Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.

Autoantibodies against type I IFNs in humans with alternative NF-κB pathway deficiency.

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs)1,2, conferring a predisposition to life-threatening COVID-19 pneumonia3. Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52LOF/IκBδGOF). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52LOF/IκBδLOF) or gain-of-function of p52 (hereafter, p52GOF/IκBδLOF). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases.

Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM.

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies.

Discovering disease-causing pathogens in resource-scarce Southeast Asia using a global metagenomic pathogen monitoring system.

SignificanceMetagenomic pathogen sequencing offers an unbiased approach to characterizing febrile illness. In resource-scarce settings with high biodiversity, it is critical to identify disease-causing pathogens in order to understand burden and to prioritize efforts for control. Here, metagenomic next-generation sequencing (mNGS) characterization of the pathogen landscape in Cambodia revealed diverse vector-borne and zoonotic pathogens irrespective of age and gender as risk factors. Identification of key pathogens led to changes in national program surveillance. This study is a "real world" example of the use of mNGS surveillance of febrile individuals, executed in-country, to identify outbreaks of vector-borne, zoonotic, and other emerging pathogens in a resource-scarce setting.

Detection of High-Risk Paraneoplastic Antibodies against TRIM9 and TRIM67 Proteins.

OBJECTIVE: Co-occurring anti-tripartite motif-containing protein 9 and 67 autoantibodies (TRIM9/67-IgG) have been reported in only a very few cases of paraneoplastic cerebellar syndrome. The value of these biomarkers and the most sensitive methods of TRIM9/67-IgG detection are not known. METHODS: We performed a retrospective, multicenter study to evaluate the cerebrospinal fluid and serum of candidate TRIM9/67-IgG cases by tissue-based immunofluorescence, peptide phage display immunoprecipitation sequencing, overexpression cell-based assay (CBA), and immunoblot. Cases in which TRIM9/67-IgG was detected by at least 2 assays were considered TRIM9/67-IgG positive. RESULTS: Among these cases (n = 13), CBA was the most sensitive (100%) and revealed that all cases had TRIM9 and TRIM67 autoantibodies. Of TRIM9/67-IgG cases with available clinical history, a subacute cerebellar syndrome was the most common presentation (n = 7/10), followed by encephalitis (n = 3/10). Of these 10 patients, 70% had comorbid cancer (7/10), 85% of whom (n = 6/7) had confirmed metastatic disease. All evaluable cancer biopsies expressed TRIM9 protein (n = 5/5), whose expression was elevated in the cancerous regions of the tissue in 4 of 5 cases. INTERPRETATION: TRIM9/67-IgG is a rare but likely high-risk paraneoplastic biomarker for which CBA appears to be the most sensitive diagnostic assay. ANN NEUROL 2023;94:1086-1101.

Zika virus alters centrosome organization to suppress the innate immune response.

Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. Inherited forms of microcephaly (MCPH) are associated with disrupted centrosome organization. Similarly, we found that ZIKV infection disrupted centrosome organization. ZIKV infection disrupted the organization of centrosomal proteins including CEP63, a MCPH-associated protein. The ZIKV nonstructural protein NS3 bound CEP63, and expression of NS3 was sufficient to alter centrosome architecture and CEP63 localization. Loss of CEP63 suppressed ZIKV-induced centrosome disorganization, indicating that ZIKV requires CEP63 to disrupt centrosome organization. ZIKV infection or CEP63 loss decreased the centrosomal localization and stability of TANK-binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection also increased the centrosomal accumulation of the CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1. Therefore, we propose that ZIKV disrupts CEP63 function to increase centrosomal DTX4 localization and destabilization of TBK1, thereby tempering the innate immune response.

Genomic analysis, immunomodulation and deep phenotyping of patients with nodding syndrome.

