Larger capacity for unconscious versus conscious episodic memory

Episodic memory is the memory for experienced events. A peak competence of episodic memory is the mental combination of events to infer commonalities. Inferring commonalities may proceed with and without consciousness of events. Yet what distinguishes conscious from unconscious inference? This question inspired nine experiments that featured strongly and weakly masked cartoon clips presented for unconscious and conscious inference. Each clip featured a scene with a visually impenetrable hiding place. Five animals crossed the scene one-by-one consecutively. One animal trajectory represented one event. The animals moved through the hiding place, where they might linger or not. The participants' task was to observe the animals' entrances and exits to maintain a mental record of which animals hid simultaneously. We manipulated information load to explore capacity limits. Memory of inferences was tested immediately, 3.5 or 6 min following encoding. The participants retrieved inferences well when encoding was conscious. When encoding was unconscious, the participants needed to respond intuitively. Only habitually intuitive decision makers exhibited a significant delayed retrieval of inferences drawn unconsciously. Their unconscious retrieval performance did not drop significantly with increasing information load, while conscious retrieval performance dropped significantly. A working memory network, including hippocampus, was activated during both conscious and unconscious inference and correlated with retrieval success. An episodic retrieval network, including hippocampus, was activated during both conscious and unconscious retrieval of inferences and correlated with retrieval success. Only conscious encoding/retrieval recruited additional brain regions outside these networks. Hence, levels of consciousness influenced the memories' behavioral impact, memory capacity, and the neural representational code

Coronavirus Non-Structural Protein 1 Is a Major Pathogenicity Factor: Implications for the Rational Design of Coronavirus Vaccines

Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1). In cell culture, nsp1 of mouse hepatitis virus (MHV), like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN) receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines

A Systems Immunology Approach to Plasmacytoid Dendritic Cell Function in Cytopathic Virus Infections

Plasmacytoid dendritic cell (pDC)-mediated protection against cytopathic virus infection involves various molecular, cellular, tissue-scale, and organism-scale events. In order to better understand such multiscale interactions, we have implemented a systems immunology approach focusing on the analysis of the structure, dynamics and operating principles of virus-host interactions which constrain the initial spread of the pathogen. Using high-resolution experimental data sets coming from the well-described mouse hepatitis virus (MHV) model, we first calibrated basic modules including MHV infection of its primary target cells, i.e. pDCs and macrophages (Mφs). These basic building blocks were used to generate and validate an integrative mathematical model for in vivo infection dynamics. Parameter estimation for the system indicated that on a per capita basis, one infected pDC secretes sufficient type I IFN to protect 103 to 104 Mφs from cytopathic viral infection. This extremely high protective capacity of pDCs secures the spleen's capability to function as a ‘sink’ for the virus produced in peripheral organs such as the liver. Furthermore, our results suggest that the pDC population in spleen ensures a robust protection against virus variants which substantially down-modulate IFN secretion. However, the ability of pDCs to protect against severe disease caused by virus variants exhibiting an enhanced liver tropism and higher replication rates appears to be rather limited. Taken together, this systems immunology analysis suggests that antiviral therapy against cytopathic viruses should primarily limit viral replication within peripheral target organs

Persistence, prevalence, and polymorphism of sequelae after COVID-19 in unvaccinated, young adults of the Swiss Armed Forces: a longitudinal, cohort study (LoCoMo)

