Porcine epidemic diarrhea virus (PEDV) is a globally distributed alphacoronavirus with economic importance that can cause severe watery diarrhea and even death in piglets. To identify host factors essential for PEDV infection, we performed a genome-wide CRISPR/Cas9 screen in human hepatocellular carcinoma cells (Huh7) using the highly virulent PEDV GIIb strain GDU. Several genes involved in the sialic acid and heparan sulfate biosynthesis pathway and cholesterol metabolism were highly enriched following PEDV selection. We validated that the host factor ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4), which catalyzes the transfer of sialic acid to sugar chains via α2,3-linked linkages, is important for PEDV infection. To systematically investigate the role of sialic acid in PEDV infection, we knocked out genes related to sialic acid synthesis. This led to a reduced abundance of sialic acid on the cell surface, which in turn inhibited PEDV adsorption and internalization. Furthermore, we found that both α2,3-linked and α2,6-linked sialic acids can serve as cellular attachment factors for PEDV. We conducted a glycan microarray screen to determine which sialoglycans are preferred by the PEDV spike protein. The results revealed that PEDV favors binding to α2,3-sialoglycans. Additionally, we found that not only current circulating PEDV strains but also other porcine coronaviruses rely on sialic acid for efficient infection. Collectively, our findings provide insights into critical host factors involved in PEDV infection and demonstrate that disrupting genes involved in sialic acid biosynthesis negatively affects the infectivity of multiple porcine enteric coronaviruses.IMPORTANCEA wide range of viruses utilize sialic acid as receptors. Sialic acid binding may serve as a key determinant of viral host range. Different viruses exhibit distinct preferences for specific types of sialic acid linkages. However, it remains unclear which specific subtypes of sialic acid are utilized during PEDV infection. In this study, we performed CRISPR-based genome-wide knockout screening and identified ST3GAL4 as a key host factor for PEDV infection. Furthermore, we found that both α2,3-linked and α2,6-linked sialic acids can function as attachment factors for PEDV infection. A glycan microarray screen revealed that PEDV S1 shows the strongest binding preference for α2,3-linked and α2,8-linked sialosides. Sialic acids were also implicated in infections by other porcine enteric coronaviruses. Overall, our findings advance our understanding of viral entry mechanisms of PEDV and other swine coronaviruses and may provide avenues for designing antiviral strategies.

Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype.

Porcine deltacoronavirus (PDCoV) is an emerging pathogen that can cause severe diarrhoea and high mortality in suckling piglets. Moreover, evidence of PDCoV infection in humans has raised concerns regarding potential public health risks. To identify potential therapeutic targets for PDCoV, we performed a genome-wide CRISPR/Cas9 library screening to find key host factors important to PDCoV infection. Several host genes in this screen were enriched, including ANPEP, which encodes the PDCoV receptor aminopeptidase N (APN). Furthermore, we discovered C16orf62, also known as the VPS35 endosomal protein sorting factor like (VPS35L), as an important host factor required for PDCoV infection. C16orf62 is an important component of the multiprotein retriever complex involved in protein recycling in the endosomal compartment and its gene knockout led to a remarkable decrease in the binding and internalization of PDCoV into host cells. While we did not find evidence for direct interaction between C16orf62 and the viral s (spike) protein, C16orf62 gene knockout was shown to downregulate APN expression at the cell surface. This study marks the first instance of a genome-wide CRISPR/Cas9-based screen tailored for PDCoV, revealing C16orf62 as a host factor required for PDCoV replication. These insights may provide promising avenues for the development of antiviral drugs against PDCoV infection.