The aetiology of nodding syndrome remains unclear, and comprehensive genotyping and phenotyping data from patients remain sparse. Our objectives were to characterize the phenotype of patients with nodding syndrome, investigate potential contributors to disease aetiology, and evaluate response to immunotherapy. This cohort study investigated members of a single-family unit from Lamwo District, Uganda. The participants for this study were selected by the Ugandan Ministry of Health as representative for nodding syndrome and with a conducive family structure for genomic analyses. Of the eight family members who participated in the study at the National Institutes of Health (NIH) Clinical Center, three had nodding syndrome. The three affected patients were extensively evaluated with metagenomic sequencing for infectious pathogens, exome sequencing, spinal fluid immune analyses, neurometabolic and toxicology testing, continuous electroencephalography and neuroimaging. Five unaffected family members underwent a subset of testing for comparison. A distinctive interictal pattern of sleep-activated bursts of generalized and multifocal epileptiform discharges and slowing was observed in two patients. Brain imaging showed two patients had mild generalized cerebral atrophy, and both patients and unaffected family members had excessive metal deposition in the basal ganglia. Trace metal biochemical evaluation was normal. CSF was non-inflammatory and one patient had CSF-restricted oligoclonal bands. Onchocerca volvulus-specific antibodies were present in all patients and skin snips were negative for active onchocerciasis. Metagenomic sequencing of serum and CSF revealed hepatitis B virus in the serum of one patient. Vitamin B6 metabolites were borderline low in all family members and CSF pyridoxine metabolites were normal. Mitochondrial DNA testing was normal. Exome sequencing did not identify potentially causal candidate gene variants. Nodding syndrome is characterized by a distinctive pattern of sleep-activated epileptiform activity. The associated growth stunting may be due to hypothalamic dysfunction. Extensive testing years after disease onset did not clarify a causal aetiology. A trial of immunomodulation (plasmapheresis in two patients and intravenous immunoglobulin in one patient) was given without short-term effect, but longer-term follow-up was not possible to fully assess any benefit of this intervention.

Low Prevalence of Interferon α Autoantibodies in People Experiencing Symptoms of Post-Coronavirus Disease 2019 (COVID-19) Conditions, or Long COVID.

Interferon (IFN)-specific autoantibodies have been implicated in severe coronavirus disease 2019 (COVID-19) and have been proposed as a potential driver of the persistent symptoms characterizing "long COVID," a type of postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report that only 2 of 215 participants with convalescent SARS-CoV-2 infection tested over 394 time points, including 121 people experiencing long COVID symptoms, had detectable IFN-α2 antibodies. Both had been hospitalized during the acute phase of the infection. These data suggest that persistent anti-IFN antibodies, although a potential driver of severe COVID-19, are unlikely to contribute to long COVID symptoms in the postacute phase of the infection.

Successful Treatment of Balamuthia mandrillaris Granulomatous Amebic Encephalitis with Nitroxoline.

A patient in California, USA, with rare and usually fatal Balamuthia mandrillaris granulomatous amebic encephalitis survived after receiving treatment with a regimen that included the repurposed drug nitroxoline. Nitroxoline, which is a quinolone typically used to treat urinary tract infections, was identified in a screen for drugs with amebicidal activity against Balamuthia.

Discovery and Genomic Characterization of a Novel Henipavirus, Angavokely Virus, from Fruit Bats in Madagascar.

The genus Henipavirus (family Paramyxoviridae) currently comprises seven viruses, four of which have demonstrated prior evidence of zoonotic capacity. These include the biosafety level 4 agents Hendra (HeV) and Nipah (NiV) viruses, which circulate naturally in pteropodid fruit bats. Here, we describe and characterize Angavokely virus (AngV), a divergent henipavirus identified in urine samples from wild, Madagascar fruit bats. We report the nearly complete 16,740-nucleotide genome of AngV, which encodes the six major henipavirus structural proteins (nucleocapsid, phosphoprotein, matrix, fusion, glycoprotein, and L polymerase). Within the phosphoprotein (P) gene, we identify an alternative start codon encoding the AngV C protein and a putative mRNA editing site where the insertion of one or two guanine residues encodes, respectively, additional V and W proteins. In other paramyxovirus systems, C, V, and W are accessory proteins involved in antagonism of host immune responses during infection. Phylogenetic analysis suggests that AngV is ancestral to all four previously described bat henipaviruses-HeV, NiV, Cedar virus (CedV), and Ghanaian bat virus (GhV)-but evolved more recently than rodent- and shrew-derived henipaviruses, Mojiang (MojV), Gamak (GAKV), and Daeryong (DARV) viruses. Predictive structure-based alignments suggest that AngV is unlikely to bind ephrin receptors, which mediate cell entry for all other known bat henipaviruses. Identification of the AngV receptor is needed to clarify the virus's potential host range. The presence of V and W proteins in the AngV genome suggest that the virus could be pathogenic following zoonotic spillover. IMPORTANCE Henipaviruses include highly pathogenic emerging zoonotic viruses, derived from bat, rodent, and shrew reservoirs. Bat-borne Hendra (HeV) and Nipah (NiV) are the most well-known henipaviruses, for which no effective antivirals or vaccines for humans have been described. Here, we report the discovery and characterization of a novel henipavirus, Angavokely virus (AngV), isolated from wild fruit bats in Madagascar. Genomic characterization of AngV reveals all major features associated with pathogenicity in other henipaviruses, suggesting that AngV could be pathogenic following spillover to human hosts. Our work suggests that AngV is an ancestral bat henipavirus that likely uses viral entry pathways distinct from those previously described for HeV and NiV. In Madagascar, bats are consumed as a source of human food, presenting opportunities for cross-species transmission. Characterization of novel henipaviruses and documentation of their pathogenic and zoonotic potential are essential to predicting and preventing the emergence of future zoonoses that cause pandemics.

