Inhibition of type I interferon induction and signalling by mosquito-borne flaviviruses

The Flavivirus genus (Flaviviridae family) contains a number of important human pathogens, including dengue and Zika viruses, which have the potential to cause severe disease. In order to efficiently establish a productive infection in mammalian cells, flaviviruses have developed key strategies to counteract host immune defences, including the type I interferon response. They employ different mechanisms to control interferon signal transduction and effector pathways, and key research generated over the past couple of decades has uncovered new insights into their abilities to actively decrease interferon antiviral activity. Given the lack of antivirals or prophylactic treatments for many flaviviral infections, it is important to fully understand how these viruses affect cellular processes to influence pathogenesis and disease outcome. This review will discuss the strategies mosquito-borne flaviviruses have evolved to antagonise type I interferon mediated immune responses

Full genome sequence and sfRNA interferon antagonist activity of Zika virus from Recife, Brazil

Background: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. Methodology/Principal findings: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. Conclusions/Significance: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions

Zika virus infection leads to demyelination and axonal injury in mature CNS cultures

Understanding how Zika virus (Flaviviridae; ZIKV) affects neural cells is paramount in comprehending pathologies associated with infection. Whilst the effects of ZIKV in neural development are well documented, impact on the adult nervous system remains obscure. Here, we investigated the effects of ZIKV infection in established mature myelinated central nervous system (CNS) cultures. Infection incurred damage to myelinated fibers, with ZIKV-positive cells appearing when myelin damage was first detected as well as axonal pathology, suggesting the latter was a consequence of oligodendroglia infection. Transcriptome analysis revealed host factors that were upregulated during ZIKV infection. One such factor, CCL5, was validated in vitro as inhibiting myelination. Transferred UV-inactivated media from infected cultures did not damage myelin and axons, suggesting that viral replication is necessary to induce the observed effects. These data show that ZIKV infection affects CNS cells even after myelination—which is critical for saltatory conduction and neuronal function—has taken place. Understanding the targets of this virus across developmental stages including the mature CNS, and the subsequent effects of infection of cell types, is necessary to understand effective time frames for therapeutic intervention

Comparison of African and Asian lineage ZIKV protein coding regions.

<p>The mean pairwise identity of all pairs at a given position is indicated by the identity bar; light blue denotes 100% pairwise identity, dark blue highlights positions possessing less than 100% pairwise identity. Positions and quantity of amino acid substitutions are indicated by black bands within grey sequence bars. Sequences 1–37, highlighted yellow, correspond to the outbreak originating in 2015 in South America. Microcephaly, adult mortality and ZIKV PE243 associated sequences are highlighted as previously described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005048#pntd.0005048.g001" target="_blank">Fig 1</a>.</p

Primers used for cloning of ZIKV 3'UTR containing sfRNA.

<p>Primers used for cloning of ZIKV 3'UTR containing sfRNA.</p

The predicted structure of ZIKV PE243 3’UTR.

<p>5’-3’ of the ZIKV PE243 3’UTR sequence, left to right. The arrow indicates the predicted start of sfRNA. Nucleotides are indicated either counted from the 3’ (indicated as negative numbers) or from the start of the 3’UTR (positive number in brackets). SL, stem loop structure; DBL, dumbbell structure; 3’SL; 3’ end stem loop structure. The dotted line represents the predicted pseudoknot.</p

Primers sequences used for 5’/3’ RACE of ZIKV viral termini.

<p>Primers sequences used for 5’/3’ RACE of ZIKV viral termini.</p

Comparison of the 5’UTR nucleotide sequences of Asian and African ZIKV isolates.

<p>The mean pairwise identity of all pairs at a given position is indicated by the identity bar; lilac is indicative of 100% pairwise identity, dark purple highlights positions possessing <100% pairwise identity. Positions and quantity of single nucleotide polymorphisms (SNPs) are represented as black bands within grey sequence bars. Sequences 1–32, highlighted orange, correspond to the outbreak originating in 2015 in Brazil. Microcephaly, adult mortality and ZIKV PE243 associated sequences are highlighted as previously described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005048#pntd.0005048.g001" target="_blank">Fig 1</a>.</p

Bayesian maximum clade credibility tree generated from coding sequence data.

<p>Bayesian posterior probabilities are given at nodes of importance. Isolates which have been implicated in particular diseases are highlighted, as is the ZIKV PE243 isolate we have sequenced. GenBank accession numbers of all sequences used are given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005048#pntd.0005048.s001" target="_blank">S1 Table</a>. EC_2007 refers to the epidemic consensus sequence generated from the Yap Island outbreak in 2007 (EU545988).</p

Activation of the IFN-β promoter by poly I:C in cells over expressing ZIKV sfRNA.

<p>A549 cells were co-transfected with either pDEST-DENV-3’UTR, pDEST-ZIKV PE243-3‘UTR or pDEST40-MBP (sfRNA over-expression plasmids and MBP-HDVr control, respectively) and p125Luc IFN-β promoter reporter (expressing Firefly luciferase) along with pRL-CMV (internal control, expressing <i>Renilla</i> luciferase). The IFN-β promoter was stimulated by transfecting poly I:C 24 h after the primary transfection. The relative luciferase activity (Firefly/<i>Renilla</i>) was analyzed at 24 h following the second transfection. The mean with standard error is shown for three independent experiments performed in triplicate; values of independent experiments were used for analysis. The data were normalized to cells transfected with pDEST40-MBP without any poly I:C treatment. Asterisk (*) indicates significance (2-way ANOVA, p<0.05).</p