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Influenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we 35 performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types 40 and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection.45 50

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word count: 165 words 21 Main text word count: 5,097 words 22 Abstract 23 Encephalomyocarditis virus (EMCV) is a picornavirus that produces lytic 24 infections in murine and human cells. Employing a genome-wide CRISPR-Cas9 25 knockout screen to find host factors required for EMCV infection, we identified a role for 26 ADAM9 in EMCV infection. CRISPR-mediated deletion of ADAM9 in multiple human cell 27 lines rendered the cells highly resistant to EMCV infection and cell death. Primary 28 fibroblasts from ADAM9 KO mice were also strongly resistant to EMCV infection and cell 29 death. In contrast, ADAM9 KO and WT cells were equally susceptible to infection with 30 other viruses, including the picornavirus Coxsackie virus B. ADAM9 KO cells failed to 31 produce viral progeny when incubated with EMCV. However, bypassing EMCV entry into 32 cells through delivery of viral RNA directly to the cytosol yielded infectious EMCV virions 33 from ADAM9 KO cells, suggesting that ADAM9 is not required for EMCV replication 34 post-entry. These findings establish that ADAM9 is required for the early stage of EMCV 35 infection, likely for virus entry or viral genome delivery to the cytosol. 36 Importance 37 Viral myocarditis is a leading cause of death in the U.S., contributing to 38 numerous unexplained deaths in people ≤ 35 years old. Enteroviruses contribute to 39 many cases of human myocarditis. Encephalomyocarditis virus (EMCV) infection causes 40 viral myocarditis in rodent models but its receptor requirements have not been fully 41 identified. CRISPR-Cas9 screens can identify host dependency factors essential for 42 EMCV infection and enhance our understanding of key events that follow viral infection, 43 potentially leading to new strategies for preventing viral myocarditis. Using a CRISPR-44 Cas9 screen, we identified A Disintegrin and Metalloproteinase 9 Domain (ADAM9) as a 45 major factor required for the early stages of EMCV infection in both human and murine 46 infection. 47 Results 90 CRISPR-Cas9 Screening Identifies EMCV Dependency Factors (EDFs) 91EMCV infection is rapidly lytic in human and murine cells (51-54). We took advantage of 92 the high lytic potential of EMCV and the power of CRISPR genetic screening (53, 55) to 93 discover virus-host interaction genes that mediated virus infection and, thus, rendered 94 the cells susceptible to EMCV-induced cell death. HeLa cells stably expressing Cas9 95 were used for screening (53, 55). In initial optimization experiments, we determined that 96 HeLa cells were killed by EMCV within 24 h of infection at a multiplicity of infection (MOI) 97 ≥ 0.1. The rapid lysis of HeLa cells with EMCV infection allowed us to screen for EDFs 98 using pooled single-guide RNAs (sgRNAs) since we could identify such mutant cells by 99 their resistance to EMCV-induced cell death, i.e., these mutants would no longer be 100 susceptible to EMCV infection and would survive EMCV challenge. 101We screened for EDFs using CRISPR-Cas9 pooled human gene screen (Fig. 1). 102 H1-HeLa cells which stably exp...