ZSCAN1 Autoantibodies Are Associated with Pediatric Paraneoplastic ROHHAD.

OBJECTIVE: Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation and Autonomic Dysregulation (ROHHAD), is a severe pediatric disorder of uncertain etiology resulting in hypothalamic dysfunction and frequent sudden death. Frequent co-occurrence of neuroblastic tumors have fueled suspicion of an autoimmune paraneoplastic neurological syndrome (PNS); however, specific anti-neural autoantibodies, a hallmark of PNS, have not been identified. Our objective is to determine if an autoimmune paraneoplastic etiology underlies ROHHAD. METHODS: Immunoglobulin G (IgG) from pediatric ROHHAD patients (n = 9), non-inflammatory individuals (n = 100) and relevant pediatric controls (n = 25) was screened using a programmable phage display of the human peptidome (PhIP-Seq). Putative ROHHAD-specific autoantibodies were orthogonally validated using radioactive ligand binding and cell-based assays. Expression of autoantibody targets in ROHHAD tumor and healthy brain tissue was assessed with immunohistochemistry and mass spectrometry, respectively. RESULTS: Autoantibodies to ZSCAN1 were detected in ROHHAD patients by PhIP-Seq and orthogonally validated in 7/9 ROHHAD patients and 0/125 controls using radioactive ligand binding and cell-based assays. Expression of ZSCAN1 in ROHHAD tumor and healthy human brain tissue was confirmed. INTERPRETATION: Our results support the notion that tumor-associated ROHHAD syndrome is a pediatric PNS, potentially initiated by an immune response to peripheral neuroblastic tumor. ZSCAN1 autoantibodies may aid in earlier, accurate diagnosis of ROHHAD syndrome, thus providing a means toward early detection and treatment. This work warrants follow-up studies to test sensitivity and specificity of a novel diagnostic test. Last, given the absence of the ZSCAN1 gene in rodents, our study highlights the value of human-based approaches for detecting novel PNS subtypes. ANN NEUROL 2022;92:279-291.

The pulmonary metatranscriptome prior to pediatric HCT identifies post-HCT lung injury.

Lung injury after pediatric allogeneic hematopoietic cell transplantation (HCT) is a common and disastrous complication that threatens long-term survival. To develop strategies to prevent lung injury, novel tools are needed to comprehensively assess lung health in HCT candidates. Therefore, this study analyzed biospecimens from 181 pediatric HCT candidates who underwent routine pre-HCT bronchoalveolar lavage (BAL) at the University Medical Center Utrecht between 2005 and 2016. BAL fluid underwent metatranscriptomic sequencing of microbial and human RNA, and unsupervised clustering and generalized linear models were used to associate microbiome gene expression data with the development of post-HCT lung injury. Microbe-gene correlations were validated using a geographically distinct cohort of 18 pediatric HCT candidates. The cumulative incidence of post-HCT lung injury varied significantly according to 4 pre-HCT pulmonary metatranscriptome clusters, with the highest incidence observed in children with pre-HCT viral enrichment and innate immune activation, as well as in children with profound microbial depletion and concomitant natural killer/T-cell activation (P < .001). In contrast, children with pre-HCT pulmonary metatranscriptomes containing diverse oropharyngeal taxa and lacking inflammation rarely developed post-HCT lung injury. In addition, activation of epithelial-epidermal differentiation, mucus production, and cellular adhesion were associated with fatal post-HCT lung injury. In a separate validation cohort, associations among pulmonary respiratory viral load, oropharyngeal taxa, and pulmonary gene expression were recapitulated; the association with post-HCT lung injury needs to be validated in an independent cohort. This analysis suggests that assessment of the pre-HCT BAL fluid may identify high-risk pediatric HCT candidates who may benefit from pathobiology-targeted interventions.

