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To evaluate SARS-CoV-2 variants we isolated SARS-CoV-2 temporally during the pandemic starting with first appearance of virus in the Western hemisphere near Seattle, WA, USA, and isolated each known major variant class, revealing the dynamics of emergence and complete take-over of all new cases by current Omicron variants. We assessed virus neutralization in a first-ever full comparison across variants and evaluated a novel monoclonal antibody (Mab). We found that convalescence greater than 5-months provides little-to-no protection against SARS-CoV-2 variants, vaccination enhances immunity against variants with the exception of Omicron BA.1, and paired testing of vaccine sera against ancestral virus compared to Omicron BA.1 shows that 3-dose vaccine regimen provides over 50-fold enhanced protection against Omicron BA.1 compared to a 2-dose regimen. We also reveal a novel Mab that effectively neutralizes Omicron BA.1 and BA.2 variants over clinically-approved Mabs. Our observations underscore the need for continued vaccination efforts, with innovation for vaccine and Mab improvement, for protection against variants of SARS-CoV-2.SummaryWe isolated SARS-CoV-2 temporally starting with emergence of virus in the Western hemisphere. Neutralization analyses across all variant lineages show that vaccine-boost regimen provides protection against Omicron BA.1. We reveal a Mab that protects against Omicron BA.1 and BA.2 variants.

Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera’s neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.

To determine how serologic antibody testing outcome links with virus neutralization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we evaluated individuals for SARS-CoV-2 antibody level and viral neutralization.We compared serum Ig levels across platforms of viral antigens and antibodies with 15 positive and 30 negative SARS-CoV-2 controls followed by viral neutralization assessment. We then applied these platforms to a clinically relevant cohort of 114 individuals with unknown histories of SARS-CoV-2 infection.In controls, the best-performing virus-specific antibody detection platforms were SARS-CoV-2 receptor binding domain (RBD) IgG (sensitivity 87%, specificity 100%, positive predictive value [PPV] 100%, negative predictive value [NPV] 94%), spike IgG3 (sensitivity 93%, specificity 97%, PPV 93%, NPV 97%), and nucleocapsid protein (NP) IgG (sensitivity 93%, specificity 97%, PPV 93%, NPV 97%). Neutralization of positive and negative control sera showed 100% agreement. Twenty individuals with unknown history had detectable SARS-CoV-2 antibodies with 16 demonstrating virus neutralization. Spike IgG3 provided the highest accuracy for predicting serologically positive individuals with virus neutralization activity (misidentified 1/20 unknowns compared to 2/20 for RBD and NP IgG).The coupling of virus neutralization analysis to a spike IgG3 antibody test is optimal to categorize patients for correlates of SARS-CoV-2 immune protection status.

Thouvenel et al. isolate BCR sequences from Plasmodium-specific IgM+ human memory B cells, expressing them as both IgM and IgG molecules. Multimerization confers superior antigen binding and parasite restriction, highlighting an important role for memory B cell–derived IgM in immunity., Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum–specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines., Graphical Abstract

BackgroundTo determine how serologic antibody testing outcome links with virus neutralization of SARS-CoV-2 to ascertain immune protection status, we evaluated a unique set of individuals for SARS-CoV-2 antibody detection and viral neutralization.MethodsHerein, we compare several analytic platforms with 15 positive and 30 negative SARS-CoV-2 infected controls followed by viral neutralization assessment. We then applied these platforms in a clinically relevant population: 114 individuals with unknown histories of SARS-CoV-2 infection.ResultsIn control populations, the best performing antibody detection assays were SARS-CoV-2 receptor binding domain (RBD) IgG (specificity 87%, sensitivity 100%, PPV 100%, NPV 93%), spike IgG3 (specificity 93%, sensitivity 97%, PPV 93%, NPV 97%), and nucleocapsid (NP) protein IgG (specificity 93%, sensitivity 97%, PPV 93%, NPV 97%). Neutralization of positive and negative control sera showed 100% agreement. 20 unknown individuals had detectable SARS-CoV-2 antibodies with 16 demonstrating virus neutralization. The antibody assays that best predicted virus neutralization were RBD IgG (misidentified 2), spike IgG3 (misidentified 1), and NP IgG (misidentified 2).ConclusionThese data suggest that meaningful evaluation of antibody assay performance requires testing in an unknown population. Further, these results indicate coupling of virus neutralization analysis to a positive antibody test is required to categorize patients based on SARS-CoV-2 immune protection status following virus exposure or vaccine administration. One of the antibody detection platforms identified in this study followed by the pseudoneutralization or focus reduction assay would provide a practical testing strategy to assess for SARS-CoV-2 antibodies with optimal prediction of correlates to neutralizing immunity.FundingSupported by NIH grants AI148684, AI151698, AI145296, and UW funds to the Center for Innate Immunity and Immune Disease.

