Inhibition of human cytomegalovirus replication by interferon alpha can involve multiple anti-viral factors.

The shortcomings of current direct-acting anti-viral therapy against human cytomegalovirus (HCMV) has led to interest in host-directed therapy. Here we re-examine the use of interferon proteins to inhibit HCMV replication utilizing both high and low passage strains of HCMV. Pre-treatment of cells with interferon alpha (IFNα) was required for robust and prolonged inhibition of both low and high passage HCMV strains, with no obvious toxicity, and was associated with an increased anti-viral state in HCMV-infected cells. Pre-treatment of cells with IFNα led to poor expression of HCMV immediate-early proteins from both high and low passage strains, which was associated with the presence of the anti-viral factor SUMO-PML. Inhibition of HCMV replication in the presence of IFNα involving ZAP proteins was HCMV strain-dependent, wherein a high passage HCMV strain was obviously restricted by ZAP and a low passage strain was not. This suggested that strain-specific combinations of anti-viral factors were involved in inhibition of HCMV replication in the presence of IFNα. Overall, this work further supports the development of strategies involving IFNα that may be useful to inhibit HCMV replication and highlights the complexity of the anti-viral response to HCMV in the presence of IFNα.

The ongoing Streptococcus pyogenes (Group A Streptococcus) outbreak in London, United Kingdom, in December 2022: a molecular epidemiology study.

OBJECTIVES: Epidemiological and whole-genome sequencing analysis of an ongoing outbreak of Streptococcus pyogenes (Group A Streptococcus) in London (United Kingdom). METHODS: Prospective identification of Group A Streptococcus cases from a diagnostic laboratory serving central and south London between 27 November and 10 December 2022. Case notes were reviewed and isolates were retrieved. Case numbers were compared with the previous 5 years. Whole-genome sequencing was performed with long-read, nanopore technology for emm typing and identification of superantigen genes. Associations of pathogen-related factors with an invasive disease were assessed by single-variable and multi-variable logistic regression. RESULTS: Case numbers began increasing in October 2022 from a baseline of 2.0 cases per day, and in December 2022, the average daily case numbers reached 10.8 cases per day, four-fold the number usually seen in winter. A total of 113 cases were identified during the prospective study period. Three quarters (86/113, 76%) were paediatric cases, including 2 deaths. Of 113 cases, 11 (10%) were invasive. In total, 56 isolates were successfully sequenced, including 10 of 11 (91%) invasive isolates. The emm12 (33/56, 59%) and emm1 (9/56, 16%) types were predominant, with 7 of 9 (78%) emm1 isolates being from the M1uk clone. The majority of invasive isolates had superantigen genes spea (7/10, 70%) and spej (8/10, 80%), whereas, in non-invasive isolates, these superantigen genes were found less frequently (spea: 5/46, 11% and spej: 7/46, 15%). By multivariable analysis of pathogen-related factors, spea (OR 8.9, CI 1.4-57, p 0.020) and spej (OR 12, CI 1.8-78, p 0.011) were associated with invasive disease. CONCLUSIONS: emm12 and emm1 types predominate in the ongoing outbreak, which mainly affects children. In this outbreak, the spea and spej superantigen genes are associated with the severity of presentation.

Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins.

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.

Pangolin merbecovirus gets down to (poly)basics.

Trafficking of live mammals is considered a major risk for emergence of zoonotic viruses. SARS-CoV-2-related coronaviruses have previously been identified in pangolins, the world's most smuggled mammal. A new study identifies a MERS-related coronavirus in trafficked pangolins with broad mammalian tropism and a newly acquired furin cleavage site in Spike.

A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods.

