A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

Abstract

An arthropod-borne virus, Zika virus (ZIKV), has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics.

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone

“…Toward that end, several reverse genetics systems have been developed to support these critical research needs. Interestingly, virus rescued from these systems has been found to be fairly significantly attenuated in cell cultures, mice, and other systems (16,17). Although the reasons for this rather severe attenuation are not fully clear at present, an important characteristic of infectious clones is the degree to which they mimic critical phenotypes of wild-type uncloned virus.…”

“…Mouse models for ZIKV neuropathogenesis and in utero transmission have recently been reported in mice deficient in innate immune responses by using African and Asian genotype isolates (12)(13)(14)(15), though there have not been in vivo pathogenesis studies using an isolate from the American outbreak. In addition, infectious clones for Asian genotype ZIKV strains have recently been described, including one from a virus circulating in the American outbreak (16,17). These clones, however, are attenuated in vitro and in vivo, somewhat limiting their utility for certain types of studies.…”

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

“…We set out to examine if N-linked glycosylation within the VNDT motif plays any role in ZIKV pathogenicity in an immunocompromised-mouse model. Although infectious clones of MR766 viruses (25,28,29), as well as several other ZIKV isolates (26,27,47), have been reported, the role of VNDT or glycosylation in this region in pathogenicity has not been explored. A recent study using ectopic expression and pseudoparticle formation with a WNV replicon showed that N-glycosylation of ZIKV E protein is necessary for efficient particle assembly and infectivity (48).…”

“…In fact, a recent study has shown that a single amino acid substitution (A188V) in the viral NS1 protein is critical for NS1 antigenemia, which in turn facilitates ZIKV acquisition by mosquitoes from viremic animals (24). With the availability of infectious clones of ZIKV (25)(26)(27)(28)(29)47) from historical and contemporary isolates, it will be possible to further examine the specific changes in the viral genome that are responsible for viral transmission, replication, virulence, and tissue tropism.…”

“…We consistently observed deletion of 1 or 2 nucleotides at positions around 2800 or 3500 in fragment A (within the NS1 coding region) of all (about 20) examined clones. Attempts to repair the deletion at one site resulted in deletion at the other site, indicating sequence instability around this region, as has been reported (25,26). Strategies such as insertion of introns into the viral genome (25,26) or maintaining the genome in multiple small fragments (27-31) have been used to overcome the inherent instability of the ZIKV and other flavivirus genomes.…”