Zika virus infection perturbs osteoblast function

Abstract

Zika virus (ZIKV) infection is typically characterized by a mild self-limiting disease presenting with fever, rash, myalgia and arthralgia and severe fetal complications during pregnancy such as microcephaly, subcortical calcifications and arthrogyropsis. Virus-induced arthralgia due to perturbed osteoblast function has been described for other arboviruses. In case of ZIKV infection, the role of osteoblasts in ZIKV pathogenesis and bone related pathology remains unknown. Here, we study the effect of ZIKV infection on osteoblast differentiation, maturation and function by quantifying activity and gene expression of key biomarkers, using human bone marrow-derived mesenchymal stromal cells (MSCs, osteoblast precursors). MSCs were induced to differentiate into osteoblasts and we found that osteoblasts were highly susceptible to ZIKV infection. While infection did not cause a cytopathic effect, a significant reduction of key osteogenic markers such as ALP, RUNX2, calcium contents and increased expression of IL6 in ZIKV-infected MSCs implicated a delay in osteoblast development and maturation, as compared to uninfected controls. In conclusion, we have developed and characterized a new in vitro model to study the role of bone development in ZIKV pathogenesis, which will help to identify possible new targets for developing therapeutic and preventive measures.

Figure 1

Replication of ZIKV in primary osteoblasts. Culture supernatant was collected at different time points after infection of primary osteoblasts by ZIKV (moi = 5). (a) Growth curve kinetics of ZIKV infection in osteoblasts from Donor 4266 (closed circles) and Donor 3520 (open squares) during differentiation over the period of 3 weeks. Error bars represent the standard error of mean (S.E.M). (b) Representative immunofluorescent images of ZIKV-infected cells stained for ZIKV antigen (green) and nuclei (blue), and (c) uninfected controls at day 4 post-infection. Magnification 200x.

Figure 2

Effect of ZIKV infection on osteoblast differentiation and maturation. Effect of ZIKV infection on osteoblast differentiation is measured by alkaline phosphatase (ALP) activity in cultures from (a) Donor 4266 and (b) Donor 3520, and effect on mineralization is measured as a concentration of calcium present in the cultures from c) Donor 4266 and (d) Donor 3520. Results are compared between ZIKV -infected (white bars) and uninfected controls (black bars) and ALP levels are normalized against total protein. Error bars represent the standard error of mean. *p < 0.05.

Figure 3

Gene expression levels of analysed genes after ZIKV infection. Gene expression of key transcription factors from ZIKV infected osteoblasts (white bars) versus uninfected controls (black bars) in (Left panel) Donor 4266 and (Right panel) Donor 3520. Gene expression was corrected for house keeping gene, GAPDH. Error bars represent the standard error of mean. *p < 0.05.