Zika Virus Infection Activates Proinflammatory Cytokines and Triggers Monocyte Differentiation

Abstract

In 2015, Zika virus (ZIKV) outbreak in several countries of South America was linked to microcephaly in newborns. Microcephaly, a severe clinical presentation, was first diagnosed in association with ZIKV, which primarily considered self-limiting infection. From infected keratinocytes, virus disseminates crossing blood-tissue barriers and is detected in various tissues including brain, muscles and placenta. One of the mechanisms utilized by viruses to cross tissue barriers involves leukocytes, where monocytes are often identified as the carriers. Recently, circulating monocytes were shown to harbor ZIKV, suggesting that these leukocyte populations could serve as vehicle to transport ZIKV to the fetus. We speculate that ZIKV infection could promote monocyte activation and release of cytokines causing tissue damage. However, little is known about monocyte susceptibility to ZIKV infection. Also, effect of ZIKV infection on transcriptional activation and production remains unclear.Materials and methods. Monocytes were isolated from cord blood buffy coats (University of Colorado Cord Blood Bank, Aurora) using Miltenyi magnetic bead separation kit (CD14 Microbeads; Miltenyi, Auburn, CA). Monocytes were infected with two ZIKV strains, PRVABC59 (South America) and IBH30656 (Nigeria), ATCC (Manassas, VA). Total RNA was extracted and used for Next generation Sequencing (Illustra RNAspin Mini kit (GE Healthcare, Marlborough, MA)). FastQ data generated by the MiSeq (Illumina, San Diego CA) was annotated and the sequence reads were analyzed by CLC workbench 9.0.1 (Qiagen, Germantown, MD) for the detection of viral and cellular genes. Differential expressions of cellular genes based on the Reads Per Kilobase of transcript per Million mapped reads were determined by comparing with mock-infected cells using RNA-seq analysis tool of CLC Workbench. Differentially regulated genes due to ZIKV infection, identified by CLC Workbench, were subjected for determining the deregulated pathways using IPA Ingenuity Pathways analysis tool (Qiagen Germantown, MD). Viral replication was determined by qPCR, Western blot and immunofluorescence. Cytokine activation was analyzed using Bio-Plex Pro Human Cytokine 27-plex Panel (Bio-Rad, Hercules, CA, USA) following manufacturer's instructions.Results. We have shown that South American and Nigerian strains of ZIKV can infect monocytes and actively replicate. Presence of capsid protein and accumulation of viral transcripts was demonstrated in vitro. Infection with PRVABC59 strain changes transcriptional activity of 332 cellular genes, while the Nigerian strain affected the transcription of 521 cellular genes compared to the mock-infected controls. Interestingly, 195 genes were commonly deregulated in cells infected with both strains. Analysis of these commonly affected genes identified multiple pathways including apoptosis, chemotaxis, cell proliferation, inflammation, acute phase response, mitochondria activation. The most amazing observation was upregulation of mitochondrial and inflammasome genes. Mitochondrial genes (MT-ND2, MT-ND5 and MT-ATP8) activated in ZIKV infected monocytes were shown to play a key role in pathogenesis of other neurodegenerative disease.Analysis of cytokine production revealed early activation of proinflammatory cytokines (IL-1β, IL-6 and IL-8) (Table 1). Upregulation of IL-1β by ZIKV infected monocytes is evident for inflammasome activation. Upregulation of IL-6, IL-10 and IL-17α suggested an activation of Th17 pathway in ZIKV infected monocytes. Besides only Nigerian ZIKV strain increased the production of IL-4 in monocytes. IL-4 together with IL-10 could promote Th2 type immune response to ZIKV. Finally, both strains increased production of FGF, while levels of VEGF were increased only in supernatants of PRVABC59 infected monocytes.Conclusions. Human monocytes are susceptible to infection with ZIKV strains, PRVABC59 and IBH30656 and support viral replication. ZIKV infection affects cellular pathways, including mitochondrial and inflammasome ones that could play significant role in pathogenesis. ZIKV infection of monocytes upregulates cytokine production, which can trigger inflammation and leukocyte migration. Analysis of cytokine production by ZIKV infected monocytes suggests activation of Th2 and Th17 leukocytes, supporting the role of this signaling in ZIKV pathogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.