Abstract
Chronic viral disease constitutes a major global health problem, with several hundred million people affected and an associated elevated number of deaths. An increasing number of disorders caused by human flaviviruses are related to their capacity to establish a persistent infection. Here we show that Usutu virus (USUV), an emerging zoonotic flavivirus linked to sporadic neurologic disease in humans, can establish a persistent infection in cell culture. Two independent lineages of Vero cells surviving USUV lytic infection were cultured over 82 days (41 cell transfers) without any apparent cytopathology crisis associated. We found elevated titers in the supernatant of these cells, with modest fluctuations during passages but no overall tendency towards increased or decreased infectivity. In addition to full-length genomes, viral RNA isolated from these cells at passage 40 revealed the presence of defective genomes, containing different deletions at the 5’ end. These truncated transcripts were all predicted to encode shorter polyprotein products lacking membrane and envelope structural proteins, and most of non-structural protein 1. Treatment with different broad-range antiviral nucleosides revealed that USUV is sensitive to these compounds in the context of a persistent infection, in agreement with previous observations during lytic infections. The exposure of infected cells to prolonged treatment (10 days) with favipiravir and/or ribavirin resulted in the complete clearance of infectivity in the cellular supernatants (decrease of ~5 log10 in virus titers and RNA levels), although modest changes in intracellular viral RNA levels were recorded (<2 log10 decrease). Drug withdrawal after treatment day 10 resulted in a relapse in virus titers. These results encourage the use of persistently-infected cultures as a surrogate system in the identification of improved antivirals against flaviviral chronic disease.
Highlights
Chronic viral infections constitute a major challenge to global public health, with several hundred million people affected and significant associated fatalities [1,2,3,4,5,6]
Further evidence supporting an association between chronic disease and persistent kidney infection has been obtained with animal models for West Nile virus (WNV) and other flaviviruses [12,14,15,16,17]
In V cells, we identified three bands of different sizes in amplifications of the 5’ end entire USUV genome isolated from persistently-infected cells after 80 days
Summary
Chronic viral infections constitute a major challenge to global public health, with several hundred million people affected and significant associated fatalities [1,2,3,4,5,6]. Viral persistence in the host generally leads to different pathogenic outcomes including the exacerbation of medical conditions [7]. Apart from their direct relationship with disease, viral persistence in animal reservoirs has been linked to the re-emergence of pathogenic viruses [7]. Besides severe neurological disorders linked to acute infection (e.g., Guillain–Barré in adults and congenital brain defects in neonates), Zika virus (ZIKV) establishes persistence in different cells and tissues, leading to an array of other medical conditions [9,18,19].
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