Objectives Antibody testing against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has been instrumental in detecting previous exposures and analyzing vaccine‐elicited immune responses. Here, we describe a scalable solution to detect and quantify SARS‐CoV‐2 antibodies, discriminate between natural infection‐ and vaccination‐induced responses, and assess antibody‐mediated inhibition of the spike‐angiotensin converting enzyme 2 (ACE2) interaction. Methods We developed methods and reagents to detect SARS‐CoV‐2 antibodies by enzyme‐linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD) and nucleocapsid (N). We automated a surrogate neutralisation (sn)ELISA that measures inhibition of ACE2‐spike or ‐RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard. Results Our single‐point IgG‐based ELISAs accurately distinguished non‐infected and infected individuals. For seroprevalence assessment (in a non‐vaccinated cohort), classifying a sample as positive if antibodies were detected for ≥ 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti‐spike and ‐RBD (but not ‐N) antibodies are observed. We present detailed protocols for serum/plasma or dried blood spots analysis performed manually and on automated platforms. The snELISA can be performed automatically at single points, increasing its scalability. Conclusions Measuring antibodies to three viral antigens and identify neutralising antibodies capable of disrupting spike‐ACE2 interactions in high‐throughput enables large‐scale analyses of humoral immune responses to SARS‐CoV‐2 infection and vaccination. The reagents are available to enable scaling up of standardised serological assays, permitting inter‐laboratory data comparison and aggregation.
The influenza A virus RNA polymerase cleaves the 5′ end of host pre-mRNAs and uses the capped RNA fragments as primers for viral mRNA synthesis. We performed deep sequencing of the 5′ ends of viral mRNAs from all genome segments transcribed in both human (A549) and mouse (M-1) cells infected with the influenza A/HongKong/1/1968 (H3N2) virus. In addition to information on RNA motifs present, our results indicate that the host primers are divergent between the viral transcripts. We observed differences in length distributions, nucleotide motifs and the identity of the host primers between the viral mRNAs. Mapping the reads to known transcription start sites indicates that the virus targets the most abundant host mRNAs, which is likely caused by the higher expression of these genes. Our findings suggest negligible competition amongst RdRp:vRNA complexes for individual host mRNA templates during cap-snatching and provide a better understanding of the molecular mechanism governing the first step of transcription of this influenza strain.
The influenza A virus RNA polymerase cleaves the 5' ends of host RNAs and uses these RNA fragments as primers for viral mRNA synthesis. We performed deep sequencing of the 5' host-derived ends of the eight viral mRNAs of influenza A/Puerto Rico/8/1934 (H1N1) virus in infected A549 cells, and compared the population to those of A/Hong Kong/1/1968 (H3N2) and A/WSN/1933 (H1N1). In the three strains, the viral RNAs target different populations of host RNAs. Host RNAs are cap-snatched based on their abundance, and we found that RNAs encoding proteins involved in metabolism are overrepresented in the cap-snatched populations. Because this overrepresentation could be a reflection of the host response early after infection, and thus of the increased availability of these transcripts, our results suggest that host RNAs are cap-snatched mainly based on their abundance without preferential targeting.
Background: Viroids, satellite RNAs, satellites viruses and the human hepatitis delta virus form the 'brotherhood' of the smallest known infectious RNA agents, known as the subviral RNAs. For most of these species, it is generally accepted that characteristics such as cell movement, replication, host specificity and pathogenicity are encoded in their RNA sequences and their resulting RNA structures. Although many sequences are indexed in publicly available databases, these sequence annotation databases do not provide the advanced searches and data manipulation capability for identifying and characterizing subviral RNA motifs.
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by an expansion of CUG repeats in the 3' UTR of the DMPK gene. The CUG repeats form aggregates of mutant mRNA, which cause misregulation and/or sequestration of RNA-binding proteins, causing aberrant alternative splicing in cells. Previously, we showed that the multi-functional RNA-binding protein Staufen1 (Stau1) was increased in skeletal muscle of DM1 mouse models and patients. We also showed that Stau1 rescues the alternative splicing profile of pre-mRNAs, e.g. the INSR and CLC1, known to be aberrantly spliced in DM1. In order to explore further the potential of Stau1 as a therapeutic target for DM1, we first investigated the mechanism by which Stau1 regulates pre-mRNA alternative splicing. We report here that Stau1 regulates the alternative splicing of exon 11 of the human INSR via binding to Alu elements located in intron 10. Additionally, using a high-throughput RT-PCR screen, we have identified numerous Stau1-regulated alternative splicing events in both WT and DM1 myoblasts. A number of these aberrant ASEs in DM1, including INSR exon 11, are rescued by overexpression of Stau1. However, we find other ASEs in DM1 cells, where overexpression of Stau1 shifts the splicing patterns away from WT conditions. Moreover, we uncovered that Stau1-regulated ASEs harbour Alu elements in intronic regions flanking the alternative exon more than non-Stau1 targets. Taken together, these data highlight the broad impact of Stau1 as a splicing regulator and suggest that Stau1 may act as a disease modifier in DM1.
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