Ifit1 regulates norovirus infection and enhances the interferon response in murine macrophage-like cells [version 1; peer review: 1 approved, 2 approved with reservations]

Background: Norovirus, also known as the winter vomiting bug, is the predominant cause of non-bacterial gastroenteritis worldwide. Disease control is predicated on a robust innate immune response during the early stages of infection. Double-stranded RNA intermediates generated during viral genome replication are recognised by host innate immune sensors in the cytoplasm, activating the strongly antiviral interferon gene programme. Ifit proteins (interferon induced proteins with tetratricopeptide repeats), which are highly expressed during the interferon response, have been shown to directly inhibit viral protein synthesis as well as regulate innate immune signalling pathways. Ifit1 is well-characterised to inhibit viral translation by sequestration of eukaryotic initiation factors or by directly binding to the 5' terminus of foreign RNA, particularly those with non-self cap structures. However, noroviruses have a viral protein, VPg, covalently linked to the 5' end of the genomic RNA, which acts as a cap substitute to recruit the translation initiation machinery. Methods: : Ifit1 knockout RAW264.7 murine macrophage-like cells were generated using CRISPR-Cas9 gene editing. These cells were analysed for their ability to support murine norovirus infection, determined by virus yield, and respond to different immune stimuli, assayed by quantitative PCR. The effect of Ifit proteins on norovirus translation was also tested in vitro . Results: : Here, we show that VPg-dependent translation is completely refractory to Ifit1-mediated translation inhibition in vitro and Ifit1 cannot bind the 5' end of VPg-linked RNA. Nevertheless, knockout of Ifit1 promoted viral replication in murine norovirus infected cells. We then demonstrate that Ifit1 promoted interferon-beta expression following transfection of synthetic double-stranded RNA but had little effect on toll-like receptor 3 and 4 signalling. Conclusions: : Ifit1 is an antiviral factor during norovirus infection but cannot directly inhibit viral translation. Instead, Ifit1 stimulates the antiviral state following cytoplasmic RNA sensing, contributing to restriction of norovirus replication

An upstream protein-coding region in enteroviruses modulates virus infection in gut epithelial cells.

Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. Until now, all of the enteroviral proteins were thought to derive from the proteolytic processing of a polyprotein encoded in a single open reading frame. Here we report that many enterovirus genomes also harbour an upstream open reading frame (uORF) that is subject to strong purifying selection. Using echovirus 7 and poliovirus 1, we confirmed the expression of uORF protein in infected cells. Through ribosome profiling (a technique for the global footprinting of translating ribosomes), we also demonstrated translation of the uORF in representative members of the predominant human enterovirus species, namely Enterovirus A, B and C. In differentiated human intestinal organoids, uORF protein-knockout echoviruses are attenuated compared to the wild-type at late stages of infection where membrane-associated uORF protein facilitates virus release. Thus, we have identified a previously unknown enterovirus protein that facilitates virus growth in gut epithelial cells-the site of initial viral invasion into susceptible hosts. These findings overturn the 50-year-old dogma that enteroviruses use a single-polyprotein gene expression strategy and have important implications for the understanding of enterovirus pathogenesis.This work was supported by Wellcome Trust grant [106207] and European Research Council grant [646891] to A.E.F; and Wellcome Trust grants 097997/Z/11/Z and 207498/Z/17/Z to I.G

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination

B cells are important in immunity to both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and vaccination, but B cell receptor (BCR) repertoire development in these contexts has not been compared. We analyze serial samples from 171 SARS-CoV-2-infected individuals and 63 vaccine recipients and find the global BCR repertoire differs between them. Following infection, immunoglobulin (Ig)G1/3 and IgA1 BCRs increase, somatic hypermutation (SHM) decreases, and, in severe disease, IgM and IgA clones are expanded. In contrast, after vaccination, the proportion of IgD/M BCRs increase, SHM is unchanged, and expansion of IgG clones is prominent. VH1-24, which targets the N-terminal domain (NTD) and contributes to neutralization, is expanded post infection except in the most severe disease. Infection generates a broad distribution of SARS-CoV-2-specific clones predicted to target the spike protein, while a more focused response after vaccination mainly targets the spike's receptor-binding domain. Thus, the nature of SARS-CoV-2 exposure differentially affects BCR repertoire development, potentially informing vaccine strategies

Evolution of enhanced innate immune evasion by SARS-CoV-2

Emergence of SARS-CoV-2 variants of concern (VOCs) suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on characterisation of spike changes in VOCs, mutations outside spike likely contribute to adaptation. Here we used unbiased abundance proteomics, phosphoproteomics, RNAseq and viral replication assays to show that isolates of the Alpha (B.1.1.7) variant3 more effectively suppress innate immune responses in airway epithelial cells, compared to first wave isolates. We found that Alpha has dramatically increased subgenomic RNA and protein levels of N, Orf9b and Orf6, all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein required for RNA sensing adaptor MAVS activation. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful Alpha transmission, and may increase in vivo replication and duration of infection4. The importance of mutations outside Spike in adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the Delta and Omicron N/Orf9b regulatory regions

Combined Point-of-Care Nucleic Acid and Antibody Testing for SARS-CoV-2 following Emergence of D614G Spike Variant

Rapid COVID-19 diagnosis in the hospital is essential, although this is complicated by 30%-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant dominates the pandemic and it is unclear how serological tests designed to detect anti-spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild-type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95% CI 57.8-92.9) by rapid NAAT alone. The combined point of care antibody test and rapid NAAT is not affected by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity

Superspreaders drive the largest outbreaks of hospital onset COVID-19 infections.

SARS-CoV-2 is notable both for its rapid spread, and for the heterogeneity of its patterns of transmission, with multiple published incidences of superspreading behaviour. Here, we applied a novel network reconstruction algorithm to infer patterns of viral transmission occurring between patients and health care workers (HCWs) in the largest clusters of COVID-19 infection identified during the first wave of the epidemic at Cambridge University Hospitals NHS Foundation Trust, UK. Based upon dates of individuals reporting symptoms, recorded individual locations, and viral genome sequence data, we show an uneven pattern of transmission between individuals, with patients being much more likely to be infected by other patients than by HCWs. Further, the data were consistent with a pattern of superspreading, whereby 21% of individuals caused 80% of transmission events. Our study provides a detailed retrospective analysis of nosocomial SARS-CoV-2 transmission, and sheds light on the need for intensive and pervasive infection control procedures

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity

The SARS-CoV-2 Omicron BA.1 variant emerged in 2021(1) and has multiple mutations in its spike protein(2). Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways(3) demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis

Combined Point-of-Care Nucleic Acid and Antibody Testing for SARS-CoV-2 following Emergence of D614G Spike Variant.

Rapid COVID-19 diagnosis in the hospital is essential, although this is complicated by 30%-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant dominates the pandemic and it is unclear how serological tests designed to detect anti-spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild-type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95% CI 57.8-92.9) by rapid NAAT alone. The combined point of care antibody test and rapid NAAT is not affected by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity

Stem cell‐derived enteroid cultures as a tool for dissecting host‐parasite interactions in the small intestinal epithelium.

Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects, or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant, and species-specific models. Increasingly, 3D stem cell-derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments