Dou D, Hernández-Neuta I, Wang H, Östbye H, Qian X, Thiele S, Resa-Infante P, Kouassi NM, Sender V, Hentrich K, Mellroth P, Henriques-Normark B, Gabriel G, Nilsson M, Daniels R
Cell Reports 20 (1) 251-263 [2017-07-05; online 2017-07-07]
Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.
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