br Material and Methods br Results br Discussion In

Material and Methods
Results
Discussion In this study, we show significant differences in the infectivity and cellular response between two Asian and African ZIKV strains. Using a combination of cell and molecular biology approaches, we observed in two human neural cell types (NSCs and astrocytes) a higher infectivity for the African strain. Similarly to the accumulating evidences reporting infection of neural stem or precursor cells by ZIKV, we report that both ZIKV AF and AS can infect IPSc-derived NSCs. However, ZIKV AF led to a stronger modulation of cellular homeostasis, in term of effects on cell cycle progression and anti-viral response. We also report that both strains of ZIKV can infect human astrocytes, opening new questions on the pathogenesis of this Flavivirus in the developing and adult cystic fibrosis chloride channel as this most abundant cell type in the brain is responsible for multiple functions such as BBB maintenance, synaptogenesis and immune regulation among others (Molofsky and Deneen, 2015). The reports of the first suspected cases of microcephaly in Brazil in 2015 (Schuler-Faccini et al., 2016; ECDC, 2015; COES, 2016) led to an unprecedented effort of the scientific community to understand the pathophysiological mechanisms of a virus barely characterized, despite its discovery almost 70years ago. Numerous studies were subsequently published describing the placental transcytosis of ZIKV (Bayer et al., 2016; Miner et al., 2016) and highlighting the strong tropism for neural precursor cells (Tang et al., 2016; Qian et al., 2016; Lazear et al., 2016; Dang et al., 2016; Cugola et al., 2016). These observations, together with cohort studies and the demonstration of viral shedding in the amniotic fluid and in fetal tissue (brain included), provide strong conclusions regarding the link between ZIKV infection and microcephaly. Retrospective studies in French Polynesia also reported neurodevelopmental disorders associated with ZIKV infection (Besnard et al., 2016), suggesting that this feature of ZIKV was not specific to the Brazilian epidemic. Regarding Africa, due to the poor surveillance and traceability in the human health system, it is difficult to apprehend and predict the circulation of this virus (Meda et al., 2016). However, Cape Verde has reported an outbreak and several cases were recently found in Guinea-Bissau (both due to ZIKV of the Asian lineage) in 2015–2016. Occasional studies or reports show that ZIKV AF strains are currently circulating and support the need to closely monitor ZIKV AF (Baraka and Kweka, 2016; Grard et al., 2014; Van Esbroeck et al., 2016). Surprisingly, we repeatedly detected a stronger infectivity, viral production and cellular response of the ZIKV AF strain ArB41644 vs ZIKV AS strain H/PF/2013 ex vivo in neural cells. Because there is strong probability that these strains have not derived due to extensive passages (no >5 passages for both strains), our observations could suggest a potential difference in the physiopathology between the two strains. A rather common trait among Flaviviruses is their ability to access the CNS and cause neuronal impairment (Neal, 2014). However, some Flaviviruses can display strain-dependent neurovirulence. For instance, the neuroinvasive potential of West Nile virus (WNV) is strain-dependent but mostly because of difference in their ability to reach the CNS (Beasley et al., 2002). However, in some case this difference could also be due to different replication abilities in cells of the BBB or directly in neural cells, as was shown for WNV (Hussmann et al., 2013) and the Alphavirus Semliki Forest virus (Fazakerley et al., 2002). One mechanism responsible for difference in neurovirulence is the glycosylation status of the envelope protein (Kawano et al., 1993; Shirato et al., 2004). Crystal structure of ZIKV demonstrated that its envelop protein contains a glycosylation site at Asn154 (Dai et al., 2016; Kostyuchenko et al., 2016), similarly to the site responsible for neuroinvasiveness of WNV (Beasley et al., 2005). Moreover, passage history likely influenced glycosylation sites in the (original) MR-766 strain (Haddow et al., 2012). This observation, among other critics such as number and way of passages, are suggesting that MR-766 may not be the ideal African reference strain. In this context, a study described a stronger replication in a Brazilian ZIKV strain in NPCs as compared to the MR-766 strain, which therefore appears to have less capacity of replication than the low passage African strain used in our study (Cugola et al., 2016).