The SARS-CoV-2 virus is causing a global pandemic and cases continue to rise. Most infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that could contribute to immunity. We performed a longitudinal assessment of individuals recovered from mild COVID-19 to determine if they develop and sustain multifaceted SARS-CoV-2-specific immunological memory. Recovered individuals developed SARS-CoV-2-specific IgG antibodies, neutralizing plasma, memory B and memory T cells that persisted for at least three months. Our data further reveal that SARS-CoV-2-specific IgG memory B cells increased over time. Additionally, SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral function: memory T cells secreted cytokines and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as monoclonal antibodies. Therefore, mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks of antiviral immunity., Highlights • Longitudinal analysis of multifaceted immune memory following mild COVID-19 • Antibodies capable of neutralizing virus persist for at least 3 months in most subjects • Virus-specific memory B and T cells display hallmarks of anti-viral immunity • MBCs increase in number and express antibodies capable of neutralizing SARS-CoV-2, Longitudinal analysis of immune memory following mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks of antiviral immunity.

SummaryThe recently emerged SARS-CoV-2 virus is currently causing a global pandemic and cases continue to rise. The majority of infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that might contribute to herd immunity. Thus, we performed a longitudinal assessment of individuals recovered from mildly symptomatic COVID-19 to determine if they develop and sustain immunological memory against the virus. We found that recovered individuals developed SARS-CoV-2-specific IgG antibody and neutralizing plasma, as well as virus-specific memory B and T cells that not only persisted, but in some cases increased numerically over three months following symptom onset. Furthermore, the SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral immunity: memory T cells secreted IFN-γ and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as antibodies. These findings demonstrate that mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks associated with antiviral protective immunity.

SummaryHumoral immunity depends upon the development of long-lived, antibody-secreting plasma cells and rapidly responsive memory B cells (MBCs). The differentiation of high affinity, class-switched MBCs after immunization is critically dependent upon BCL6 expression in germinal center (GC) B cells and CD4+ T follicular helper (Tfh) cells. It is less well understood how more recently described MBC subsets are generated, including the CD73+CD80+ IgM+ MBCs that initially form antibody-secreting effector cells in response to a secondary Plasmodium infection. Herein, we interrogated how BCL6 expression in both B and CD4+ T cells influenced the formation of heterogeneous Plasmodium-specific MBC populations. All Plasmodium-specific CD73+CD80+ MBCs required BCL6 expression for their formation, suggesting germinal center dependence. Further dissection of the CD4+ T and B cell interactions however revealed that somatically hypermutated CD73+CD80+ IgM+ MBCs can form not only in the absence of germinal centers, but also in the absence of CXCR5+ CD4+ Tfh cells.

The mouse model of West Nile virus (WNV), which is a leading cause of mosquito-borne encephalitis worldwide, has provided fundamental insights into the host and viral factors that regulate viral pathogenesis and infection outcome. In particular, CD8 + T cells are critical for controlling WNV replication and promoting protection against infection. Here, we present the characterization of a T cell receptor (TCR)-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein (here referred to as transgenic WNV-I mice). Using an adoptive-transfer model, we found that WNV-I CD8 + T cells behave similarly to endogenous CD8 + T cell responses, with an expansion phase in the periphery beginning around day 7 postinfection (p.i.) followed by a contraction phase through day 15 p.i. Through the use of in vivo intravascular immune cell staining, we determined the kinetics, expansion, and differentiation into effector and memory subsets of WNV-I CD8 + T cells within the spleen and brain. We found that red-pulp WNV-I CD8 + T cells were more effector-like than white-pulp WNV-I CD8 + T cells, which displayed increased differentiation into memory precursor cells. Within the central nervous system (CNS), we found that WNV-I CD8 + T cells were polyfunctional (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), displayed tissue-resident characteristics (CD69 + and CD103 + ), persisted in the brain through day 15 p.i., and reduced the viral burden within the brain. The use of these TCR-transgenic WNV-I mice provides a new resource to dissect the immunological mechanisms of CD8 + T cell-mediated protection during WNV infection. IMPORTANCE West Nile Virus (WNV) is the leading cause of mosquito-borne encephalitis worldwide. There are currently no approved therapeutics or vaccines for use in humans to treat or prevent WNV infection. CD8 + T cells are critical for controlling WNV replication and protecting against infection. Here, we present a comprehensive characterization of a novel TCR-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein. In this study, we determine the kinetics, proliferation, differentiation into effector and memory subsets, homing, and clearance of WNV in the CNS. Our findings provide a new resource to dissect the immunological mechanisms of CD8 + T cell-mediated protection during WNV infection.

