Abstract
Zika virus (ZIKV) emerged in the Americas in 2015, presenting unique challenges to public health. Unlike other arboviruses of the Flaviviridae family, it is transmissible by sexual contact, which facilitates the spread of the virus into new geographic areas. Additionally, ZIKV can be transmitted from mother to fetus, causing microcephaly and other severe developmental abnormalities. Reliable and easy-to-work-with clones of ZIKV expressing heterologous genes will significantly facilitate studies aimed at understanding the virus pathogenesis and tissue tropism. Here, we developed and characterized two novel approaches for expression of heterologous genes of interest in the context of full-length ZIKV genome and compared them to two previously published strategies for ZIKV-mediated gene expression. We demonstrated that among the four tested viruses expressing nLuc gene, the virus constructed using a newly developed approach of partial capsid gene duplication (PCGD) attained the highest titer in Vero cells and resulted in the highest level of nLuc expression. Suitability of the PCGD approach for expression of different genes of interest was validated by replacing nLuc sequence with that of eGFP gene. The generated constructs were further characterized in cell culture. Potential applications of ZIKV clones stably expressing heterologous genes include development of detection assays, antivirals, therapeutics, live imaging systems, and vaccines.
Highlights
Zika virus (ZIKV) is a mosquito-borne virus from the Flaviviridae family, genus Flavivirus
We showed that the duplicated capsid gene region (dCGR), but not the E/NS1 region, is the most suitable site in the ZIKV genome for heterologous gene insertion and that the combination of codon optimization and frame shifting mutations is necessary and sufficient to prevent the ejection of the reporter gene during prolonged cell passaging
Since the objective of this study was to develop ZIKV clones expressing gene(s) of interest in the region of capsid gene duplication, we began our analysis by mapping the region containing regulatory elements of the C gene of ZIKV
Summary
ZIKV is a mosquito-borne virus from the Flaviviridae family, genus Flavivirus. Since its discovery in 1947 in Uganda, the virus has remained relatively dormant throughout the most of the 20th century.in the first decade of the 21st century, ZIKV reemerged in a series of large-scale outbreaks on several islands in the Pacific Ocean. The follow-up studies of the apparently unaffected babies born from the mothers who contracted ZIKV during pregnancy indicated a high risk for development of neurological abnormalities later in life (reviewed in [3,4]). This information, coupled with the fact that ZIKV is able to be transmitted through non-vector routes of infection (sexual, intrauterine, perinatal), makes research of ZIKV pathogenesis and development of vaccines, antiviral therapeutics, and sensitive and specific assays for detection of the virus a high priority [5,6]
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