N6-methyladenosine (m6A) is a RNA modification that can regulate post-transcriptional processes including RNA stability, translation, splicing, and nuclear export. In CD4+ lymphocytes, m6A modifications have been demonstrated to play a role in early differentiation processes. The role of m6A in CD4+ T cell activation and effector function remains incompletely understood. To assess the role of m6A in CD4+ T lymphocyte activation and function, we assessed the transcriptome-wide m6A landscape of human primary CD4+ T cells by methylated RNA immunoprecipitation sequencing. Stimulation of the T cells impacted the m6A pattern of hundreds of transcripts including tumor necrosis factor (TNF). m6A methylation was increased on TNF messenger RNA (mRNA) after activation, predominantly in the 3' untranslated region of the transcript. Manipulation of m6A levels in primary human T cells, the directly affected the expression of TNF. Furthermore, we identified that the m6A reader protein YTHDF2 binds m6A-methylated TNF mRNA, and promotes its degradation. Taken together, this study demonstrates that TNF expression in CD4+ T lymphocytes is regulated via m6A and YTHDF2, thereby providing novel insight into the regulation of T cell effector functions.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection in young children and the second leading cause of infant death worldwide. While global circulation has been extensively studied for respiratory viruses such as seasonal influenza, and more recently also in great detail for SARS-CoV-2, a lack of global multi-annual sampling of complete RSV genomes limits our understanding of RSV molecular epidemiology. Here, we capitalise on the genomic surveillance by the INFORM-RSV study and apply phylodynamic approaches to uncover how selection and neutral epidemiological processes shape RSV diversity. Using complete viral genome sequences, we show similar patterns of site-specific diversifying selection among RSVA and RSVB and recover the imprint of non-neutral epidemic processes on their genealogies. Using a phylogeographic approach, we provide evidence for air travel governing the global patterns of RSVA and RSVB spread, which results in a considerable degree of phylogenetic mixing across countries. Our findings highlight the potential of systematic global RSV genomic surveillance for transforming our understanding of global RSV spread.

T cell activation is a highly regulated process, modulated via the expression of various immune regulatory proteins including cytokines, surface receptors and co-stimulatory proteins. N6-methyladenosine (m6A) is an RNA modification that can directly regulate RNA expression levels and it is associated with various biological processes. However, the function of m6A in T cell activation remains incompletely understood. We identify m6A as a novel regulator of the expression of the CD40 ligand (CD40L) in human CD4+ lymphocytes. Manipulation of the m6A 'eraser' fat mass and obesity-associated protein (FTO) and m6A 'writer' protein methyltransferase-like 3 (METTL3) directly affects the expression of CD40L. The m6A 'reader' protein YT521-B homology domain family-2 (YTHDF2) is hypothesized to be able to recognize and bind m6A specific sequences on the CD40L mRNA and promotes its degradation. This study demonstrates that CD40L expression in human primary CD4+ T lymphocytes is regulated via m6A modifications, elucidating a new regulatory mechanism in CD4+ T cell activation that could possibly be leveraged in the future to modulate T cell responses in patients with immune-related diseases.