We used a newly generated T-cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10-mer) or SSVWNATTA (9-mer) in complex with class I MHC antigen H-2Db, were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8+ T cells can detect 10- and 9-mer length variants similarly. This result varies from the idea that a given T-cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10-mer was presented on the surface of uninfected and infected CD8α+CD11c+ dendritic cells, which suggests the use of direct and cross-presentation pathways. In contrast, CD11b+CD11c− cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell-type specific mechanisms of antigen presentation in vivo.

CD4 and CD8 T lymphocytes are adaptive immune cells that play a key role in the immune response to pathogens. They have been extensively studied in a variety of model systems and the mechanisms by which they function are well described. However, the responses by these cell types vary widely from pathogen to pathogen. In this review, we will discuss the role of CD8 and CD4 T cells in the immune response to West Nile virus infection.

Summary The RIG-I-like receptors (RLRs) signal innate immune defenses upon RNA virus infection, but their roles in adaptive immunity have not been clearly defined. Here, we showed that the RLR LGP2 was not essential for induction of innate immune defenses, but rather was required for controlling antigen-specific CD8 + T cell survival and fitness during peripheral T cell-number expansion in response to virus infection. Adoptive transfer and biochemical studies demonstrated that T cell-receptor signaling induced LGP2 expression wherein LGP2 operated to regulate death-receptor signaling and imparted sensitivity to CD95-mediated cell death. Thus, LGP2 promotes an essential prosurvival signal in response to antigen stimulation to confer CD8 + T cell-number expansion and effector functions against divergent RNA viruses, including West Nile virus and lymphocytic choriomeningitis virus.

The failure to account for differences between adolescent males who offend against children and those who offend against peers may account for the similarities found between sex-offending samples and non-sexual delinquents. Psychopathy traits (grandiosity, impulsivity, lack of empathy, interpersonally exploitative and risk-taking) and antisocial behaviour, including behaviours and age of onset for delinquent behaviours and drug use, were explored in male adolescents with sex offences against children, sex offences against peer/adults, sexual offences against both and non-sex delinquents. Youths who committed sexual offences were similar on psychopathic traits and level of antisocial behaviour. Higher levels of grandiosity and lack of empathy were found in those whose offences were non-sexual. Parental dysfunction was consistent across all groups, but maternal psychiatric difficulties were more frequent in youths with histories of sexual offending and a history of maternal substance abuse was more...

The severe acute respiratory syndrome coronavirus (SARS-CoV) accessory protein 6 (p6) is a 63-amino-acid multifunctional Golgi-endoplasmic reticulum (ER) membrane-associated protein, with roles in enhancing virus replication and in evading the innate immune response to infection by inhibiting STAT1 (signal transducer and activator of transcription factor 1) translocation to the nucleus. Here, we demonstrate that p6 has an N-terminal region-cytoplasm-C-terminal region-cytoplasm configuration with residues 2 to 37 likely membrane embedded. Expression of p6, or of its N-terminal 41-amino-acid region, in the absence of other viral proteins, induced the formation of membranous structures, some of which were similar to double membrane vesicles involved in virus replication. Consistent with a role in virus replication, p6 partially colocalized with nonstructural protein 3 (nsp3), a marker for virus replication complexes. Further, while the C-terminal region is required for preventing STAT1 translocation to the nucleus, our results also indicated that the N-terminal 18 amino acids were necessary for maximal inhibition. Collectively, these results support the notion that p6 is a two-domain protein, although the function of each is not completely independent of the other.

