Dataset Information

A NanoLuc Luciferase Reporter Pseudorabies Virus for Live Imaging and Quantification of Viral Infection.

ABSTRACT: Pseudorabies (PR), also known as Aujeszky's disease, is an acute infectious disease of pigs, resulting in significant economic losses to the pig industry in many countries. Since 2011, PR outbreaks have occurred in many Bartha-K61-vaccinated pig farms in China. The emerging pseudorabies virus (PRV) variants possess higher pathogenicity in pigs and mice than the strains isolated before. Here, a recombinant PRV (rPRVTJ-NLuc) stably expressing the NanoLuc (NLuc) luciferase fusion with the red fluorescent protein (DsRed) was constructed to trace viral replication and spread in mice. Moreover, both DsRed and NLuc luciferases were stably expressed in the infected cells, and there was no significant difference between wild-type and recombinant viruses in both growth kinetics and pathogenicity. Seven-week-old BALB/c mice were infected with 103 50% tissue culture infective dose rPRVTJ-NLuc and subjected to daily imaging. The mice infected with rPRVTJ-NLuc displayed robust bioluminescence that started 4 days postinfection (dpi), bioluminescence signal increased over time, peaked at 5 dpi, remained detectable for at least 6 dpi, and disappeared at 7 dpi, meanwhile, the increased flux accompanied by the spread of the virus from the injection site to the superior respiratory tract. However, the signal was also observed in the spinal cord, trigeminal ganglion, and partial region of the brain from separated tissues, not in living mice. Our results depicted a new approach to rapidly access the replication and pathogenicity of emerging PRVs in mice.

SUBMITTER: Wang Y

PROVIDER: S-EPMC7537659 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

A NanoLuc Luciferase Reporter Pseudorabies Virus for Live Imaging and Quantification of Viral Infection.

Wang Yalin Y Wu Hongxia H Wang Bing B Qi Hansong H Jin Zhao Z Qiu Hua-Ji HJ Sun Yuan Y

Frontiers in veterinary science 20200922

Pseudorabies (PR), also known as Aujeszky's disease, is an acute infectious disease of pigs, resulting in significant economic losses to the pig industry in many countries. Since 2011, PR outbreaks have occurred in many Bartha-K61-vaccinated pig farms in China. The emerging pseudorabies virus (PRV) variants possess higher pathogenicity in pigs and mice than the strains isolated before. Here, a recombinant PRV (rPRVTJ-NLuc) stably expressing the NanoLuc (NLuc) luciferase fusion with the red fluor ...[more]

PMID: 33195544

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Project description:Infection by flaviviruses leads to dramatic remodeling of the endoplasmic reticulum (ER). Viral replication occurs within virus-induced vesicular invaginations in the ER membrane. A hallmark of flavivirus infection is expansion of the ER membrane which can be observed at specific time points post infection. However, this process has not been effectively visualized in living cells throughout the course of infection at the single cell resolution. In this study, we developed a plasmid-based reporter system to monitor flavivirus infection and simultaneous virus-induced manipulation of single cells throughout the course of infection in real-time. This system requires viral protease cleavage to release an ER-anchored fluorescent protein infection reporter that is fused to a nuclear localization signal (NLS). This proteolytic cleavage allows for the translocation of the infection reporter signal to the nucleus while an ER-specific fluorescent marker remains localized in the lumen. Thus, the construct allows for the visualization of virus-dependent changes to the ER throughout the course of infection. In this study, we show that our reporter was efficiently cleaved upon the expression of multiple flavivirus proteases, including dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). We also found that the DENV protease-dependent cleavage of our ER-anchored reporter exhibited more stringent cleavage sequence specificity than what has previously been shown with biochemical assays. Using this system for long term time-lapse imaging of living cells infected with DENV, we observed nuclear translocation of the reporter signal beginning approximately 8 hours post-infection, which continued to increase throughout the time course. Interestingly, we found that increased reporter signal translocation correlated with increased ER signal intensity, suggesting a positive association between DENV infection and ER expansion in a time-dependent manner. Overall, this report demonstrates that the FlavER platform provides a useful tool for monitoring flavivirus infection and simultaneously observing virus-dependent changes to the host cell ER, allowing for study of the temporal nature of virus-host interactions.

Dataset Information

A NanoLuc Luciferase Reporter Pseudorabies Virus for Live Imaging and Quantification of Viral Infection.

Publications

A NanoLuc Luciferase Reporter Pseudorabies Virus for Live Imaging and Quantification of Viral Infection.

Similar Datasets

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