Characterization of a novel bioluminescent reporter protein for the non-invasive preclinical imaging of viral infection
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Abstract
RNA viruses pose significant public health concerns due to their high mutation rates. This ability enables the rapid evolution of endemic viruses and the emergence of new strains with enhanced virulence. Many RNA viruses originate from animals, facilitating zoonotic transmission and the potential for pandemics. RNA viruses exhibit a wide range of hosts, leading to interspecies transmission and the emergence of novel diseases. Treatment options are often limited, and vaccine development can be challenging due to genetic variability [1,2]. An exception that’s overcome this challenge is the Yellow Fever Virus (YFV). In the early 20th century, a live attenuated strain of the YFV (17D) was deemed as an effective vaccine against the mosquito-borne virus. Despite this progress, the virus persists in endemic regions of Africa and South America, necessitating ongoing study and surveillance. Continued research into the virus's biology, epidemiology, and transmission dynamics is essential for maintaining global health security against this enduring threat [3,4].
The integration of bioluminescent reporter genes into viral genomes presents a cutting-edge approach in understanding how a virus propagates throughout the host. This allows for researchers to non-invasively track the infection's progression from start to finish in laboratory animal models [5]. The aim of this study is to characterize a novel bioluminescent reporter protein with increased tissue penetration ability for the comprehensive non-invasive preclinical imaging of YFV infection. Such tool could open novel avenues for the identification of critical host and viral determinants of viral disease, and the evaluation of antiviral countermeasures.
In-vitro characterization of the novel reporter, NanoLSS1, was first achieved by transducing A549 (Adenocarcinoma Human Alveolar Basal Epithelial) cells using a lentiviral transduction system to demonstrate how the reporter emits signal at different wavelengths [6]. Using a Bioluminescent Resonance Energy Transfer (BRET) system, the reporter exhibits both excitation and emission peaks, with the excitation peak at 460nm and an emission peak of 615nm [7]. Both spectrophotometer and in vivo imaging system (IVIS) platform were used to capture and quantify the emitted signals from the transduced cells lines [8].
Specifically, different conformational variations of the NanoLSS1 were analyzed to determine which exhibited the highest photon intensities emitted at multiple wavelengths. The most optimal variation of the reporter was used in later experiments [11]. By mimicking tissue depth using slices of ham overlayed over the transduced cell lines, we also confirmed that NanoLSS1 had the highest degree of tissue penetrance when emitting signal [10] compared to conventional bioluminescent reporter, such as Nanoluc [11].
Utilizing a reverse genetics recombinant virus system, reporter proteins can be introduced into a viral genome through PCR, resulting in viral particles expressing the desired reporter genes upon infection and viral replication [9]. Using such system, we successfully developed and rescued a NanoLSS1-expressing 17D virus. This newly generated virus can infect 293T (Human Embryonic Kidney) and Huh7.5 (Hepatocyte-Derived Carcinoma) cells, infection is associated with BRET function and a bioluminescence emission peak of 615nm. The recombinant virus system was also implemented to generate YFV-17D expressing Nanoluc to be used as a control for future mouse infection studies.
Collectively, my work successfully characterized a novel bioluminescent reporter protein for investigating viral dissemination into an infected host. My work open avenues for unravelling novel facets of viral pathogenesis and developing innovative antiviral countermeasures.
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2024