The severe acute respiratory syndrome coronavirus (SARS-CoV) caused substantial morbidity and mortality in 2002-2003. Deletion of the envelope (E) protein modestly diminished virus growth in tissue culture but abrogated virulence in animals. Here, we show that immunization with rSARS-CoV-ΔE or SARS-CoV-Δ[E,6-9b] (deleted in accessory proteins (6, 7a, 7b, 8a, 8b, 9b) in addition to E) nearly completely protected BALB/c mice from fatal respiratory disease caused by mouse-adapted SARS-CoV and partly protected hACE2 Tg mice from lethal disease. hACE2 Tg mice, which express the human SARS-CoV receptor, are extremely susceptible to infection. We also show that rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b] induced anti-virus T cell and antibody responses. Further, the E-deleted viruses were stable after 16 blind passages through tissue culture cells, with only a single mutation in the surface glycoprotein detected. The passaged virus remained avirulent in mice. These results suggest that rSARS-CoV-ΔE is an efficacious vaccine candidate that might be useful if SARS recurred. © 2009 Elsevier Inc. All rights reserved., This work was supported by grants from the National Institutes of Health (US) (PO1 AI060699-01) (SP); RO1 AI079424-01A1 (SP and LE), the Ministry of Education and Science of Spain (BIO2007-60978) (LE), and the European Commission (EMPERIE PROJECT, Ref. No. 223498) (LE). J.N. was supported by an NIH training grant (T32 AI007533).

In this single-participant case study, the clinical experiences of a Somali Muslim man are presented and reviewed with special attention to cross-cultural, religious, or ethnic factors. Paradoxically, this man's religious convictions serve as both a protective and vital part of who he is while contributing to his psychotic experience. We frame our conceptualization of this case using the guidelines suggested by the DSM-IV-TR Outline for Cultural Formulation (American Psychiatric Association, 2000) and discuss our summary impressions of the case and the lessons we take from it.

The theory of reasoned action and planned behavior (TRA/PB) is a model of behavior change that has been extensively studied in the health sciences but has had limited exposure in the counseling psychology literature. The model offers counseling psychologists a framework to conceptualize prevention research and practice. The model is important to consider since the training of counseling psychologists is heavily dominated by theories of psychotherapy that emphasize remediation rather than prevention. This article discusses a brief history and explanation of TRA/PB along with relevant research, limitations, and multicultural considerations. Elicitation research, an important component of the model, receives emphasis because it solicits population-specific cognitions and social influences relevant to targeted behaviors, thus strengthening prevention interventions. An example demonstrates how TRA/PB can be used as a theoretical framework to support prevention research. Recommendations for prevention training of counseling psychologists are presented.

Severe acute respiratory syndrome coronavirus encodes several accessory proteins of unknown function. We previously showed that one such protein, encoded by ORF6, enhanced the growth of mouse hepatitis virus in tissue culture cells and in mice. Protein 6 consists of an N-terminal hydrophobic peptide and a C-terminal region containing intracellular protein sorting motifs. Herein, we show that mutation of the hydrophobic region but not the sorting motifs affected the ability of protein 6 to enhance virus growth. Collectively, these results support the notion that the 6 protein interacts with membrane-bound viral replication or assembly machinery to directly enhance virus replication and virulence in animals.

One or more of the unique 3′-proximal open reading frames (ORFs) of the severe acute respiratory syndrome (SARS) coronavirus may encode determinants of virus virulence. A prime candidate is ORF6, which encodes a 63-amino-acid membrane-associated peptide that can dramatically increase the lethality of an otherwise attenuated JHM strain of murine coronavirus (L. Pewe, H. Zhou, J. Netland, C. Tangudu, H. Olivares, L. Shi, D. Look, T. Gallagher, and S. Perlman, J. Virol. 79:11335-11342, 2005). To discern virulence mechanisms, we compared the in vitro growth properties of rJ.6, a recombinant JHM expressing the SARS peptide, with isogenic rJ.6-KO, which has an inactive ORF containing a mutated initiation codon and a termination codon at internal position 27. The rJ.6 infections proceeded rapidly, secreting progeny about 1.5 h earlier than rJ.6-KO infections did. The rJ.6 infections were also set apart by early viral protein accumulation and by robust expansion via syncytia, a characteristic feature of JHM virus dissemination. We found no evidence for protein 6 operating at the virus entry or assembly stage, as virions from either infection were indistinguishable. Rather, protein 6 appeared to operate by fostering viral RNA and protein synthesis, as RNA quantifications by reverse transcription-quantitative PCR revealed viral RNA levels in the rJ.6 cultures that were five to eight times higher than those lacking protein 6. Furthermore, protein 6 coimmunoprecipitated with viral RNAs and colocalized on cytoplasmic vesicles with replicating viral RNAs. The SARS coronavirus encodes a novel membrane protein 6 that can accelerate replication of a related mouse virus, a property that may explain its ability to increase in vivo virus virulence.

The severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in substantial morbidity, mortality, and economic losses during the 2003 epidemic. While SARS-CoV infection has not recurred to a significant extent since 2003, it still remains a potential threat. Understanding of SARS and development of therapeutic approaches have been hampered by the absence of an animal model that mimics the human disease and is reproducible. Here we show that transgenic mice that express the SARS-CoV receptor (human angiotensin-converting enzyme 2 [hACE2]) in airway and other epithelia develop a rapidly lethal infection after intranasal inoculation with a human strain of the virus. Infection begins in airway epithelia, with subsequent alveolar involvement and extrapulmonary virus spread to the brain. Infection results in macrophage and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. This model of lethal infection with SARS-CoV should be useful for studies of pathogenesis and for the development of antiviral therapies.

Several oligosaccharide fragments, ranging from 2 to 9 contiguous residues, have been isolated from purified tamarind xyloglucan using enzymatic digestion and partial acid hydrolysis. Structures were determined using matrix assisted laser adsorption ionization-time of flight (MALDI-TOF) mass spectrometry, gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), and Dionex high pH anion exchange–high performance liquid chromatography (HPAE–HPLC). These fragments will be used to identify reaction products from xyloglucan xylosyltransferase and glucosyltransferase enzyme assays and as possible acceptor molecules for these enzymes.

This study explores in an adolescent sample hypotheses about child sexual abuse perpetration drawn from contemporary theories that implicate insecure attachment and adolescent social development. Specifically, three 13- to 18-year-old adolescent male samples (sex offenders with child victims, sex offenders with peer/adult victims, and nonsex delinquent youth) were compared in a cross-sectional design. Participants completed a computer-administered self-report questionnaire and a semistructured attachment style interview. Attachment style was coded by two independent raters blind to study hypotheses and group membership. The results indicated an indirect effect for attachment style. Attachment anxiety affected involvement with peers and interpersonal adequacy. Feelings of interpersonal inadequacy, combined with oversexualization and positive attitudes toward others distinguished sex offenders with child victims from nonsex delinquents and from sex offenders with peer/adult victims. These data provide a preliminary model of sexual abuse perpetration consistent with contemporary theories. Attachment anxiety with a lack of misanthropic attitudes toward others appears to lead to isolation from peers and feelings of interpersonal inadequacy. Individuals with this constellation of factors may turn to children to meet their intimacy and sexual needs, both of which seem to be exaggerated compared with other troubled youth.

While coronaviruses were first identified nearly 60 years ago, they received notoriety in 2003 when one of their members was identified as the etiological agent of the Severe Acute Respiratory Syndrome (SARS-CoV) 1. Previously these viruses were known to be important agents of respiratory and enteric infections of domestic and companion animals and to cause approximately 15% of all cases of the common cold. This Review focuses on recent advances in our understanding of the mechanisms of coronavirus replication, interactions with the host immune response and disease pathogenesis and highlights the recent identification of numerous novel coronaviruses and the propensity of this virus family to cross species barriers.

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several accessory proteins of unknown function. One of these proteins, protein 6 (p6), which is encoded by ORF6, enhances virus replication when introduced into a heterologous murine coronavirus (mouse hepatitis virus [MHV]) but is not essential for optimal SARS-CoV replication after infection at a relatively high multiplicity of infection (MOI). Here, we reconcile these apparently conflicting results by showing that p6 enhances SARS-CoV replication to nearly the same extent as when expressed in the context of MHV if cells are infected at a low MOI and accelerates disease in mice transgenic for the human SARS-CoV receptor. Copyright © 2009, American Society for Microbiology. All Rights Reserved., This research was supported in part by NIH grant PO1 AI060699 and by the European Community Frame VI, DISSECT PROJECT, SP22-CT-2004-511060.

