Development and characterization of infectious clones of two strains of Usutu virus

Bates, Tyler; Chuong, Christina; Hawks, Seth A.; Rai, Pallavi; Salgado, Rebecca; Duggal, Nisha K.; Weger-Lucarelli, James

doi:10.1101/2020.08.05.238543

Cited by 2 publications

(3 citation statements)

References 39 publications

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“…We assembled the products using the NEB Builder HiFi DNA Assembly Master Mix (E2621L). The assemblies were digested with DpnI (R0176S), Lambda exonuclease (M0262S), and Exonuclease I (M0293S) and amplified using SuperPhi RCA Premix Kit with Random Primers (Evomics catalog number PM100) [59]. As previously described, RCA products were then transfected into HEK293A cells to produce p0 stocks of the virus [51, 60].…”

Section: Methodsmentioning

confidence: 99%

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

Marano

Weger-Lucarelli

2022

Preprint

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Flaviviruses 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. We used anin vitrodirected evolution system, passaging ZIKV in the presence of serum from humans previously infected with DENV (anti-DENV) or serum from DENV-na&iumlve 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. Both viruses had increased fitness in Vero cells with and without the addition of anti-DENV serum and in human lung epithelial and monocyte cells. InAedes aegyptimosquitoes — 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.

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Section: Methodsmentioning

confidence: 99%

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

Marano

Weger-Lucarelli

2022

Preprint

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“…Thus, intending to use a model that is more representative of human CHIKV infection, we treated mice with a low dose (0.1 mg) of type I IFN receptor (IFNAR) antagonizing antibody MAR1-5A3 (herein referred to as MAR1) the day before infection. The MAR1 antibody has been used to establish widely accepted mouse models for Zika virus 48 , dengue virus 49 , Usutu virus 50 , and several alphaviruses [51][52][53] . We hypothesized that using a more susceptible model would reveal differences between vaccine candidates in terms of safety and protective e cacy.…”

Section: Cytokine-expressing Vaccine Candidates Protect Against Chik ...mentioning

confidence: 99%

“…As expected, mock-vaccinated mice had no signi cant nAbs against CHIKV. All vaccinated mice neutralized CHIKV as shown by mean PRNT 50 Cytokine-expressing vaccine candidates protect against WT CHIKV challenge in transiently immunocompromised C57BL/6 mice To evaluate the protective e cacy in mice vaccinated following MAR1 administration, we challenged the mice with 10 3 PFU WT CHIKV following an additional treatment of 0.1 mg of MAR1 to produce a more potent challenge. The study design is presented in Fig.…”

Section: Cytokine-expressing Vaccine Candidates Protect Against Chik ...mentioning

confidence: 99%

Enhanced attenuation of chikungunya vaccines expressing antiviral cytokines

Weger-Lucarelli

Chuong

Cereghino

et al. 2023

Preprint

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Chikungunya virus (CHIKV) is an emerging virus responsible for millions of infections globally within the last 15 years and has the potential to become endemic in the US. CHIK disease is characterized by severe febrile illness, with 30–60% of cases leading to debilitating chronic joint pain. No licensed treatments are available to protect against CHIK disease; thus, there is a tremendous need to generate a safe and effective vaccine. Live-attenuated vaccines (LAVs) are an appealing immunization strategy because they typically generate long-term protection from a single dose. However, LAVs often cause post-vaccination side effects and produce systemic viral replication, which can potentially lead to reversion to a pathogenic phenotype or transmission to mosquitoes; thus, safer LAV platforms are needed. To that end, we sought to improve the traditional LAV platform by combining attenuating strategies; as a vaccine backbone, we used a previously developed chimera of CHIKV and the closely related Semliki Forest virus (SFV) where we replaced the E2 domain C region of CHIKV with the corresponding domain from SFV (CHIKV-SFV/DomC) that was highly attenuated in mice and mosquitoes. To further attenuate the backbone, we inserted IFN-γ or IL-21, important antiviral cytokine genes, into the viral genome. The IFN-γ- and IL-21-expressing candidates were significantly attenuated post-vaccination, generating reduced footpad swelling with minimal systemic replication and dissemination capacity compared to the parental vaccine. Additionally, these candidates provided complete protection to mice challenged with WT CHIKV. This novel dual attenuation strategy combining an attenuated chimeric backbone and an antiviral cytokine has the possibility to be applied for the attenuation of any RNA virus.

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Development and characterization of infectious clones of two strains of Usutu virus

Cited by 2 publications

References 39 publications

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

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

Enhanced attenuation of chikungunya vaccines expressing antiviral cytokines

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