Replication in the presence of dengue convalescent serum impacts Zika virus neutralization sensitivity and fitness

Abstract

IntroductionFlaviviruses like dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne viruses that cause febrile, hemorrhagic, and neurological diseases in humans, resulting in 400 million infections annually. Due to their co-circulation in many parts of the world, flaviviruses must replicate in the presence of pre-existing adaptive immune responses targeted at serologically closely related pathogens, which can provide protection or enhance disease. However, the impact of pre-existing cross-reactive immunity as a driver of flavivirus evolution, and subsequently the implications on the emergence of immune escape variants, is poorly understood. Therefore, we investigated how replication in the presence of convalescent dengue serum drives ZIKV evolution.MethodsWe used an in vitro directed evolution system, passaging ZIKV in the presence of serum from humans previously infected with DENV (anti-DENV) or serum from DENV-naïve patients (control serum). Following five passages in the presence of serum, we performed next-generation sequencing to identify mutations that arose during passaging. We studied two non-synonymous mutations found in the anti-DENV passaged population (E-V355I and NS1-T139A) by generating individual ZIKV mutants and assessing fitness in mammalian cells and live mosquitoes, as well as their sensitivity to antibody neutralization.Results and discussionBoth viruses had increased fitness in Vero cells with and without the addition of anti-DENV serum and in human lung epithelial and monocyte cells. In Aedes aegypti mosquitoes—using blood meals with and without anti-DENV serum—the mutant viruses had significantly reduced fitness compared to wild-type ZIKV. These results align with the trade-off hypothesis of constrained mosquito-borne virus evolution. Notably, only the NS1-T139A mutation escaped neutralization, while E-V335I demonstrated enhanced neutralization sensitivity to neutralization by anti-DENV serum, indicating that neutralization escape is not necessary for viruses passaged under cross-reactive immune pressures. Future studies are needed to assess cross-reactive immune selection in humans and relevant animal models or with different flaviviruses.

Widespread exposure to SARS-CoV-2 in wildlife communities

“…Serum samples were then heat inactivated at 55°C for 1 hour. Plaque reduction neutralization assays (PRNTs) were performed similarly to prior reports 52,53 . Briefly, samples were diluted 1:10 in RPMI-1640 with 10 mM HEPES and 2% FetalPure bovine serum (Genesee Scientific 25-525H).…”

“…Serum samples were then heat inactivated at 55°C for 1 hour. Plaque reduction neutralization assays (PRNTs) were performed similarly to prior reports 52,53 .…”

Experimental SARS-CoV-2 Infection of Elk and Mule Deer

“…We then sequenced the isolate by using a next-generation pipeline and detected a single synonymous consensus change at amino acid 410 in nonstructural protein 14 (C to T transition) (GenBank accession no. OR758451) ( 14 ). We then intranasally inoculated 2 animals/room with 3.7–4.5 log 10 PFU of virus diluted in phosphate-buffered saline (confirmed by back-titration of inoculum on Vero cells); the third animal in each room served as a contact.…”

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“…Viral RNA was isolated from infectious clones of Zika virus. 34 Viral supernatants were treated with benzonase (250 units per mL) diluted in 10× benzonase buffer 35,36 and RNAse A (250 units per mL) to remove host nucleic acids. 37 Samples were then incubated for 37 °C for three hours.…”