances. According to PCR analyses, no murine parasitic, viral, and bacterial pathogens excluded at the institution were detected in any floor, exhaust air dust, or sentinel samples at any time or location, regardless of the floor contamination control method in use. These findings show that floor contamination control methods help to reduce the organic load in rodent IVC facilities but do not enhance protection from environmental contamination due to murine pathogens.

Interferon (IFN) regulatory factors (IRFs) are important determinants of the innate response to infection. We evaluated the role(s) of combined and individual IRF deficiencies in the outcome of infection of C57BL/6 mice with Sindbis virus, an alphavirus that infects neurons and causes encephalomyelitis. The brain and spinal cord levels of Irf7, but not Irf3 mRNAs, were increased after infection. IRF3/5/7-/- and IRF3/7-/- mice died within 3-4 days with uncontrolled virus replication, similar to IFNα receptor-deficient mice, while all wild-type (WT) mice recovered. IRF3-/- and IRF7-/- mice had brain levels of IFNα that were lower, but brain and spinal cord levels of IFNβ and IFN-stimulated gene mRNAs that were similar to or higher than WT mice without detectable serum IFN or increases in Ifna or Ifnb mRNAs in the lymph nodes, indicating that the differences in outcome were not due to deficiencies in the central nervous system (CNS) type I IFN response. IRF3-/- mice developed persistent neurological deficits and had more spinal cord inflammation and higher CNS levels of Il1b and Ifnγ mRNAs than WT mice, but all mice survived. IRF7-/- mice died 5-8 days after infection with rapidly progressive paralysis and differed from both WT and IRF3-/- mice in the induction of higher CNS levels of IFNβ, tumour necrosis factor (TNF) α and Cxcl13 mRNA, delayed virus clearance and more extensive cell death. Therefore, fatal disease in IRF7-/- mice is likely due to immune-mediated neurotoxicity associated with failure to regulate the production of inflammatory cytokines such as TNFα in the CNS.

Sindbis virus (SINV) infection of neurons in the brain and spinal cord in mice provides a model system for investigating recovery from encephalomyelitis and antibody-mediated clearance of virus from the central nervous system (CNS). To determine the roles of IgM and IgG in recovery, we compared the responses of immunoglobulin-deficient activation-induced adenosine deaminase-deficient (AID -/- ), secretory IgM-deficient (sIgM -/- ), and AID -/- sIgM -/- double-knockout (DKO) mice with those of wild-type (WT) C57BL/6 mice for disease, clearance of infectious virus and viral RNA from brain and spinal cord, antibody responses, and B cell infiltration into the CNS. Because AID is essential for immunoglobulin class switch recombination and somatic hypermutation, AID -/- mice produce only germ line IgM, while sIgM -/- mice secrete IgG but no IgM and DKO mice produce no secreted immunoglobulin. After intracerebral infection with the TE strain of SINV, most mice recovered. Development of neurologic disease occurred slightly later in sIgM -/- mice, but disease severity, weight loss, and survival were similar between the groups. AID -/- mice produced high levels of SINV-specific IgM, while sIgM -/- mice produced no IgM and high levels of IgG2a compared to WT mice. All mice cleared infectious virus from the spinal cord, but DKO mice failed to clear infectious virus from brain and had higher levels of viral RNA in the CNS late after infection. The numbers of infected cells and the amount of cell death in brain were comparable. We conclude that antibody is required and that either germ line IgM or IgG is sufficient for clearance of virus from the CNS. IMPORTANCE Mosquito-borne alphaviruses that infect neurons can cause fatal encephalomyelitis. Recovery requires a mechanism for the immune system to clear virus from infected neurons without harming the infected cells. Antiviral antibody has previously been shown to be a noncytolytic means for alphavirus clearance. Antibody-secreting cells enter the nervous system after infection and produce antiviral IgM before IgG. Clinical studies of human viral encephalomyelitis suggest that prompt production of IgM is associated with recovery, but it was not known whether IgM is effective for clearance. Our studies used mice deficient in production of IgM, IgG, or both to characterize the antibody necessary for alphavirus clearance. All mice developed similar signs of neurologic disease and recovered from infection. Antibody was necessary for virus clearance from the brain, and either early germ line IgM or IgG was sufficient. These studies support the clinical observation that prompt production of antiviral antibody is a determinant of outcome., (Copyright © 2018 American Society for Microbiology.)

