Background

Zika virus (ZIKV), a mosquito-borne Flavivirus within the Flaviviridae virus family, was first isolated from a sentinel Rhesus monkey in 1947 in the Ziika Forest in Uganda and is closely related to dengue virus (DENV) (World Health Organization, 2018). Since then, it became famous in the last decade for its outbreaks in some Pacific islands (2007 and 2013) and in the Americas (2015). Symptomatic patients (25%-20% of the cases) may usually show rather mild clinical manifestations such as fevers, rashes, conjunctivitis, muscle/joint pains and/or headaches. However, since ZIKV re-emergence, severe neurological symptoms including (but not restricted to) Guillain-Barre syndrome in adults and congenitally transmitted newborn microcephaly were identified in infected individuals (Lazear and Diamond, 2016; Grubaugh et al., 2018). Unfortunately, there are currently no available treatments or vaccines against Zika virus, and this is partly due to our poor understanding of its biology.

In terms of phylogeny, there are 2 distinct lineages of Zika virus (Haddow et al., 2012). The so-called “historical” African lineage including the prototype strain MR766 and the “contemporary” Asian lineage derived from the African lineage after decades of mutations. For instance, the Asian strain H/PF/2013 is responsible for the outbreak in French Polynesia in 2013. Similarly, the virus strain responsible for the Brazilian outbreak also phylogenetically derives from the Asian lineage. Indeed, experiments in mouse and mosquito infection models support the idea that the Asian strain has acquired several key mutations which led to microcephaly in infected newborns in Brazil (Yuan et al., 2017) and to enhanced viral infectivity in the arthropod transmission vector Aedes aegypti (Liu et al., 2017).

Upon entry in the host cell, the viral genome, a positive single-stranded RNA, is translated into a single polyprotein at the endoplasmic reticulum (ER), which is subsequently cleaved into 3 structural proteins and 7 non-structural proteins by host and viral proteases. After replication, the genome is encapsidated into neosynthesized virions which are subsequently released from the cell (Neufeldt et al., 2018). Like all other tested flaviviruses, ZIKV remodels the endomembranes of the infected cell to generate a cytoplasmic environment which is favorable to ZIKV life cycle (Cortese et al., 2017). These membranous compartments, called viral replication factories are all ER-derived and can be sub-divided into three classes of ultrastructures (Cortese et al., 2017): 1. vesicle packets, which are ER invaginations believed to be the site of viral RNA replication, 2. virus bags in which assembled immature viruses accumulate in regular arrays, and 3. convoluted membranes whose roles remain unclear although it was proposed for DENV that they modulate host processes for the benefit of replication (Chatel-Chaix et al., 2016).

Before 2016, little was known about the general biology of ZIKV, and most of our knowledge relied on a direct transposition of fundamental discoveries about other flaviviruses closely related to this pathogen like DENV. However, it remains unclear what makes ZIKV so unique in terms of neuropathogenesis as compared to the other members of the Flavivirus genus. Hence, since ZIKV re-emergence and the recent outbreaks, the scientific community around the world has begun to decipher the mysteries surrounding this virus and to develop tools such as molecular clones as well as cell culture and animal models (Schwarz et al., 2016; Shan et al., 2016; Xie et al., 2016; Morrison and Diamond, 2017; Mutso et al., 2017; Munster et al., 2018). Knowing how to culture ZIKV and to measure its infectivity constitutes key methods to perform any descriptive or functional studies about this virus both in cellulo and in vivo. Moreover, given that Asian and African lineages may differ in terms of symptom severity in infected mouse fetuses (Cugola et al., 2016), it is sometimes relevant to compare model strains of these two lineages. In this bio-protocol, we describe basic cell culture methods to produce ZIKV stocks from Asian and African lineages and to quantify their concentration in infectious virus particles (more generally referred to as “titer”) as well as to image this pathogen inside infected cells using immunofluorescence-based microscopy.