Elevated N-Linked Glycosylation of IgG V Regions in Myasthenia Gravis Disease Subtypes.

Elevated N-linked glycosylation of IgG V regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-Glyc, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design focused on examining the B cell repertoire and total IgG. It specifically included adaptive immune receptor repertoire sequencing to quantify and characterize N-linked glycosylation sites in the circulating BCR repertoire, proteomics to examine glycosylation patterns of the total circulating IgG, and an exploration of human-derived recombinant autoantibodies, which were studied with mass spectrometry and Ag binding assays to respectively confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-VN-Glyc motifs was increased in the total BCR repertoire of patients with MG when compared with healthy donors. The elevated frequency was attributed to both biased V gene segment usage and somatic hypermutation. IgG-VN-Glyc could be observed in the total circulating IgG in a subset of patients with MG. Autoantigen binding, by four patient-derived MG autoantigen-specific mAbs with experimentally confirmed presence of IgG-VN-Glyc, was not altered by the glycosylation. Our findings extend prior work on patterns of Ig V region N-linked glycosylation in autoimmunity to MG subtypes.

Deep profiling of protease substrate specificity enabled by dual random and scanned human proteome substrate phage libraries.

Proteolysis is a major posttranslational regulator of biology inside and outside of cells. Broad identification of optimal cleavage sites and natural substrates of proteases is critical for drug discovery and to understand protease biology. Here, we present a method that employs two genetically encoded substrate phage display libraries coupled with next generation sequencing (SPD-NGS) that allows up to 10,000-fold deeper sequence coverage of the typical six- to eight-residue protease cleavage sites compared to state-of-the-art synthetic peptide libraries or proteomics. We applied SPD-NGS to two classes of proteases, the intracellular caspases, and the ectodomains of the sheddases, ADAMs 10 and 17. The first library (Lib 10AA) allowed us to identify 104 to 105 unique cleavage sites over a 1,000-fold dynamic range of NGS counts and produced consensus and optimal cleavage motifs based position-specific scoring matrices. A second SPD-NGS library (Lib hP), which displayed virtually the entire human proteome tiled in contiguous 49 amino acid sequences with 25 amino acid overlaps, enabled us to identify candidate human proteome sequences. We identified up to 104 natural linear cut sites, depending on the protease, and captured most of the examples previously identified by proteomics and predicted 10- to 100-fold more. Structural bioinformatics was used to facilitate the identification of candidate natural protein substrates. SPD-NGS is rapid, reproducible, simple to perform and analyze, inexpensive, and renewable, with unprecedented depth of coverage for substrate sequences, and is an important tool for protease biologists interested in protease specificity for specific assays and inhibitors and to facilitate identification of natural protein substrates.

A pathogenic and clonally expanded B cell transcriptome in active multiple sclerosis.

