Abstract
The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively.
Highlights
The genus Enterovirus belongs to the family of Picornaviridae, and they are classified into 15 species [1]
During the optimization of the bDNA fluorescent ISH (FISH) method, we discovered that the −RNA was barely visible under the confocal microscope, possibly due to the +RNA and −RNA strands making double-stranded RNA intermediates, effectively inhibiting the binding of our RNA probes to the strands
We have shown that the amounts of both +RNA and −RNA are negligible before 3–4 h p.i. (Figures 3 and 4) using ViewRNA FISH and RT-quantitative real-time PCR (qPCR), meaning that the entry and uncoating of the virus particle and the initial translation of the input +RNA strand takes around 3–4 h
Summary
The genus Enterovirus belongs to the family of Picornaviridae, and they are classified into 15 species [1]. Over 280 different enterovirus types can infect humans, and they all belong to the species Enterovirus A-D (≈115 types) or Rhinovirus A-C (≈170 types) according to their molecular and/or antigenic characteristics. Enteroviruses include, for example, coxsackieviruses, polioviruses, rhinoviruses, and echoviruses, and they can cause a range of diseases from minor common colds and rashes to severe conditions such as myocarditis, meningitis, and encephalitis [2]. Some chronic diseases, such as atherosclerosis and type 1 diabetes, have been linked to enteroviruses [3,4]. Some antiviral drugs have been or are being developed against enteroviruses, but none have made it to commercial markets yet
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