Background: Persistent COVID-19 sequelae could have global, public health ramifications. We therefore aimed to describe sequelae presenting more than 180 days after COVID-19-focussing on several organ systems, general health, and laboratory parameters-in non-hospitalised, unvaccinated, young adults. Methods: We did a longitudinal cohort study of all army bases in Switzerland. Eligible participants were personnel of the Swiss Armed Forces (SAF) who were aged 18-30 years with a positive or negative RT-PCR test for SARS-CoV-2 during their service between March 1, 2020, and Dec 31, 2020. Exclusion criteria were unwillingness to participate in testing. Females or men with a known reproductive anomaly were excluded from the optional component of male fertility testing. COVID-19 was defined as a positive diagnostic RT-PCR test result for SARS-CoV-2 with concurrent symptoms compatible with COVID-19. Participants were subdivided into four groups: control group (ie, serologically negative), asymptomatic infection group (ie, serologically positive but with no symptoms), non-recent COVID-19 group (>180 days since positive PCR test), and recent COVID-19 group (≤180 days since positive PCR test). Outcomes of interest were part of a comprehensive, intensive test battery that was administered during a single day. The test battery quantified the effect of SARS-CoV-2 infection on cardiovascular, pulmonary, neurological, renal, ophthalmological, male reproductive, psychological, general health, and laboratory parameters. This study was registered with ClinicalTrials.gov, number NCT04942249. Findings: Between May 20, 2021, and Nov 26, 2021, we enrolled 501 participants. 29 (6%) of 501 were female and 464 (93%) were male, and the median age was 21 years (IQR 21-23). Eight (2%) of 501 had incomplete data and were not included into the study groups. 177 participants had previous COVID-19 that was more than 180 days (mean 340 days) since diagnosis (ie, the non-recent COVID-19 group) compared with 251 serologically negative individuals (ie, the control group). We included 19 participants in the recent COVID-19 group and 46 in the asymptomatic infection group. We found a significant trend towards metabolic disorders in participants of the non-recent COVID-19 group compared with those in the control group: higher BMI (median 24·0 kg/m2 [IQR 22·0-25·8] vs 23·2 kg/m2 [27·1-25·0]; p=0·035), lower aerobic threshold (39% [36-43] vs 41% [37-46]; p=0·012), and higher blood cholesterol (4·2 μM [3·7-4·7] vs 3·9 μM [3·5-4·5]; p<0·0001) and LDL concentrations (2·4 μM [1·9-2·9] vs 2·2 μM [1·7-2·7]; p=0·001). The only significant psychosocial difference was found in the results of the Chalder Fatigue scale with the non-recent COVID-19 group reporting higher fatigue scores than the control group (median 12 points [IQR 11-15] vs 11 [9-14]; p=0·027). No significant differences in other psychosocial questionnaire scores, ophthalmological outcomes, and sperm quality or motility were reported between the control group and non-recent COVID-19 group. Interpretation: Young, previously healthy, individuals largely recover from SARS-CoV-2 infection. However, the constellation of higher BMI, dyslipidaemia, and lower physical endurance 180 days after COVID-19 is suggestive of a higher risk of developing metabolic disorders and possible cardiovascular complications. These findings will guide future investigations and follow-up management

Titers of Neutralizing Antibodies against SARS-CoV-2 Are Independent of Symptoms of Non-Severe COVID-19 in Young Adults.

Neutralizing antibodies are an important part of the humoral immune response to SARS-CoV-2. It is currently unclear to what extent such antibodies are produced after non-severe disease or asymptomatic infection. We studied a cluster of SARS-CoV-2 infections among a homogeneous population of 332 predominantly male Swiss soldiers and determined the neutralizing antibody response with a serum neutralization assay using a recombinant SARS-CoV-2-GFP. All patients with non-severe COVID-19 showed a swift humoral response within two weeks after the onset of symptoms, which remained stable for the duration of the study. One month after the outbreak, titers in COVID-19 convalescents did not differ from the titers of asymptomatically infected individuals. Furthermore, symptoms of COVID-19 did not correlate with neutralizing antibody titers. Therefore, we conclude that asymptomatic infection can induce the same humoral immunity as non-severe COVID-19 in young adults

