RESUMO
The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Marcação de Genes/métodos , Estabilidade de RNA , Vírus de RNA/enzimologia , RNA Viral/metabolismo , Células A549 , Animais , Proteínas Associadas a CRISPR/genética , Chlorocebus aethiops , Cães , Escherichia coli/enzimologia , Escherichia coli/genética , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Vírus de RNA/genética , RNA Viral/genética , Células VeroRESUMO
Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests.
Assuntos
Filogenia , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia , Zika virus/genética , Zika virus/isolamento & purificação , Animais , Brasil/epidemiologia , Colômbia/epidemiologia , Culicidae/virologia , Surtos de Doenças/estatística & dados numéricos , Genoma Viral/genética , Mapeamento Geográfico , Honduras/epidemiologia , Humanos , Metagenoma/genética , Epidemiologia Molecular , Mosquitos Vetores/virologia , Mutação , Vigilância em Saúde Pública , Porto Rico/epidemiologia , Estados Unidos/epidemiologia , Zika virus/classificação , Zika virus/patogenicidade , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/epidemiologiaRESUMO
In one patient over time, we found that concentration of Ebola virus RNA in semen during recovery is remarkably higher than blood at peak illness. Virus in semen is replication-competent with no change in viral genome over time. Presence of sense RNA suggests replication in cells present in semen.