The zinc finger antiviral protein (ZAP) inhibits viral replication by directly binding CpG dinucleotides in cytoplasmic viral RNA to inhibit protein synthesis and target the RNA for degradation. ZAP evolved in tetrapods and there are clear orthologs in reptiles, birds, and mammals. When ZAP emerged, other proteins may have evolved to become cofactors for its antiviral activity. KHNYN is a putative endoribonuclease that is required for ZAP to restrict retroviruses. To determine its evolutionary path after ZAP emerged, we compared KHNYN orthologs in mammals and reptiles to those in fish, which do not encode ZAP. This identified residues in KHNYN that are highly conserved in species that encode ZAP, including several in the CUBAN domain. The CUBAN domain interacts with NEDD8 and Cullin-RING E3 ubiquitin ligases. Deletion of the CUBAN domain decreased KHNYN antiviral activity, increased protein expression and increased nuclear localization. However, mutation of residues required for the CUBAN domain-NEDD8 interaction increased KHNYN abundance but did not affect its antiviral activity or cytoplasmic localization, indicating that Cullin-mediated degradation may control its homeostasis and regulation of protein turnover is separable from its antiviral activity. By contrast, the C-terminal residues in the CUBAN domain form a CRM1-dependent nuclear export signal (NES) that is required for its antiviral activity. Deletion or mutation of the NES increased KHNYN nuclear localization and decreased its interaction with ZAP. The final 2 positions of this NES are not present in fish KHNYN orthologs and we hypothesize their evolution allowed KHNYN to act as a ZAP cofactor. IMPORTANCE The interferon system is part of the innate immune response that inhibits viruses and other pathogens. This system emerged approximately 500 million years ago in early vertebrates. Since then, some genes have evolved to become antiviral interferon-stimulated genes (ISGs) while others evolved so their encoded protein could interact with proteins encoded by ISGs and contribute to their activity. However, this remains poorly characterized. ZAP is an ISG that arose during tetrapod evolution and inhibits viral replication. Because KHNYN interacts with ZAP and is required for its antiviral activity against retroviruses, we conducted an evolutionary analysis to determine how specific amino acids in KHNYN evolved after ZAP emerged. This identified a nuclear export signal that evolved in tetrapods and is required for KHNYN to traffic in the cell and interact with ZAP. Overall, specific residues in KHNYN evolved to allow it to act as a cofactor for ZAP antiviral activity.

Real-Time Whole Genome Sequencing to Guide Patient-Tailored Therapy of Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

The management of coronavirus disease 2019 has become more complex due to the expansion of available therapies. The presence of severe acute respiratory syndrome coronavirus 2 variants and mutations further complicates treatment due to their differing susceptibilities to therapies. Here we outline the use of real-time whole genome sequencing to detect persistent infection, evaluate for mutations confering resistance to treatments, and guide treatment decisions.

The P681H Mutation in the Spike Glycoprotein of the Alpha Variant of SARS-CoV-2 Escapes IFITM Restriction and Is Necessary for Type I Interferon Resistance.

The appearance of new dominant variants of concern (VOC) of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) threatens the global response to the coronavirus disease 2019 (COVID-19) pandemic. Of these, the alpha variant (also known as B.1.1.7), which appeared initially in the United Kingdom, became the dominant variant in much of Europe and North America in the first half of 2021. The spike (S) glycoprotein of alpha acquired seven mutations and two deletions compared to the ancestral virus, including the P681H mutation adjacent to the polybasic cleavage site, which has been suggested to enhance S cleavage. Here, we show that the alpha spike protein confers a level of resistance to beta interferon (IFN-ß) in human lung epithelial cells. This correlates with resistance to an entry restriction mediated by interferon-induced transmembrane protein 2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is essential for resistance to IFN-ß and context-dependent resistance to IFITMs in the alpha S. P681H reduces dependence on endosomal cathepsins, consistent with enhanced cell surface entry. However, reversion of H681 does not reduce cleaved spike incorporation into particles, indicating that it exerts its effect on entry and IFN-ß downstream of furin cleavage. Overall, we suggest that, in addition to adaptive immune escape, mutations associated with VOC may well also confer a replication and/or transmission advantage through adaptation to resist innate immune mechanisms. IMPORTANCE Accumulating evidence suggests that variants of concern (VOC) of SARS-CoV-2 evolve to evade the human immune response, with much interest focused on mutations in the spike protein that escape from antibodies. However, resistance to the innate immune response is essential for efficient viral replication and transmission. Here, we show that the alpha (B.1.1.7) VOC of SARS-CoV-2 is substantially more resistant to type I interferons than the parental Wuhan-like virus. This correlates with resistance to the antiviral protein IFITM2 and enhancement by its paralogue IFITM3. The key determinant of this is a proline-to-histidine change at position 681 in S adjacent to the furin cleavage site, which in the context of the alpha spike modulates cell entry pathways of SARS-CoV-2. Reversion of the mutation is sufficient to restore interferon and IFITM2 sensitivity, highlighting the dynamic nature of the SARS CoV-2 as it adapts to both innate and adaptive immunity in the humans.

TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction.

Ebola virus (EBOV) causes highly pathogenic disease in primates. Through screening a library of human interferon-stimulated genes (ISGs), we identified TRIM25 as a potent inhibitor of EBOV transcription-and-replication-competent virus-like particle (trVLP) propagation. TRIM25 overexpression inhibited the accumulation of viral genomic and messenger RNAs independently of the RNA sensor RIG-I or secondary proinflammatory gene expression. Deletion of TRIM25 strongly attenuated the sensitivity of trVLPs to inhibition by type-I interferon. The antiviral activity of TRIM25 required ZAP and the effect of type-I interferon was modulated by the CpG dinucleotide content of the viral genome. We find that TRIM25 interacts with the EBOV vRNP, resulting in its autoubiquitination and ubiquitination of the viral nucleoprotein (NP). TRIM25 is recruited to incoming vRNPs shortly after cell entry and leads to dissociation of NP from the vRNA. We propose that TRIM25 targets the EBOV vRNP, exposing CpG-rich viral RNA species to restriction by ZAP.

Homebrew: Protocol for glassmilk-based nucleic-acid extraction for SARS-CoV-2 diagnostics.

The gold standard protocol for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection detection remains reverse transcription quantitative polymerase chain reaction (qRT-PCR), which detects viral RNA more sensitively than any other approach. Here, we present Homebrew, a low-cost protocol to extract RNA using widely available reagents. Homebrew is as sensitive as commercially available RNA extraction kits. Homebrew allows for sample pooling and can be adapted for automation in high-throughput settings. For complete details on the use and execution of this protocol, please refer to Page et al. (2022).

Homebrew: An economical and sensitive glassmilk-based nucleic-acid extraction method for SARS-CoV-2 diagnostics.

Management of COVID-19 and other epidemics requires large-scale diagnostic testing. The gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains reverse transcription quantitative PCR (qRT-PCR) analysis, which detects viral RNA more sensitively than any other method. However, the resource use and supply-chain requirements of RT-PCR have continued to challenge diagnostic laboratories worldwide. Here, we establish and characterize a low-cost method to detect SARS-CoV-2 in clinical combined nose and throat swabs, allowing for automation in high-throughput settings. This method inactivates virus material with sodium dodecylsulfate (SDS) and uses silicon dioxide as the RNA-binding matrix in combination with sodium chloride (NaCl) and isopropanol. With similar sensitivity for SARS-CoV-2 viral targets but a fraction of time and reagent expenditure compared with commercial kits, our method also enables sample pooling without loss of sensitivity. We suggest that this method will facilitate more economical widespread testing, particularly in resource-limited settings.

Disrupted Peyer's Patch Microanatomy in COVID-19 Including Germinal Centre Atrophy Independent of Local Virus.