The SARS-coronavirus-host interactome

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock

N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition

The ongoing coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is associated with substantial morbidity and mortality. Understanding the immunological and pathological processes of coronavirus diseases is crucial for the rational design of effective vaccines and therapies for COVID-19. Previous studies showed that 2′-O-methylation of the viral RNA cap structure is required to prevent the recognition of viral RNAs by intracellular innate sensors. Here, we demonstrate that the guanine N7-methylation of the 5′ cap mediated by coronavirus nonstructural protein 14 (nsp14) contributes to viral evasion of the type I interferon (IFN-I)-mediated immune response and pathogenesis in mice. A Y414A substitution in nsp14 of the coronavirus mouse hepatitis virus (MHV) significantly decreased N7-methyltransferase activity and reduced guanine N7-methylation of the 5′ cap in vitro. Infection of myeloid cells with recombinant MHV harboring the nsp14-Y414A mutation (rMHVnsp14-Y414A) resulted in upregulated expression of IFN-I and ISG15 mainly via MDA5 signaling and in reduced viral replication compared to that of wild-type rMHV. rMHVnsp14-Y414A replicated to lower titers in livers and brains and exhibited an attenuated phenotype in mice. This attenuated phenotype was IFN-I dependent because the virulence of the rMHVnsp14-Y414A mutant was restored in Ifnar−/− mice. We further found that the comparable mutation (Y420A) in SARS-CoV-2 nsp14 (rSARS-CoV-2nsp14-Y420A) also significantly decreased N7-methyltransferase activity in vitro, and the mutant virus was attenuated in K18-human ACE2 transgenic mice. Moreover, infection with rSARS-CoV-2nsp14-Y420A conferred complete protection against subsequent and otherwise lethal SARS-CoV-2 infection in mice, indicating the vaccine potential of this mutant. IMPORTANCE Coronaviruses (CoVs), including SARS-CoV-2, the cause of COVID-19, use several strategies to evade the host innate immune responses. While the cap structure of RNA, including CoV RNA, is important for translation, previous studies indicate that the cap also contributes to viral evasion from the host immune response. In this study, we demonstrate that the N7-methylated cap structure of CoV RNA is pivotal for virus immunoevasion. Using recombinant MHV and SARS-CoV-2 encoding an inactive N7-methyltransferase, we demonstrate that these mutant viruses are highly attenuated in vivo and that attenuation is apparent at very early times after infection. Virulence is restored in mice lacking interferon signaling. Further, we show that infection with virus defective in N7-methylation protects mice from lethal SARS-CoV-2, suggesting that the N7-methylase might be a useful target in drug and vaccine development

Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5'-cap-structures of higher eukaryote mRNAs are ribose 2'-O-methylated. Likewise, a number of viruses replicating in the cytoplasm of eukayotes have evolved 2'-O-methyltransferases to modify autonomously their mRNAs. However, a defined biological role of mRNA 2'-O-methylation remains elusive. Here we show that viral mRNA 2'-O-methylation is critically involved in subversion of type-I-interferon (IFN-I) induction. We demonstrate that human and murine coronavirus 2'-O-methyltransferase mutants induce increased IFN-I expression, and are highly IFN-I sensitive. Importantly, IFN-I induction by 2'-O-methyltransferase-deficient viruses is dependent on the cytoplasmic RNA sensor melanoma differentiation-associated gene 5 (MDA5). This link between MDA5-mediated sensing of viral RNA and mRNA 2'-O-methylation suggests that RNA modifications, such as 2'-O-methylation, provide a molecular signature for the discrimination of self and non-self mRNA

Dendritic cell-specific delivery of Flt3L by coronavirus vectors secures induction of therapeutic antitumor immunity

Efficacy of antitumor vaccination depends to a large extent on antigen targeting to dendritic cells (DCs). Here, we assessed antitumor immunity induced by attenuated coronavirus vectors which exclusively target DCs in vivo and express either lymphocyte- or DC-activating cytokines in combination with a GFP-tagged model antigen. Tracking of in vivo transduced DCs revealed that vectors encoding for Fms-like tyrosine kinase 3 ligand (Flt3L) exhibited a higher capacity to induce DC maturation compared to vectors delivering IL-2 or IL-15. Moreover, Flt3L vectors more efficiently induced tumor-specific CD8(+) T cells, expanded the epitope repertoire, and provided both prophylactic and therapeutic tumor immunity. In contrast, IL-2- or IL-15-encoding vectors showed a substantially lower efficacy in CD8(+) T cell priming and failed to protect the host once tumors had been established. Thus, specific in vivo targeting of DCs with coronavirus vectors in conjunction with appropriate conditioning of the microenvironment through Flt3L represents an efficient strategy for the generation of therapeutic antitumor immunity

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses