Generation and Characterization of a Replication-Competent Human Adenovirus Type 55 Encoding EGFP

Abstract

Human adenovirus 55 (HAdV-55) has recently caused outbreaks of acute respiratory disease (ARD), posing a significant public threat to civilians and military trainees. Efforts to develop antiviral inhibitors and quantify neutralizing antibodies require an experimental system to rapidly monitor viral infections, which can be achieved through the use of a plasmid that can produce an infectious virus. Here, we used a bacteria-mediated recombination approach to construct a full-length infectious cDNA clone, pAd55-FL, containing the whole genome of HadV-55. Then, the green fluorescent protein expression cassette was assembled into pAd55-FL to replace the E3 region to obtain a recombinant plasmid of pAd55-dE3-EGFP. The rescued recombinant virus rAdv55-dE3-EGFP is genetically stable and replicates similarly to the wild-type virus in cell culture. The virus rAdv55-dE3-EGFP can be used to quantify neutralizing antibody activity in sera samples, producing results in concordance with the cytopathic effect (CPE)-based microneutralization assay. Using an rAdv55-dE3-EGFP infection of A549 cells, we showed that the assay could be used for antiviral screening. Our findings suggest that the rAdv55-dE3-EGFP-based high-throughput assay provides a reliable tool for rapid neutralization testing and antiviral screening for HAdV-55.

Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication

“…Replication competent reporter viruses producing bioluminescent or fluorescent signals, have been constructed for a variety of viruses and proven immensely helpful for high-throughput screening, monitoring of viral replication dynamics in vitro and in vivo , and studying basic or clinical virology ( 37 – 42 ). Adenovirus reporters have also been generated and shown to demonstrate strong utility ( 43 – 49 ). In this work, we expand the adenovirus reporter toolbox with a suite of nine additional replication competent viruses engineered in a WT HAdV-C5 genomic background to investigate immediate early gene expression (AdNTE, AdGTE, and AdCTE), late gene expression (AdNTV, AdGTV, and AdCTV), virion attachment (AdNV), VRC visualization (AdGDBP), and the temporal dynamics between early and late infection (AdDFEV).…”

“…Further, using both reporters would allow one to tease out non-redundant inhibitors that specifically affect the late or early phases of infection ( 47 ). Similarly, the mCherry or EGFP fluorescent reporter viruses could be used to quickly and easily visualize changes in the number of infected cells treated with therapeutics for infections done at low MOIs ( 43 ).…”

“…The AdNV reporter virus could represent a rapid system for testing engineered antiviral antibodies or other therapeutics that target viral attachment and entry ( 76 ), because this technique can be completed in under an hour and does not require the virus to replicate. In addition, this reporter virus offers a streamlined approach to microneutralization assays, which could also benefit studies of HAdV-C5 with the host immune system ( 43 , 70 ).…”

Expanding the Scope of Adenoviral Vectors by Utilizing Novel Tools for Recombination and Vector Rescue

“…An operational reverse genetic workflow based on infectious genomic clones has been available for HAdVs of species C since the 1990s [6]. It was further developed and applied to model types representing most species of HAdV, allowing the generation of virus mutants and developing vectors by rational design based on specific HAdVs [7][8][9][10]. However, to date, these technologies have not reached high efficiencies, which are available for HAdV-C5-based platforms, and still maintain a lag compared to the technologies available for concurrent vector platforms, especially regarding the library-based applications established for recombinant AAVs [11].…”

An Expeditious Neutralization Assay for Porcine Reproductive and Respiratory Syndrome Virus Based on a Recombinant Virus Expressing Green Fluorescent Protein

“…(1) the sensitive PRRSV-specific antibody used for IFA detection is difficult to obtain and expensive; (2) both of these assays are time-consuming, as it takes four days to obtain a diagnosis of cytopathy with a CPE assay, while the FFN assay utilizes the time-consuming process of an indirect immune fluorescent assay (IFA) [22]. By contrast, neutralization assays based on reporter viruses can save time, simplify the experimental process, and provide sensitive and reliable results [23,24].…”