To the Editors: Respiratory syncytial virus (RSV) is the most common cause of severe lower respiratory tract infection in the first 6 months of life with more than 97% of mortality occurring in low- and middle-income countries (LMICs).1 RSV mortality data from these geographic regions are limited, and if available, they mainly reflect in-hospital deaths resulting in an underestimate of the global burden of fatal RSV. Because of poor access to healthcare and low-quality healthcare, a sizable proportion of RSV-related deaths among infants in LMICs occurs in the community. For most infectious diseases, including influenza, genetic diversity of viruses affects mortality risk.2 Previously, we demonstrated that RSV is a major cause of overall infant mortality in Zambia.3 In the Zambia Pertussis and RSV Infant Mortality Estimation (ZPRIME) study, we measured facility and community RSV deaths among infants in Lusaka, Zambia through a systematic postmortem surveillance project at the University Teaching Hospital morgue. Between August 2017 and 2020, we found that RSV was present in 7% of all deceased infants and 32% of the RSV+ infant deaths occurred in the community. RSV deaths were concentrated in infants younger than 3 months and in infants from densely populated Lusaka townships. The key distinguishing feature of the ZPRIME study, compared with most studies that have measured the impact of RSV, is that all the participants were deceased, and therefore represented the most extreme of infection outcomes. We aimed to establish whether fatal RSV infection is related to specific RSV genetic sequences, or they could reflect nonvirologic factors such as the vulnerability of the infant population and/or ease of access to supportive medical care. To test the former hypothesis, we performed whole-genome sequencing as described previously4 on a subset of nasopharyngeal samples (n = 116) collected under the ZPRIME study resulting in 71 full-genome RSV sequences (success rate of 62.2%). Of these 71 sequences, 62 were subtyped RSV-A and 9 RSV-B. We complemented ZPRIME sequences with publicly available sequences from other African countries (South Africa and Kenya) and with not yet published sequences generated by the INFORM study from 17 countries globally.4 We inferred phylogenetic trees and the migration history for both subtypes in a Bayesian framework (Fig. 1).5,6FIGURE 1.: Phylogenetic reconstruction of ZPRIME postmortem sequences, and sequences from other African and non-African locations. Tips and internal branches are colored according to the most probable reconstructed ancestral state (location). The correspondence between the colors and locations is as in the legend.Here, we demonstrate that infants in Zambia are dying of RSV linked to diverse viral strains that are intermixed across the globe. Clusters of Zambian RSV sequences obtained from postmortem samples were identified throughout the phylogenetic trees, making it highly unlikely that there was a virologic factor involved in mortality (Fig. 1). In terms of global diversity of RSV, we found no single lineage specific for Zambia: Zambian sequences cluster with sequences from elsewhere. Zambian sequences are closely related to South African sequences and to a lesser extent to Kenyan sequences, indicating that RSV strains cocirculate within Africa. We therefore suggest that there does not appear to be anything distinct about the Zambian RSV strains per se compared with other African locations. We found limited local persistence of RSV within African countries, as sequences from African countries also cluster with those obtained at non-African locations. We did not find evidence of molecular nirsevimab resistance among the RSV strains from the ZPRIME study. The Ile206Met:Gln209Arg polymorphism