Central nervous system (CNS) infection of C57BL/6 mice with the TE strain of Sindbis virus (SINV) provides a valuable animal model for studying the pathogenesis of alphavirus encephalomyelitis. While SINV TE inoculated intracranially causes little mortality, 20-30 % of mice inoculated intranasally (IN) died 8 to 11 days after infection, the period during which immune cells typically infiltrate the brain and clear infectious virus. To examine the mechanism behind the mortality, mice infected IN with SINV TE were monitored for evidence of neurological disease, and those with signs of severe disease (moribund) were sacrificed and tissues collected. Mice showing the usual mild signs of encephalomyelitis were concurrently sacrificed to serve as time-matched controls (sick). Sixty-eight per cent of the moribund mice, but none of the sick mice, showed upper gastrointestinal bleeding due to gastric ulceration. Clinical disease and gastrointestinal pathology could not be attributed to direct viral infection of tissues outside of the CNS, and brain pathology and inflammation were comparable in sick and moribund mice. However, more SINV antigen was present in the brains of moribund mice, and clearance of infectious virus from the CNS was delayed compared to sick mice. Lower levels of SINV-specific IgM and fewer B220 + B cells were present in the brains of moribund mice compared to sick mice, despite similar levels of antiviral IgM and IgG in serum. These findings highlight the importance of the local antibody response in determining the outcome of viral encephalomyelitis and offer a model system for understanding individual variation in this response.

Measles is an acute viral disease associated both with immune suppression and development of life-long immunity. Clearance of measles virus (MeV) involves rapid elimination of infectious virus during the rash followed by slow elimination of viral RNA. To characterize cellular immune responses during recovery, we analyzed the appearance, specificity and function of MeV-specific T cells for 6 months after respiratory infection of rhesus macaques with wild type MeV. IFN-γ and IL-17-producing cells specific for the hemagglutinin and nucleocapsid proteins appeared in circulation in multiple waves approximately 2-3, 8 and 18-24 weeks after infection. IFN-γ-secreting cells were most abundant early and IL-17-secreting cells late. Both CD4 + and CD8 + T cells were sources of IFN-γ and IL-17, and IL-17-producing cells expressed RORγt. Therefore, the cellular immune response evolves during MeV clearance to produce functionally distinct subsets of MeV-specific CD4 + and CD8 + T cells at different times after infection.

Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that is partially prevented by treatment with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist (Potter et al., J Neurovirol 21:159, 2015). To determine the mechanism(s) of protection, lymph node and central nervous system (CNS) tissues from SINV-infected mice treated daily for 1 week with low (0.3mg/kg) or high (0.6mg/kg) dose DON were examined. DON treatment suppressed lymphocyte proliferation in cervical lymph nodes resulting in reduced CNS immune cell infiltration, inflammation, and cell death compared to untreated SINV-infected mice. Production of SINV-specific antibody and interferon-gamma were also impaired by DON treatment with a delay in virus clearance. Cessation of treatment allowed activation of the antiviral immune response and viral clearance, but revived CNS pathology, demonstrating the ability of the immune response to mediate both CNS damage and virus clearance., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Zika virus (ZIKV) crosses the placenta and causes congenital disease. Here we develop an animal model utilizing direct ZIKV inoculation into the uterine wall of pregnant, immunocompetent mice to evaluate transplacental transmission. Intrauterine inoculation at embryonic day (E) 10, but not E14, with African, Asian or American strains of ZIKV reduces fetal viability and increases infection of placental and fetal tissues. ZIKV inoculation at E10 causes placental inflammation, placental dysfunction and reduces neonatal brain cortical thickness, which is associated with increased activation of microglia. Viral antigen localizes in trophoblast and endothelial cells in the placenta, and endothelial, microglial and neural progenitor cells in the fetal brain. ZIKV infection of the placenta increases production of IFNβ and expression of IFN-stimulated genes 48 h after infection. This mouse model provides a platform for identifying factors at the maternal-fetal interface that contribute to adverse perinatal outcomes in a host with an intact immune system.