Confirmed SARS-coronavirus-2 infection with gastrointestinal symptoms and changes in microbiota associated with coronavirus disease 2019 (COVID-19) severity have been previously reported, but the disease impact on the architecture and cellularity of ileal Peyer's patches (PP) remains unknown. Here we analysed post-mortem tissues from throughout the gastrointestinal (GI) tract of patients who died with COVID-19. When virus was detected by PCR in the GI tract, immunohistochemistry identified virus in epithelium and lamina propria macrophages, but not in lymphoid tissues. Immunohistochemistry and imaging mass cytometry (IMC) analysis of ileal PP revealed depletion of germinal centres (GC), disruption of B cell/T cell zonation and decreased potential B and T cell interaction and lower nuclear density in COVID-19 patients. This occurred independent of the local viral levels. The changes in PP demonstrate that the ability to mount an intestinal immune response is compromised in severe COVID-19, which could contribute to observed dysbiosis.

Combined epidemiological and genomic analysis of nosocomial SARS-CoV-2 infection early in the pandemic and the role of unidentified cases in transmission.

OBJECTIVES: To analyse nosocomial transmission in the early stages of the coronavirus 2019 (COVID-19) pandemic at a large multisite healthcare institution. Nosocomial incidence is linked with infection control interventions. METHODS: Viral genome sequence and epidemiological data were analysed for 574 consecutive patients, including 86 nosocomial cases, with a positive PCR test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the first 19 days of the pandemic. RESULTS: Forty-four putative transmission clusters were found through epidemiological analysis; these included 234 cases and all 86 nosocomial cases. SARS-CoV-2 genome sequences were obtained from 168/234 (72%) of these cases in epidemiological clusters, including 77/86 nosocomial cases (90%). Only 75/168 (45%) of epidemiologically linked, sequenced cases were not refuted by applying genomic data, creating 14 final clusters accounting for 59/77 sequenced nosocomial cases (77%). Viral haplotypes from these clusters were enriched 1-14x (median 4x) compared to the community. Three factors implicated unidentified cases in transmission: (a) community-onset or indeterminate cases were absent in 7/14 clusters (50%), (b) four clusters (29%) had additional evidence of cryptic transmission, and (c) in three clusters (21%) diagnosis of the earliest case was delayed, which may have facilitated transmission. Nosocomial cases decreased to low levels (0-2 per day) despite continuing high numbers of admissions of community-onset SARS-CoV-2 cases (40-50 per day) and before the impact of introducing universal face masks and banning hospital visitors. CONCLUSION: Genomics was necessary to accurately resolve transmission clusters. Our data support unidentified cases-such as healthcare workers or asymptomatic patients-as important vectors of transmission. Evidence is needed to ascertain whether routine screening increases case ascertainment and limits nosocomial transmission.

TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction.

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

S-farnesylation is essential for antiviral activity of the long ZAP isoform against RNA viruses with diverse replication strategies.

The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNAs and, through the recruitment of TRIM25, KHNYN and other cofactors, target them for degradation or prevent their translation. The long and short isoforms of ZAP (ZAP-L and ZAP-S) have different intracellular localization and it is unclear how this regulates their antiviral activity against viruses with different sites of replication. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), which transcribe the viral RNA in the nucleus and assemble virions at the plasma membrane, we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain in ZAP-L is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the residues in place of the catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks antiviral activity for CpG-enriched HIV-1 despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together jointly modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, which replicates in double-membrane vesicles derived from the endoplasmic reticulum. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in ZAP-L-mediated antiviral activity against divergent viruses with different subcellular replication sites.

Minimal impact of ZAP on lentiviral vector production and transduction efficiency.

