Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disabling disease that can cause long-term severe arthritis. Since the last large CHIKV outbreak in 2015, the reemergence of the virus represents a serious public health concern. The morbidity associated with viral infection emphasizes the need for the development of specific anti-CHIKV drugs. Herein, we describe the development and characterization of a CHIKV reporter replicon cell line and its use in replicon-based screenings. We tested 960 compounds from MMV/DNDi Open Box libraries and identified four candidates with interesting antiviral activities, which were confirmed in viral infection assays employing CHIKV-nanoluc and BHK-21 cells. The most noteworthy compound identified was itraconazole (ITZ), an orally available, safe, and cheap antifungal, that showed high selectivity indexes of >312 and >294 in both replicon-based and viral infection assays, respectively. The antiviral activity of this molecule has been described against positive-sense single stranded RNA viruses (+ssRNA) and was related to cholesterol metabolism that could affect the formation of the replication organelles. Although its precise mechanism of action against CHIKV still needs to be elucidated, our results demonstrate that ITZ is a potent inhibitor of the viral replication that could be repurposed as a broad-spectrum antiviral.

Section: Discussionmentioning

confidence: 99%

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication

Policastro

Dolci

Godoy

et al. 2022

“…The data showed that rHCLV-uspCap and rHCLV-pspCap rather than rHCLV-2ACap elicited detectable anti-Cap antibodies in rabbits, which demonstrated that C-strain could be used as a viral vector to develop bivalent vaccines [ 122 ]. Furthermore, it has been shown that the recombinant BVDV expressing the PEDV spike protein, as a recombinant virus vector, can induce higher titers of NAbs and provide protection against virulent challenge [ 128 , 134 ].…”

Section: Development Of Multivalent Vaccines Based On Pestivirusesmentioning

confidence: 99%

Different Types of Vaccines against Pestiviral Infections: “Barriers” for “Pestis”

Yuan

Yang

Zhang

et al. 2022

The genus Pestivirus of the family Flaviviridae mainly comprises classical swine fever virus (CSFV), bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, border disease virus (BDV), and multiple new pestivirus species such as atypical porcine pestivirus (APPV), giraffe pestivirus, and antelope pestivirus. Pestiviruses cause infectious diseases, resulting in tremendous economic losses to animal husbandry. Different types of pestivirus vaccines have been developed to control and prevent these important animal diseases. In recent years, pestiviruses have shown great potential as viral vectors for developing multivalent vaccines. This review analyzes the advantages and disadvantages of various pestivirus vaccines, including live attenuated pestivirus strains, genetically engineered marker pestiviruses, and pestivirus-based multivalent vaccines. This review provides new insights into the development of novel vaccines against emerging pestiviruses, such as APPV and ovine pestivirus.

“…For positive-sense single-stranded RNA viruses, there are two key steps for successful viral reverse genetics to generate infectious virus particles with engineered genomes: production of genomic RNA and its delivery to susceptible cells. The former was often achieved by in vitro transcription (IVT) of DNA copies of viral genomes using T7 RNA polymerase [ 29 , 30 , 31 , 32 ] or SP6 RNA polymerase [ 33 , 34 , 35 ]. The latter was usually achieved by transfecting the genomic RNA using electroporation or cationic detergent transfection reagents, such as Lipofectamine.…”

Section: Introductionmentioning

confidence: 99%

Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion

Ding

Brown

Glass

2021

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.