Chikungunya virus (CHIKV), an Old World alphavirus, is transmitted to humans by infected mosquitoes and causes acute rash and arthritis, occasionally complicated by neurologic disease and chronic arthritis. One determinant of alphavirus virulence is nonstructural protein 3 (nsP3) that contains a highly conserved MacroD-type macrodomain at the N terminus, but the roles of nsP3 and the macrodomain in virulence have not been defined. Macrodomain is a conserved protein fold found in several plus-strand RNA viruses that binds to the small molecule ADP-ribose. Prototype MacroD-type macrodomains also hydrolyze derivative linkages on the distal ribose ring. Here, we demonstrated that the CHIKV nsP3 macrodomain is able to hydrolyze ADP-ribose groups from mono(ADP-ribosyl)ated proteins. Using mass spectrometry, we unambiguously defined its substrate specificity as mono(ADP-ribosyl)ated aspartate and glutamate but not lysine residues. Mutant viruses lacking hydrolase activity were unable to replicate in mammalian BHK-21 cells or mosquito Aedes albopictus cells and rapidly reverted catalytically inactivating mutations. Mutants with reduced enzymatic activity had slower replication in mammalian neuronal cells and reduced virulence in 2-day-old mice. Therefore, nsP3 mono(ADP-ribosyl)hydrolase activity is critical for CHIKV replication in both vertebrate hosts and insect vectors, and for virulence in mice.

Infection of mice with Sindbis virus (SINV) produces encephalomyelitis and provides a model for examination of the central nervous system (CNS) immune response to alphavirus infection. Clearance of infectious virus is accomplished through a cooperative effort between SINV-specific antibody and IFN-γ, but the regulatory interactions are poorly understood. To determine the effects of IFN-γ on clinical disease and the antiviral immune response, C57BL/6 mice lacking IFN-γ (Ifng-/-) or IFN-γ receptor (Ifngr1-/-) were studied in comparison to WT mice. Maximum production of Ifng mRNA and IFN-γ protein in the CNS of WT and Ifngr1-/- mice occurred 5-7 days after infection, with higher levels of IFN-γ in Ifngr1-/- mice. Onset of clinical disease was earlier in mice with impaired IFN-γ signalling, although Ifngr1-/- mice recovered more rapidly. Ifng-/- and Ifngr1-/- mice maintained body weight better than WT mice, associated with better food intake and lower brain levels of inflammatory cytokines. Clearance of infectious virus from the spinal cords was slower, and CNS, but not serum, levels of SINV-specific IgM, IgG2a and IgG2b were lower in Ifngr1-/- and Ifng-/- mice compared to WT mice. Decreased CNS antiviral antibody was associated with lower expression of mRNAs for B-cell attracting chemokines CXCL9, CXCL10 and CXCL13 and fewer B cells in the CNS. Therefore, IFN-γ signalling increases levels of CNS pro-inflammatory cytokines, leading to clinical disease, but synergistically clears virus with SINV-specific antibody at least in part by increasing chemokine production important for infiltration of antibody-secreting B cells into the CNS.

Unlabelled: Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system (CNS). A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in adult C57BL/6 mice during clearance of infectious virus from the CNS, and the virus-specific immune response is implicated as a mediator of neuronal damage. Previous studies have shown that survival is improved in T-cell-deficient mice and in mice with pharmacological inhibition of the inflammatory response and glutamate excitotoxicity. Because glutamine metabolism is important in the CNS for the generation of glutamate and in the immune system for lymphocyte proliferation, we tested the effect of the glutamine antagonist DON (6-diazo-5-oxo-l-norleucine) on the outcome of NSV infection in mice. DON treatment for 7 days from the time of infection delayed the onset of paralysis and death. Protection was associated with reduced lymphocyte proliferation in the draining cervical lymph nodes, decreased leukocyte infiltration into the CNS, lower levels of inflammatory cytokines, and delayed viral clearance. In vitro studies showed that DON inhibited stimulus-induced proliferation of lymphocytes. When in vivo treatment with DON was stopped, paralytic disease developed along with the inflammatory response and viral clearance. These studies show that fatal NSV-induced encephalomyelitis is immune mediated and that antagonists of glutamine metabolism can modulate the immune response and protect against virus-induced neuroinflammatory disease., Importance: Encephalomyelitis due to infection with mosquito-borne alphaviruses is an important cause of death and of long-term neurological disability in those who survive infection. This study demonstrates the role of the virus-induced immune response in the generation of neurological disease. DON, a glutamine antagonist, inhibited the proliferation of lymphocytes in response to infection, prevented the development of brain inflammation, and protected mice from paralysis and death during treatment. However, because DON inhibited the immune response to infection, clearance of the virus from the brain was also prevented. When treatment was stopped, the immune response was generated, brain inflammation occurred, virus was cleared, and mice developed paralysis and died. Therefore, more definitive treatment for alphaviral encephalomyelitis should inhibit virus replication as well as neuroinflammatory damage., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Aspiculuris tetraptera continues to be a problem in rodent vivaria, in part due to difficulties in parasite detection. Although PCR testing is highly sensitive, it is expensive and does not always provide immediate results. Consequently, many institutions rely on passive fecal flotation as a quick inhouse exam for diagnosing A. tetraptera infections. To increase the sensitivity of this test, we examined multiple parameters to determine the optimal test protocol. A 30-min soaking period prior to fecal flotation for 15 min allowed fecal pellets to soften and facilitated efficient egg isolation. We also evaluated the effect of time of day, sample size, age, sex, and housing status on egg isolation. No evidence of cyclical egg shedding was found, and although larger fecal sample sizes did not result in more eggs isolated, their use reduced the incidence of false-negative exams. The most eggs were isolated from 8- and 12-wk-old mice, and as mice aged, the number of eggs isolated declined. Overall, neither sex nor housing status influenced the number of eggs isolated. Finally, examination of multiple diagnostic tests (fecal flotation exam, direct examination of cecal and colonic contents, and fecal PCR) revealed that no single test was definitive, thus indicating that multiple tests might be required to successfully screen mice with low pinworm burdens. These findings provide guidance regarding sample selection, collection, and processing to efficiently detect A. tetraptera.

Immunocompetent weanling mice infested with Myocoptes musculinus harbor high mite loads, yet burdens decrease with age. The development of immunity to the parasite may explain this observation. In this study, we followed M. musculinus burdens in Rag1(-/-) mice and immunocompetent C57BL/6 controls from 4 to 36 wk of age and compared the clinical signs and body weights of noninfested and infested mice of both strains over time. In addition, histopathology of skin lesions and expression of cytokines and transcription factors associated with Th1- and Th2-type immune responses were assessed. Myocoptes burdens decreased and remained low in B6 mice over time, whereas Rag1(-/-) mice showed an initial decrease in burdens after 4 wk of age followed by an increase from 24 to 36 wk. In addition, Rag1(-/-) mice had higher burdens than B6 mice over time. Both strains of infested mice exhibited clinical signs of fur mite infestation-including alopecia, poor weight gain, mite-associated debris, and pruritus-and clinical signs positively correlated with the severity of the Myocoptes burden. Histopathology of skin from both strains of infested mice showed decreased lesion severity with age, likely a result of declining mite populations. Finally, compared with noninfested controls, infested B6 mice had increased expression of markers associated with the Th2-type immune response, which increased in magnitude with increasing age and duration of infestation. These results suggest that development of adaptive immunity plays a role in control of fur mite populations and that heavier infestations may result in more severe clinical signs and skin lesions.

The common marmoset (Callithrix jacchus) is a New World primate that is used in biomedical research due to its small size and relative ease of handling compared with larger primates. Although bone disease in common marmosets is well recognized, there are very few detailed descriptions in the literature that cover the range of lesions seen in these animals. For all animals used to model human disease, it is important to be aware of background lesions that may affect the interpretation of study findings. This retrospective study details bone diseases encountered in marmoset breeding colonies at 2 different institutions. Affected marmosets at Johns Hopkins University had lesions compatible with diagnoses of rickets, fibrous osteodystrophy and osteopenia. Affected marmosets at the Wisconsin National Primate Research Center exhibited severe lesions of osteoclastic bone resorption and remodeling that had an unusual distribution and were not easily categorized into a known disease entity. The purpose of this report is to document these naturally occurring skeletal lesions of common marmosets and suggest an approach to evaluating skeletal disease in prospective studies of these animals that will allow the most accurate diagnoses., (© The Author(s) 2015.)