The antiviral protein ZAP binds CpG dinucleotides in viral RNA to inhibit replication. This has likely led to the CpG suppression observed in many RNA viruses, including retroviruses. Sequences added to retroviral vector genomes, such as internal promoters, transgenes, or regulatory elements, substantially increase CpG abundance. Because these CpGs could allow retroviral vector RNA to be targeted by ZAP, we analyzed whether it restricts vector production, transduction efficiency, and transgene expression. Surprisingly, even though CpG-high HIV-1 was efficiently inhibited by ZAP in HEK293T cells, depleting ZAP did not substantially increase lentiviral vector titer using several packaging and genome plasmids. ZAP overexpression also did not inhibit lentiviral vector titer. In addition, decreasing CpG abundance in a lentiviral vector genome did not increase its titer, and a gammaretroviral vector derived from murine leukemia virus was not substantially restricted by ZAP. Overall, we show that the increased CpG abundance in retroviral vectors relative to the wild-type retroviruses they are derived from does not intrinsically sensitize them to ZAP. Further understanding of how ZAP specifically targets transcripts to inhibit their expression may allow the development of CpG sequence contexts that efficiently recruit or evade this antiviral system.

The origins of SARS-CoV-2: A critical review.

Since the first reports of a novel severe acute respiratory syndrome (SARS)-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the human population. Recent debate has coalesced around two competing ideas: a "laboratory escape" scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2.

Comparative performance of SARS-CoV-2 lateral flow antigen tests and association with detection of infectious virus in clinical specimens: a single-centre laboratory evaluation study.

BACKGROUND: Lateral flow devices (LFDs) for rapid antigen testing are set to become a cornerstone of SARS-CoV-2 mass community testing, although their reduced sensitivity compared with PCR has raised questions of how well they identify infectious cases. Understanding their capabilities and limitations is, therefore, essential for successful implementation. We evaluated six commercial LFDs and assessed their correlation with infectious virus culture and PCR cycle threshold (Ct) values. METHODS: In a single-centre, laboratory evaluation study, we did a head-to-head comparison of six LFDs commercially available in the UK: Innova Rapid SARS-CoV-2 Antigen Test, Spring Healthcare SARS-CoV-2 Antigen Rapid Test Cassette, E25Bio Rapid Diagnostic Test, Encode SARS-CoV-2 Antigen Rapid Test Device, SureScreen COVID-19 Rapid Antigen Test Cassette, and SureScreen COVID-19 Rapid Fluorescence Antigen Test. We estimated the specificities and sensitivities of the LFDs using stored naso-oropharyngeal swabs collected at St Thomas' Hospital (London, UK) for routine diagnostic SARS-CoV-2 testing by real-time RT-PCR (RT-rtPCR). Swabs were from inpatients and outpatients from all departments of St Thomas' Hospital, and from health-care staff (all departments) and their household contacts. SARS-CoV-2-negative swabs from the same population (confirmed by RT-rtPCR) were used for comparative specificity determinations. All samples were collected between March 23 and Oct 27, 2020. We determined the limit of detection (LOD) for each test using viral plaque-forming units (PFUs) and viral RNA copy numbers of laboratory-grown SARS-CoV-2. Additionally, LFDs were selected to assess the correlation of antigen test result with RT-rtPCR Ct values and positive viral culture in Vero E6 cells. This analysis included longitudinal swabs from five infected inpatients with varying disease severities. Furthermore, the sensitivities of available LFDs were assessed in swabs (n=23; collected from Dec 4, 2020, to Jan 12, 2021) confirmed to be positive (RT-rtPCR and whole-genome sequencing) for the B.1.1.7 variant, which was the dominant genotype in the UK at the time of study completion. FINDINGS: All LFDs showed high specificity (≥98·0%), except for the E25Bio test (86·0% [95% CI 77·9-99·9]), and most tests reliably detected 50 PFU/test (equivalent SARS-CoV-2 N gene Ct value of 23·7, or RNA copy number of 3 × 106/mL). Sensitivities of the LFDs on clinical samples ranged from 65·0% (55·2-73·6) to 89·0% (81·4-93·8). These sensitivities increased to greater than 90% for samples with Ct values of lower than 25 for all tests except the SureScreen fluorescence (SureScreen-F) test. Positive virus culture was identified in 57 (40·4%) of 141 samples; 54 (94·7%) of the positive cultures were from swabs with Ct values lower than 25. Among the three LFDs selected for detailed comparisons (the tests with highest sensitivity [Innova], highest specificity [Encode], and alternative technology [SureScreen-F]), sensitivity of the LFDs increased to at least 94·7% when only including samples with detected viral growth. Longitudinal studies of RT-rtPCR-positive samples (tested with Innova, Encode, and both SureScreen-F and the SureScreen visual [SureScreen-V] test) showed that most of the tests identified all infectious samples as positive. Test performance (assessed for Innova and SureScreen-V) was not affected when reassessed on swabs positive for the UK variant B.1.1.7. INTERPRETATION: In this comprehensive comparison of antigen LFDs and virus infectivity, we found a clear relationship between Ct values, quantitative culture of infectious virus, and antigen LFD positivity in clinical samples. Our data support regular testing of target groups with LFDs to supplement the current PCR testing capacity, which would help to rapidly identify infected individuals in situations in which they would otherwise go undetected. FUNDING: King's Together Rapid COVID-19, Medical Research Council, Wellcome Trust, Huo Family Foundation, UK Department of Health, National Institute for Health Research Comprehensive Biomedical Research Centre.