Central nervous system B cells have several potential roles in multiple sclerosis (MS): secretors of proinflammatory cytokines and chemokines, presenters of autoantigens to T cells, producers of pathogenic antibodies, and reservoirs for viruses that trigger demyelination. To interrogate these roles, single-cell RNA sequencing (scRNA-Seq) was performed on paired cerebrospinal fluid (CSF) and blood from subjects with relapsing-remitting MS (RRMS; n = 12), other neurologic diseases (ONDs; n = 1), and healthy controls (HCs; n = 3). Single-cell immunoglobulin sequencing (scIg-Seq) was performed on a subset of these subjects and additional RRMS (n = 4), clinically isolated syndrome (n = 2), and OND (n = 2) subjects. Further, paired CSF and blood B cell subsets (RRMS; n = 7) were isolated using fluorescence activated cell sorting for bulk RNA sequencing (RNA-Seq). Independent analyses across technologies demonstrated that nuclear factor kappa B (NF-κB) and cholesterol biosynthesis pathways were activated, and specific cytokine and chemokine receptors were up-regulated in CSF memory B cells. Further, SMAD/TGF-ß1 signaling was down-regulated in CSF plasmablasts/plasma cells. Clonally expanded, somatically hypermutated IgM+ and IgG1+ CSF B cells were associated with inflammation, blood-brain barrier breakdown, and intrathecal Ig synthesis. While we identified memory B cells and plasmablast/plasma cells with highly similar Ig heavy-chain sequences across MS subjects, similarities were also identified with ONDs and HCs. No viral transcripts, including from Epstein-Barr virus, were detected. Our findings support the hypothesis that in MS, CSF B cells are driven to an inflammatory and clonally expanded memory and plasmablast/plasma cell phenotype.

Kelch-like Protein 11 Antibodies in Seminoma-Associated Paraneoplastic Encephalitis.

A 37-year-old man with a history of seminoma presented with vertigo, ataxia, and diplopia. An autoantibody specific for kelch-like protein 11 (KLHL11) was identified with the use of programmable phage display. Immunoassays were used to identify KLHL11 IgG in 12 other men with similar neurologic features and testicular disease. Immunostaining of the patient's IgG on mouse brain tissue showed sparse but distinctive points of staining in multiple brain regions, with enrichment in perivascular and perimeningeal tissues. The onset of the neurologic syndrome preceded the diagnosis of seminoma in 9 of the 13 patients. An age-adjusted estimate of the prevalence of autoimmune KLHL11 encephalitis in Olmsted County, Minnesota, was 2.79 cases per 100,000 men. (Funded by the Rochester Epidemiology Project and others.).

Clinical Metagenomic Sequencing for Diagnosis of Meningitis and Encephalitis.

BACKGROUND: Metagenomic next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has the potential to identify a broad range of pathogens in a single test. METHODS: In a 1-year, multicenter, prospective study, we investigated the usefulness of metagenomic NGS of CSF for the diagnosis of infectious meningitis and encephalitis in hospitalized patients. All positive tests for pathogens on metagenomic NGS were confirmed by orthogonal laboratory testing. Physician feedback was elicited by teleconferences with a clinical microbial sequencing board and by surveys. Clinical effect was evaluated by retrospective chart review. RESULTS: We enrolled 204 pediatric and adult patients at eight hospitals. Patients were severely ill: 48.5% had been admitted to the intensive care unit, and the 30-day mortality among all study patients was 11.3%. A total of 58 infections of the nervous system were diagnosed in 57 patients (27.9%). Among these 58 infections, metagenomic NGS identified 13 (22%) that were not identified by clinical testing at the source hospital. Among the remaining 45 infections (78%), metagenomic NGS made concurrent diagnoses in 19. Of the 26 infections not identified by metagenomic NGS, 11 were diagnosed by serologic testing only, 7 were diagnosed from tissue samples other than CSF, and 8 were negative on metagenomic NGS owing to low titers of pathogens in CSF. A total of 8 of 13 diagnoses made solely by metagenomic NGS had a likely clinical effect, with 7 of 13 guiding treatment. CONCLUSIONS: Routine microbiologic testing is often insufficient to detect all neuroinvasive pathogens. In this study, metagenomic NGS of CSF obtained from patients with meningitis or encephalitis improved diagnosis of neurologic infections and provided actionable information in some cases. (Funded by the National Institutes of Health and others; PDAID ClinicalTrials.gov number, NCT02910037.).

Persistence of Ambigrammatic Narnaviruses Requires Translation of the Reverse Open Reading Frame.

Narnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods, and nematodes. Though initially described as simple single-gene nonsegmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as "ambigrammatic" harbor a unique genomic configuration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquito-infecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2-kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8-kb "Robin" RNA. We developed a system to launch CxNV1 in a naive mosquito cell line and then showed that functional RdRp is required for persistence of both segments, and an intact reverse ORF is required on the RdRp segment for persistence. Mass spectrometry of persistently CxNV1-infected cells provided evidence for translation of this reverse ORF. Finally, ribosome profiling yielded a striking pattern of footprints for all four CxNV1 RNA strands that was distinct from actively translating ribosomes on host mRNA or coinfecting RNA viruses. Taken together, these data raise the possibility that the process of translation itself is important for persistence of ambigrammatic narnaviruses, potentially by protecting viral RNA with ribosomes, thus suggesting a heretofore undescribed viral tactic for replication and transmission. IMPORTANCE Fundamental to our understanding of RNA viruses is a description of which strand(s) of RNA are transmitted as the viral genome relative to which encode the viral proteins. Ambigrammatic narnaviruses break the mold. These viruses, found broadly in fungi, plants, and insects, have the unique feature of two overlapping genes encoded on opposite strands, comprising nearly the full length of the viral genome. Such extensive overlap is not seen in other RNA viruses and comes at the cost of reduced evolutionary flexibility in the sequence. The present study is motivated by investigating the benefits which balance that cost. We show for the first time a functional requirement for the ambigrammatic genome configuration in Culex narnavirus 1, which suggests a model for how translation of both strands might benefit this virus. Our work highlights a new blueprint for viral persistence, distinct from strategies defined by canonical definitions of the coding strand.

Prevalence of type-1 interferon autoantibodies in adults with non-COVID-19 acute respiratory failure.

Auto-antibodies (Abs) to type I interferons (IFNs) are found in up to 25% of patients with severe COVID-19, and are implicated in disease pathogenesis. It has remained unknown, however, whether type I IFN auto-Abs are unique to COVID-19, or are also found in other types of severe respiratory illnesses. To address this, we studied a prospective cohort of 284 adults with acute respiratory failure due to causes other than COVID-19. We measured type I IFN auto-Abs by radio ligand binding assay and screened for respiratory viruses using clinical PCR and metagenomic sequencing. Three patients (1.1%) tested positive for type I IFN auto-Abs, and each had a different underlying clinical presentation. Of the 35 patients found to have viral infections, only one patient tested positive for type I IFN auto-Abs. Together, our data suggest that type I IFN auto-Abs are uncommon in critically ill patients with acute respiratory failure due to causes other than COVID-19.

Functional characterization of 5' UTR cis-acting sequence elements that modulate translational efficiency in Plasmodium falciparum and humans.

BACKGROUND: The eukaryotic parasite Plasmodium falciparum causes millions of malarial infections annually while drug resistance to common anti-malarials is further confounding eradication efforts. Translation is an attractive therapeutic target that will benefit from a deeper mechanistic understanding. As the rate limiting step of translation, initiation is a primary driver of translational efficiency. It is a complex process regulated by both cis and trans acting factors, providing numerous potential targets. Relative to model organisms and humans, P. falciparum mRNAs feature unusual 5' untranslated regions suggesting cis-acting sequence complexity in this parasite may act to tune levels of protein synthesis through their effects on translational efficiency. METHODS: Here, in vitro translation is deployed to compare the role of cis-acting regulatory sequences in P. falciparum and humans. Using parasite mRNAs with high or low translational efficiency, the presence, position, and termination status of upstream "AUG"s, in addition to the base composition of the 5' untranslated regions, were characterized. RESULTS: The density of upstream "AUG"s differed significantly among the most and least efficiently translated genes in P. falciparum, as did the average "GC" content of the 5' untranslated regions. Using exemplars from highly translated and poorly translated mRNAs, multiple putative upstream elements were interrogated for impact on translational efficiency. Upstream "AUG"s were found to repress translation to varying degrees, depending on their position and context, while combinations of upstream "AUG"s had non-additive effects. The base composition of the 5' untranslated regions also impacted translation, but to a lesser degree. Surprisingly, the effects of cis-acting sequences were remarkably conserved between P. falciparum and humans. CONCLUSIONS: While translational regulation is inherently complex, this work contributes toward a more comprehensive understanding of parasite and human translational regulation by examining the impact of discrete cis-acting features, acting alone or in context.