Abstract
Zika virus (ZIKV) is a mosquito-borne Flavivirus. Previous studies have shown that mosquito-transmitted flaviviruses, including yellow fever, Japanese encephalitis, and West Nile viruses, could be attenuated by serial passaging in human HeLa cells. Therefore, it was hypothesized that wild-type ZIKV would also be attenuated after HeLa cell passaging. A human isolate from the recent ZIKV epidemic was subjected to serial HeLa cell passaging, resulting in attenuated in vitro replication in both Vero and A549 cells. Additionally, infection of AG129 mice with 10 plaque forming units (pfu) of wild-type ZIKV led to viremia and mortality at 12 days, whereas infection with 103 pfu of HeLa-passage 6 (P6) ZIKV led to lower viremia, significant delay in mortality (median survival: 23 days), and increased cytokine and chemokine responses. Genomic sequencing of HeLa-passaged virus identified two amino acid substitutions as early as HeLa-P3: pre-membrane E87K and nonstructural protein 1 R103K. Furthermore, both substitutions were present in virus harvested from HeLa-P6-infected animal tissue. Together, these data show that, similarly to other mosquito-borne flaviviruses, ZIKV is attenuated following passaging in HeLa cells. This strategy can be used to improve understanding of substitutions that contribute to attenuation of ZIKV and be applied to vaccine development across multiple platforms.
Highlights
Beginning in 2015, a Zika epidemic spread throughout the Americas, leading to congenital Zika syndrome
Zika virus (ZIKV) strain FSS13025 failed to adapt for growth in human cervical cancer (HeLa) cells (Figure 1b), and was not recovered during the passage series
Studies of empirically-derived live-attenuated vaccine candidates for related flaviviruses, such as the yellow fever (YFV) 17D and JEV SA14-14-2 vaccines, have shown that multiple attenuating mutations may be introduced into the genome as a result of passaging
Summary
Beginning in 2015, a Zika epidemic spread throughout the Americas, leading to congenital Zika syndrome. There is no licensed ZIKV vaccine, and candidate vaccines are in pre-clinical and clinical development [1,2]. ZIKV belongs to the Flaviviridae family, which includes other medically important mosquito-transmitted viruses such as yellow fever (YFV), dengue, Japanese encephalitis (JEV), and West Nile (WNV) viruses. Flaviviruses contain a positive-sense, single-stranded RNA genome that encodes a single polyprotein that is processed into 10 viral proteins. (pre)-membrane, and envelope—are present in the infectious, mature virion. Seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) have functions in viral replication
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