Targeted Restriction of Viral Gene Expression and Replication by the ZAP Antiviral System.

The zinc finger antiviral protein (ZAP) restricts the replication of a broad range of RNA and DNA viruses. ZAP directly binds viral RNA, targeting it for degradation and inhibiting its translation. While the full scope of RNA determinants involved in mediating selective ZAP activity is unclear, ZAP binds CpG dinucleotides, dictating at least part of its target specificity. ZAP interacts with many cellular proteins, although only a few have been demonstrated to be essential for its antiviral activity, including the 3'-5' exoribonuclease exosome complex, TRIM25, and KHNYN. In addition to inhibiting viral gene expression, ZAP also directly and indirectly targets a subset of cellular messenger RNAs to regulate the innate immune response. Overall, ZAP protects a cell from viral infection by restricting viral replication and regulating cellular gene expression. Further understanding of the ZAP antiviral system may allow for novel viral vaccine and anticancer therapy development.

More than the Eye Can See: Shedding New Light on SARS-CoV-2 Lateral Flow Device-Based Immunoassays.

Containing the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented challenge due to high horizontal transmissivity and asymptomatic carriage rates. Lateral flow device (LFD) immunoassays were introduced in late 2020 to detect SARS-CoV-2 infection in asymptomatic or presymptomatic individuals rapidly. While LFD technologies have been used for over 60 years, their widespread use as a public health tool during a pandemic is unprecedented. By the end of 2020, data from studies into the efficacy of the LFDs emerged and showed these point-of-care devices to have very high specificity (ability to identify true negatives) but inadequate sensitivity with high false-negative rates. The low sensitivity (<50%) shown in several studies is a critical public health concern, as asymptomatic or presymptomatic carriers may wrongly be assumed to be noninfectious, posing a significant risk of further spread in the community. Here, we show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a biosample. We quantified significant immobilized antigen-antibody-labeled conjugate complexes within the LFDs visually scored as negative using high-sensitivity synchrotron X-ray fluorescence imaging. Correlating quantitative X-ray fluorescence measurements and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) determined numbers of viral copies, we identified that negatively scored samples could contain up to 100 PFU (equivalent here to ∼10 000 RNA copies/test). The study demonstrates where the shortcomings arise in many of the current direct-readout SARS-CoV-2 LFDs, namely, being a deficiency in the readout as opposed to the potential level of detection of the test, which is orders of magnitude higher. The present findings are of importance both to public health monitoring during the Coronavirus Disease 2019 (COVID-19) pandemic and to the rapid refinement of these tools for immediate and future applications.

Neutralization potency of monoclonal antibodies recognizing dominant and subdominant epitopes on SARS-CoV-2 Spike is impacted by the B.1.1.7 variant.

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.