Infection of humans with the severe acute respiratory syndrome coronavirus (SARS-CoV) results in substantial morbidity and mortality, with death resulting primarily from respiratory failure. While the lungs are the major site of infection, the brain is also infected in some patients. Brain infection may result in long-term neurological sequelae, but little is known about the pathogenesis of SARS-CoV in this organ. We previously showed that the brain was a major target organ for infection in mice that are transgenic for the SARS-CoV receptor (human angiotensin-converting enzyme 2). Herein, we use these mice to show that virus enters the brain primarily via the olfactory bulb, and infection results in rapid, transneuronal spread to connected areas of the brain. This extensive neuronal infection is the main cause of death because intracranial inoculation with low doses of virus results in a uniformly lethal disease even though little infection is detected in the lungs. Death of the animal likely results from dysfunction and/or death of infected neurons, especially those located in cardiorespiratory centers in the medulla. Remarkably, the virus induces minimal cellular infiltration in the brain. Our results show that neurons are a highly susceptible target for SARS-CoV and that only the absence of the host cell receptor prevents severe murine brain disease.

Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.

Most animal species that can be infected with the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) do not reproducibly develop clinical disease, hindering studies of pathogenesis. To develop an alternative system for the study of SARS-CoV, we introduced individual SARS-CoV genes (open reading frames [ORFs]) into the genome of an attenuated murine coronavirus. One protein, the product of SARS-CoV ORF6, converted a sublethal infection to a uniformly lethal encephalitis and enhanced virus growth in tissue culture cells, indicating that SARS-CoV proteins function in the context of a heterologous coronavirus infection. Furthermore, these results suggest that the attenuated murine coronavirus lacks a virulence gene residing in SARS-CoV. Recombinant murine coronaviruses cause a reproducible and well-characterized clinical disease, offer virtually no risk to laboratory personnel, and should be useful for elucidating the role of SARS-CoV nonstructural proteins in viral replication and pathogenesis.

We measured CD8 T cell clonotypic diversity to three epitopes recognized in C57BL/6 mice infected with mouse hepatitis virus, strain JHM, or lymphocytic choriomeningitis virus. We isolated epitope-specific T cells with an IFN-γ capture assay or MHC class I/peptide tetramers and identified different clonotypes by Vβ chain sequence analysis. In agreement with our previous results, the number of different clonotypes responding to all three epitopes fit a log-series distribution. From these distributions, we estimated that >1000 different clonotypes responded to each immunodominant CD8 T cell epitope; the response to a subdominant CD8 T cell epitope was modestly less diverse. These results suggest that T cell response diversity is greater by 1–2 orders of magnitude than predicted previously.

West Nile Virus (WNV) is an RNA virus that is the leading cause of mosquito-borne encephalitis in the United States. Recently, we demonstrated that MAVS, the central adaptor molecule of the RIG-I like receptor (RLR) signaling pathway, is essential role in programming innate and adaptive immune defenses to control WNV infection. However, the role of the individual RLRs in directing immunity during virus infection has not been clearly defined. WNV infection of Lgp2-/- mice showed increased mortality which corresponded to enhanced virus replication and reduced antigen-specific CD8+ T cells in the CNS at late times during infection. While LGP2 was determined to be non-essential in innate immune responses to WNV infection, LGP2 was required for controlling antigen-specific CD8+ T cell survival and effector functions during virus infection. Adoptive transfer studies of WT and Lgp2-/- CD8+ T cells into Rag-/- mice demonstrated that LGP2 functions in a cell-intrinsic manner to regulate T cell sensitivity to CD95-mediated cell death. Biochemical studies showed that LGP2 expression in CD8+ T cells is regulated through crosstalk between the T cell receptor signaling and interferon signaling pathways. Mechanisms by which LGP2 regulates extrinsic apoptosis signaling and T cell survival will be discussed. Overall, these studies demonstrate a novel for LGP2 in regulating T cell immunity and reveals a broader role for RLRs in programming adaptive immune responses during virus infection.