Unlabelled: Susceptibility to alphavirus encephalomyelitis is dependent on a variety of factors, including the genetic background of the host. Neuroadapted Sindbis virus (NSV) causes uniformly fatal disease in adult C57BL/6 (B6) mice, but adult BALB/c (Bc) mice recover from infection. In B6 mice, fatal encephalomyelitis is immune mediated rather than a direct result of virus infection. To identify the immunological determinants of host susceptibility to fatal NSV-induced encephalomyelitis, we compared virus titers and immune responses in adult B6 and Bc mice infected intranasally with NSV. B6 mice had higher levels of virus replication, higher levels of type I interferon (IFN), and slower virus clearance than did Bc mice. B6 mice had more neuronal apoptosis, more severe neurologic disease, and higher mortality than Bc mice. B6 mice had more infiltration of inflammatory cells and higher levels of IL1b, IL-6, TNFa, Csf2, and CCL2 mRNAs and interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IFN-γ, and C-C motif ligand 2 (CCL2) protein in brains than Bc mice. However, Bc mice had more brain antibody at day 7 and a higher percentage of CD4(+) T cells. CD4(+) T cells in the brains of Bc mice included fewer Th17 cells and more regulatory T cells (Tregs) producing IL-10 than B6 mice, accompanied by higher levels of Il2 and Cxcl10 mRNAs. In the absence of IL-10, resistant Bc mice became susceptible to fatal encephalomyelitis after NSV infection. These studies demonstrate the importance of the immune response and its regulation in determining host survival during alphavirus encephalomyelitis., Importance: Mosquito-borne alphavirus infections are an important cause of encephalomyelitis in humans. The severity of disease is dependent both on the strain of the virus and on the age and genetic background of the host. A neurovirulent strain of Sindbis virus causes immune-mediated fatal encephalomyelitis in adult C57BL/6 mice but not in BALB/c mice. To determine the host-dependent immunological mechanisms underlying the differences in susceptibility between these two strains of mice, we compared their immune responses to infection. Resistance to fatal disease in BALB/c mice was associated with better antibody responses, more-rapid virus clearance, fewer Th17 cells, and more-potent regulatory T cell responses than occurred in susceptible C57BL/6 mice. In the absence of interleukin-10, a component of the regulatory immune response, resistant mice became susceptible to lethal disease. This study demonstrates the importance of the immune response and its regulation for host survival during alphavirus encephalomyelitis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Recovery from encephalomyelitis induced by infection with mosquito-borne alphaviruses is associated with a high risk of lifelong debilitating neurological deficits. Infection of mice with the prototypic alphavirus, Sindbis virus, provides an animal model with which to study disease mechanisms and examine potential therapeutics. Infectious virus is cleared from the brain within a week after infection, but viral RNA is cleared slowly and persists for the life of the animal. However, no studies have examined the effect of infection on neurocognitive function over time. In the present study, we examined neurocognitive function at different phases of infection in 5-week-old C57BL/6 mice intranasally inoculated with Sindbis virus. At the peak of active virus infection, mice demonstrated hyperactivity, decreased anxiety, and marked hippocampal-dependent memory deficits, the latter of which persisted beyond clearance of infectious virus and resolution of clinical signs of disease. Previous studies indicate that neuronal damage during alphavirus encephalomyelitis is primarily due to inflammatory cell infiltration and glutamate excitotoxicity rather than directly by virus infection. Therefore, mice were treated with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist that can suppress both the immune response and excitotoxicity. Treatment with DON decreased inflammatory cell infiltration and cell death in the hippocampus and partially prevented development of clinical signs and neurocognitive impairment despite the presence of infectious virus and high viral RNA levels. This study presents the first report of neurocognitive sequelae in mice with alphavirus encephalomyelitis and provides a model system for further elucidation of the pathogenesis of virus infection and assessment of potential therapies.

Mosquito-borne alphaviruses are important causes of epidemic encephalomyelitis. Neuronal cell death during fatal alphavirus encephalomyelitis is immune-mediated; however, the types of cells involved and their regulation have not been determined. We show that the virus-induced inflammatory response was accompanied by production of the regulatory cytokine IL-10, and in the absence of IL-10, paralytic disease occurred earlier and mice died faster. To determine the reason for accelerated disease in the absence of IL-10, immune responses in the CNS of IL-10(-/-) and wild-type (WT) mice were compared. There were no differences in the amounts of brain inflammation or peak virus replication; however, IL-10(-/-) animals had accelerated and increased infiltration of CD4(+)IL-17A(+) and CD4(+)IL-17A(+)IFNγ(+) cells compared with WT animals. Th17 cells infiltrating the brain demonstrated a pathogenic phenotype with the expression of the transcription factor, Tbet, and the production of granzyme B, IL-22, and GM-CSF, with greater production of GM-CSF in IL-10(-/-) mice. Therefore, in fatal alphavirus encephalomyelitis, pathogenic Th17 cells enter the CNS at the onset of neurologic disease and, in the absence of IL-10, appear earlier, develop into Th1/Th17 cells more often, and have greater production of GM-CSF. This study demonstrates a role for pathogenic Th17 cells in fatal viral encephalitis.

