In Vivo Imaging with Bioluminescent Enterovirus 71 Allows for Real-Time Visualization of Tissue Tropism and Viral Spread

Abstract

Hand, foot, and mouth disease (HFMD) is a reemerging illness caused by a variety of enteroviruses. The main causative agents are enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and, most recently, coxsackievirus A6 (CVA6). Enterovirus infections can vary from asymptomatic infections to those with a mild fever and blisters on infected individuals' hands, feet, and throats to infections with severe neurological complications. Viral persistence for weeks postinfection (wpi) has also been documented by the demonstration of virus in children's stools. However, little is known about disease progression, viral spread, and tissue tropism of these viruses. These types of studies are limited because many recently developed mouse models mimic the severe neurological complications that occur in a small percentage of enterovirus infections. In the present study, we documented real-time EV71 infection in two different mouse strains by the use of in vivo imaging. Infection of BALB/c mice with a bioluminescent mouse-adapted EV71 construct (mEV71-NLuc) resulted in a lack of clinical signs of disease but in relatively high viral replication, as visualized by luminescence, for 2 wpi. In contrast, mEV71-NLuc infection of AG129 mice (alpha/beta and gamma interferon receptor deficient) showed rapid spread and long-term persistence of the virus in the brain. Interestingly, AG129 mice that survived infection maintained luminescence in the brain for up to 8 wpi. The results we present here will allow future studies on EV71 antiviral drug susceptibility, vaccine efficacy, transmissibility, and pathogenesis. IMPORTANCE We report here that a stable full-length enterovirus 71 (EV71) reporter construct was used to visualize real-time viral spread in AG129 and BALB/c mice. To our knowledge, this is the first report of in vivo imaging of infection with any member of the Picornaviridae family. The nanoluciferase (NLuc) gene, one of the smallest luciferase genes currently available, was shown to be stable in the EV71 genome for eight passages on rhabdomyosarcoma cells. Real-time visualization of EV71 infection in mice identified areas of tropism that would have been missed by traditional methods, including full characterization of EV71 replication in BALB/c mice. Additionally, the bioluminescent construct allowed for increased speed and sensitivity of cell culture assays and will allow future studies involving various degrees of enterovirus infection in mice, not just severe infections. Our data suggest that interferon plays an important role in controlling EV71 infection in the central nervous system of mice.

A novel nanoluciferase-based system to monitor Trypanosoma cruzi infection in mice by bioluminescence imaging

“…We found no significant differences when we measured the total ventral bioluminescence intensity ( S1B Fig ). Based on our experimental observations, and previous reports [ 33 , 34 ] describing the successful delivery of furimazine in the peritoneal cavity, the i. p. route was the method of choice in all subsequent experiments.…”

“…Nanoluciferase is a relatively new luciferase compared to the widely used firefly, Renilla or Gaussia luciferases and its application for in vivo imaging remains to be fully investigated [ 24 , 28 ]. To date, NLuc and its furimazine substrate have been effectively employed as bioluminescence system to study spread and tissue tropism of pathogens in real time imaging [ 23 , 34 , 42 , 43 ] with light signal outputs greater than that of Gaussia and firefly luciferases [ 30 ]. However, the use of NLuc to track T .…”

Enterovirus and Encephalitis

“…The CNS is relatively inaccessible to immune surveillance compared with other tissues, which makes it particularly vulnerable to persistent infection. Using in vivo imaging, a recent study has demonstrated that EV-A71 infection of AG129 mice, in which alpha/beta and gamma interferon receptors are deficient, shows rapid spread and long-term persistence of the virus in the brain of surviving animals (Caine and Osorio, 2017). Interestingly, high viral loads are maintained in the brain even at 6 weeks following infection, whereas viral loads in other tissues including heart, lung, liver, spleen and intestine are gradually reduced to a lower level.…”

A Simple and Robust Approach for Evaluation of Antivirals Using a Recombinant Influenza Virus Expressing Gaussia Luciferase

“…The establishment of viral reverse genetics and reporter viruses has greatly facilitated the development of animal models as tools for the evaluation of candidate vaccines and therapeutic drugs. So far, luciferase-expressing recombinant viruses have been well demonstrated for herpes simplex virus [ 29 , 30 ], enterovirus 71 [ 31 ], Japanese encephalitis virus [ 32 ], and Sendai virus [ 33 ], as well as for IAV [ 1 , 16 , 24 ]. Infection of these viruses allows for real-time monitoring in virus replication and dissemination in living animals by noninvasive in vivo imaging [ 34 ], facilitating the evaluation of candidate vaccines and therapeutic antibodies.…”