in the nirsevimab binding site of RSV B became globally dominant with a prevalence of 1843 of 2800 (65.8%) among RSV B strains observed between 2015 and 2021.7 This RSV B polymorphism was also highly prevalent (7/9 sequences; 77.8%) in Zambia between 2017 and 2020. In sum, the newly obtained lineages suggest that Zambian RSV is typical of global RSV. Using analysis of viral genetics, we found no evidence supporting viral genetic risk to mortality. This finding is important for understanding the impact of RSV on infant deaths in Africa. RSV in Zambia seems entirely typical. Mortality may not be virus-related, but explained by the poor healthcare system, population (within-host diversity) or both. To date, host factors for RSV mortality have been poorly defined. Our virologic sequence study showed no substantial differences in RSV sequences from Zambia as compared with elsewhere. We therefore conclude that the fatal outcomes in these cases are not explained by genetic factors, but more likely nonvirologic factors, such as challenges in timely access to supportive care as we have documented previously,8 limited availability of supportive treatments at facilities or intrinsic vulnerabilities in the Zambian infant population. Mutation analysis of the nirsevimab binding site showed that currently available immunoprophylaxis strategies may be effective to prevent RSV mortality in LMICs. ACKNOWLEDGMENTS All 71 ZPRIME sequences reported in the manuscript were submitted to GISAID with the following accession numbers: hRSV/A/Zambia/Lusaka-4082/2018, hRSV/A/Zambia/Lusaka-1708/2018, hRSV/A/Zambia/Lusaka-1925/2018, hRSV/A/Zambia/Lusaka-3230/2019, hRSV/A/Zambia/Lusaka-3099/2018, hRSV/A/Zambia/Lusaka-2617/2019, hRSV/A/Zambia/Lusaka-1210/2019, hRSV/A/Zambia/Lusaka-2615/2019, hRSV/A/Zambia/Lusaka-3935/2018, hRSV/A/Zambia/Lusaka-1937/2018, hRSV/A/Zambia/Lusaka-4073/2018, hRSV/A/Zambia/Lusaka-1948/2018, hRSV/A/Zambia/Lusaka-4520/2019, hRSV/A/Zambia/Lusaka-4593/2018, hRSV/A/Zambia/Lusaka-1129/2018, hRSV/B/Zambia/Lusaka-5070/2019, hRSV/A/Zambia/Lusaka-2141/2019, hRSV/A/Zambia/Lusaka-3103/2018, hRSV/A/Zambia/Lusaka-2057/2018, hRSV/A/Zambia/Lusaka-2133/2019, hRSV/A/Zambia/Lusaka-4089/2018, hRSV/A/Zambia/Lusaka-1983/2018, hRSV/A/Zambia/Lusaka-3117/2018, hRSV/A/Zambia/Lusaka-2557/2019, hRSV/A/Zambia/Lusaka-1103/2018, hRSV/A/Zambia/Lusaka-3922/2018, hRSV/A/Zambia/Lusaka-3924/2018, hRSV/A/Zambia/Lusaka-2074/2018, hRSV/B/Zambia/Lusaka-3221/2019, hRSV/A/Zambia/Lusaka-3195/2019, hRSV/B/Zambia/Lusaka-1207/2019, hRSV/A/Zambia/Lusaka-1932/2018, hRSV/A/Zambia/Lusaka-3145/2018, hRSV/A/Zambia/Lusaka-1122/2018, hRSV/A/Zambia/Lusaka-3239/2019, hRSV/A/Zambia/Lusaka-3204/2019, hRSV/A/Zambia/Lusaka-3215/2019, hRSV/B/Zambia/Lusaka-5051/2019, hRSV/A/Zambia/Lusaka-3934/2018, hRSV/A/Zambia/Lusaka-4508/2018, hRSV/B/Zambia/Lusaka-1212/2019, hRSV/A/Zambia/Lusaka-3939/2018, hRSV/A/Zambia/Lusaka-3938/2018, hRSV/B/Zambia/Lusaka-3219/2019, hRSV/A/Zambia/Lusaka-1970/2018, hRSV/A/Zambia/Lusaka-1139/2018, hRSV/A/Zambia/Lusaka-4654/2019, hRSV/A/Zambia/Lusaka-3210/2019, hRSV/A/Zambia/Lusaka-1238/2019, hRSV/A/Zambia/Lusaka-1953/2018, hRSV/A/Zambia/Lusaka-1119/2018, hRSV/A/Zambia/Lusaka-4627/2018, hRSV/A/Zambia/Lusaka-4113/2019, hRSV/A/Zambia/Lusaka-1112/2018, hRSV/B/Zambia/Lusaka-1201/2019, hRSV/A/Zambia/Lusaka-4652/2019, hRSV/B/Zambia/Lusaka-3224/2019, hRSV/B/Zambia/Lusaka-3209/2019, hRSV/A/Zambia/Lusaka-1218/2019, hRSV/A/Zambia/Lusaka-4116/2019, hRSV/A/Zambia/Lusaka-3923/2018, hRSV/A/Zambia/Lusaka-1704/2018, hRSV/A/Zambia/Lusaka-1117/2018, hRSV/A/Zambia/Lusaka-5034/2019, hRSV/A/Zambia/Lusaka-1105/2018, hRSV/A/Zambia/Lusaka-1936/2018, hRSV/A/Zambia/Lusaka-1126/2018, hRSV/A/Zambia/Lusaka-4573/2018, hRSV/A/Zambia/Lusaka-1200/2019, hRSV/A/Zambia/Lusaka-3102/2018, hRSV/A/Zambia/Lusaka-1197/2019.

Respiratory syncytial virus (RSV) is a major health problem. A better understanding of the geographical and temporal dynamics of RSV circulation will assist in tracking resistance against therapeutics currently under development. Since 2015, the field of RSV molecular epidemiology has evolved rapidly with around 20-30 published articles per year. The objective of this systematic review is to identify knowledge gaps in recent RSV genetic literature to guide global molecular epidemiology research. We included 78 studies published between 2015 and 2020 describing 12,998 RSV sequences of which 8,233 (63%) have been uploaded to GenBank. Seventeen (22%) studies were performed in low- and middle-income countries (LMICs), and seven (9%) studies sequenced whole-genomes. Although most reported polymorphisms for monoclonal antibodies in clinical development (nirsevimab, MK-1654) have not been tested for resistance in neutralisation essays, known resistance was detected at low levels for the nirsevimab and palivizumab binding site. High resistance was found for the suptavumab binding site. We present the first literature review of an enormous amount of RSV genetic data. The need for global monitoring of RSV molecular epidemiology becomes increasingly important in evaluating the effectiveness of monoclonal antibody candidates as they reach their final stages of clinical development. We have identified the following three knowledge gaps: whole-genome data to study global RSV evolution, data from LMICs and data from global surveillance programs.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 500 million infections and more than six million deaths worldwide. Although the viral genomes of SARS-CoV-1 and SARS-CoV-2 share high sequence homology, the clinical and pathological features of COVID-19 differ profoundly from those of SARS. It is apparent that changes in viral genes contribute to the increased transmissibility of SARS-CoV-2 and pathology of COVID-19. Cytotoxic T lymphocytes play a key role in the elimination of virus-infected cells, mediated by recognition of virus-derived peptides that are presented on MHC class I molecules. Here, we show that SARS-CoV-2 can interfere with antigen presentation thereby evading immune surveillance. SARS-CoV-2 infection of monkey and human cell lines resulted in reduced cell-surface expression of MHC class I molecules. We identified a single viral gene product, the accessory factor open reading frame 7a (ORF7a), that mediates this effect. ORF7a interacts with HLA class I molecules in the ER, resulting in ER retention or impaired HLA heavy chain (HC) trafficking to the Golgi. Ultimately, these actions result in reduced HLA class I surface expression on infected cells. Whereas ORF7a from SARS-CoV-2 reduces surface HLA class I levels, the homologous ORF7a from the 2002 pandemic SARS-CoV-1 did not, suggesting that SARS-CoV-2 ORF7a acquired the ability to downregulate HLA-I during evolution of the virus. We identified a single amino acid in the SARS-CoV-1 ORF7a luminal domain that, upon mutating to the corresponding SARS-CoV-2 ORF7a sequence, induced a gain-of-function in HLA surface downregulation. By abrogating HLA class I antigen presentation via ORF7a, SARS-CoV-2 may evade host immune responses by inhibiting anti-viral cytotoxic T cell activity, thereby contributing to the pathology of COVID-19.

Background Respiratory syncytial virus (RSV) is a global cause of severe respiratory morbidity and mortality in infants. While preventive and therapeutic interventions are being developed, including antivirals, vaccines and monoclonal antibodies, little is known about the global molecular epidemiology of RSV. INFORM is a prospective, multicenter, global clinical study performed by ReSViNET to investigate the worldwide molecular diversity of RSV isolates collected from children less than 5 years of age. Methods The INFORM study is performed in 17 countries spanning all inhabited continents and will provide insight into the molecular epidemiology of circulating RSV strains worldwide. Sequencing of > 4000 RSV-positive respiratory samples is planned to detect temporal and geographical molecular patterns on a molecular level over five consecutive years. Additionally, RSV will be cultured from a subset of samples to study the functional implications of specific mutations in the viral genome including viral fitness and susceptibility to different monoclonal antibodies. Discussion The sequencing and functional results will be used to investigate susceptibility and resistance to novel RSV preventive or therapeutic interventions. Finally, a repository of globally collected RSV strains and a database of RSV sequences will be created.