In experimental animals and humans, aflatoxin B1 (AFB1) is a potent hepatic toxin and carcinogen. The synthetic oleanane triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a powerful activator of Keap1-Nrf2 signaling, protects against AFB1-induced toxicity and preneoplastic lesion formation (GST-P-positive foci). This study assessed and mechanistically characterized the chemoprotective efficacy of CDDO-Im against AFB1-induced hepatocellular carcinoma (HCC). A lifetime cancer bioassay was undertaken in F344 rats dosed with AFB1 (200 μg/kg rat/day) for four weeks and receiving either vehicle or CDDO-Im (three times weekly), one week before and throughout the exposure period. Weekly, 24-hour urine samples were collected for analysis of AFB1 metabolites. In a subset of rats, livers were analyzed for GST-P foci. The comparative response of a toxicogenomic RNA expression signature for AFB1 was examined. CDDO-Im completely protected (0/20) against AFB1-induced liver cancer compared with a 96% incidence (22/23) observed in the AFB1 group. With CDDO-Im treatment, integrated level of urinary AFB1-N(7)-guanine was significantly reduced (66%) and aflatoxin-N-acetylcysteine, a detoxication product, was consistently elevated (300%) after the first AFB1 dose. In AFB1-treated rats, the hepatic burden of GST-P-positive foci increased substantially (0%-13.8%) over the four weeks, but was largely absent with CDDO-Im intervention. The toxicogenomic RNA expression signature characteristic of AFB1 was absent in the AFB1 + CDDO-Im-treated rats. The remarkable efficacy of CDDO-Im as an anticarcinogen is established even in the face of a significant aflatoxin adduct burden. Consequently, the absence of cancer requires a concept of a threshold for DNA damage for cancer development., (©2014 American Association for Cancer Research.)

The increasing use of the common marmoset (Callithrix jacchus) in research makes it important to diagnose spontaneous disease that may confound experimental studies. Bone disease and gastrointestinal disease are two major causes of morbidity and mortality in captive marmosets, but currently no effective antemortem tests are available to identify affected animals prior to the terminal stage of disease. In this study we propose that bone disease and gastrointestinal disease are associated disease entities in marmosets and aim to establish the efficacy of several economical antemortem tests in identifying and predicting disease. Tissues from marmosets were examined to define affected animals and unaffected controls. Complete blood count, serum chemistry values, body weight, quantitative radiographs, and tissue-specific biochemical markers were evaluated as candidate biomarkers for disease. Bone and gastrointestinal disease were associated, with marmosets being over seven times more likely to have either concurrent bone and gastrointestinal disease or neither disease as opposed to lesions in only one organ system. When used in tandem, serum albumin <3.5 g/dL and body weight <325 g identified 100% of the marmosets affected with concurrent bone and gastrointestinal disease. Progressive body weight loss of 0.05% of peak body weight per day predicted which marmosets would develop disease prior to the terminal stage. Bone tissue-specific tests, such as quantitative analysis of radiographs and serum parathyroid hormone levels, were effective for distinguishing between marmosets with bone disease and those without. These results provide an avenue for making informed decisions regarding the removal of affected marmosets from studies in a timely manner, preserving the integrity of research results.

Sindbis virus (SINV) infection of neurons results in nonfatal viral encephalomyelitis and provides a model system for understanding recovery from virus infection of the central nervous system (CNS). Infection is followed by clearance of infectious virus, a gradual decrease in viral RNA, and then long-term maintenance of low levels of viral RNA. Antibody to the E2 glycoprotein is important for virus clearance, and B cells enter the CNS along with CD4(+) and CD8(+) T cells during the early clearance phase. Antibody-secreting cells (ASCs) are present in the CNS and become enriched for SINV-specific ASCs. We have evaluated the factors within the CNS that facilitate continued local antibody production after infection. Expression of CXCL9, CXCL10, CCL1, CCL2, and CCL5 chemokine mRNAs increased early, and infiltrating B cells expressed CXCR3, CXCR5, and CCR7. The mRNAs for IL-10 and IL-21, cytokines important for B cell proliferation and differentiation, rose rapidly and remained elevated long after clearance of infectious virus. Active proliferation of B cells, as indicated by Ki-67 expression, continued for months. Bromodeoxyuridine (BrdU) labeling of proliferating cells showed that ASCs produced in the draining cervical lymph nodes during the early germinal center response were preferentially retained in the CNS. Sustained increase in B-cell-activating factor (BAFF) mRNA in the CNS and BAFF receptor expression by B cells coincided with the long-term maintenance of SINV-specific ASCs in the brain. We conclude that multiple changes in the brain microenvironment facilitate B-cell entry and support proliferation and differentiation and long-term survival of antiviral ASCs during recovery from alphaviral encephalomyelitis.