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.

Keywords: enterovirus 71; in vivo imaging; mouse models; nanoluciferase; pathogenesis; tissue tropism.

FIG 1

In vitro characterization of bioluminescent EV71 constructs. (A) NLuc was inserted after the 5′ UTR in vEV71 and mEV71, followed by an EV71 2A cleavage site. The arrows mark the nucleotide (red) and amino acid (black) changes in the mEV71 construct. (B) vEV71-NLuc and mEV71-NLuc reporter viruses were stable when passaged eight times in RD cell culture. RT-PCR was performed on RNA extracted from each passage. Agarose gel electrophoresis was used to analyze the products. A single band representing the full-length PCR product was detected for each passage. vEV71 without NLuc was used as a control. The gel image is for mEV71-NLuc. Similar results were obtained with vEV71-NLuc. (C) NLuc constructs were attenuated on RD cells compared to parent viruses. A multistep growth curve (MOI = 0.1) was done to determine in vitro growth characteristics. Titers of bioluminescent viruses were determined with a traditional TCID₅₀ assay and a Nano-Glo assay. The Nano-Glo assay reduced the amount of time needed before reading plates from 5 days to 2 days. (D) Optimization of the Nano-Glo TCID₅₀ assay. Forty-eight hours postinfection was the optimal time that luminescence from the Nano-Glo TCID₅₀ assay matched the virus titer determined with a traditional TCID₅₀ assay (horizontal dotted line) for both mEV71-NLuc and vEV71-NLuc. (E) The genome/TCID₅₀ ratio was higher for reporter constructs than for the parental controls. There was a significant difference between vEV71 and vEV71-NLuc (*, P ≤ 0.05).

FIG 2

In vivo characterization of bioluminescent viruses. Three-week-old AG129 and BALB/c mice received 10⁶ TCID₅₀ units of virus i.p. (A) All AG129 mice succumbed to infection with mEV71. mEV71-NLuc was attenuated in AG129 mice, with only one mouse developing clinical signs of disease. All BALB/c mice survived the challenge. Numbers in parentheses represent the number of mice in each group. (B) Mice were bled at 1, 3, and 5 dpi to test for viral copies by real-time RT-PCR. All mice had viral loads in their blood at 1 dpi. There were significant differences in viral loads at 3 and 5 dpi when AG129 mice infected with mEV71 were compared to the other two groups (*, P ≤ 0.05). There was a significant difference at 1 dpi only when AG129 mice infected with mEV71 were compared to AG129 mice infected with mEV71-NLuc. The dotted line shows the limit of detection of the assay. ns, not significant.

FIG 3

Higher levels of luminescence were detected during in vivo imaging of AG129 mice after hair removal. Initial in vivo imaging studies determined that the dark fur of AG129 mice hindered the visualization of luminescence. A significantly higher radiance was detected for AG129 mice after hair removal at 1 dpi (*, P ≤ 0.05). Hair remover was used to remove the stomach fur of the animals, and a razor was used to shave their heads.

FIG 4

Ventral view of AG129 and BALB/c mice after infection with mEV71-NLuc. Viral spread was monitored in real time after 2.4 × 10⁶ TCID₅₀ units of virus was injected i.p. into 3-week-old animals. The highest radiance was observed at the site of injection in both animal models, with a wider dispersion of luminescence in the AG129 mice. Luminescence was undetected in BALB/c mice at 21 dpi. N(−), negative control.

FIG 5

Dorsal view of AG129 and BALB/c mice after infection with mEV71-NLuc. Viral spread was monitored in real time after 2.4 × 10⁶ TCID₅₀ units of virus was injected i.p. into 3-week-old animals. Luminescence was detected in the brain region of AG129 mice at 1 dpi. Luminescence was also visualized in the footpads of infected animals, which parallels natural HFMD symptoms in humans. Luminescence was undetectable in BALB/c mice by 11 dpi, but persistent luminescence occurred in AG129 mice until the end of the study.

FIG 6

Ventral and dorsal views of AG129 mice at 28 to 56 dpi for infection with mEV71-NLuc. Luminescence was visualized in the brains of infected AG129 mice at 56 dpi. Mice 5 and 6 were euthanized at 35 dpi immediately following imaging. No luminescence was observed in BALB/c mice, suggesting that these mice cleared the infection (images not shown).

FIG 7

Average radiances of AG129 and BALB/c mice after infection with mEV71-NLuc. The average radiance was determined by placing a red box over a region of interest. (A) Radiance on the ventral side peaked at 3 dpi for both animal models. (B) Radiance continued to increase in the brain region of AG129 mice over the course of infection. Numbers in parentheses represent the number of mice used for in vivo imaging. Vertical dotted lines indicate the days that tissue samples were collected. Horizontal dotted lines show the limit of detection.

FIG 8

Comparison of mEV71-NLuc infections of AG129 and BALB/c mice. (A) Survival of AG129 and BALB/c mice infected with 2.4 × 10⁶ TCID₅₀ units of mEV71-NLuc. The number of mice in each group is indicated in parentheses. (B) Weight changes of AG129 and BALB/c mice after infection with mEV71-NLuc. (C) Viral loads in serum were tested by real-time RT-PCR at 1, 3, and 5 dpi. A significant difference between viral loads was detected on day 3. The dotted line shows the limit of detection of the assay. (D) Neutralizing antibody titers at 35 and 56 dpi were tested using a Nano-Glo assay. Significant differences in antibody titers between mouse strains were detected on both days (*, P ≤ 0.05).

FIG 9

Histological analysis of brains from AG129 and BALB/c mice after infection with mEV71-NLuc. Tissue samples were taken at the end of the study (56 dpi). None of the mice were losing weight or had clinical signs of disease. AG129 mice showed slight mononuclear meningitis (A) and perivascular cuffing (B) compared to controls. No lesions were observed in BALB/c mice. Magnification, ×20.

FIG 10

Viral loads in tissues of AG129 and BALB/c mice after infection with mEV71-NLuc. Three mice each were sacrificed at 3, 6, 12, and 42 dpi, and viral loads in tissues were tested by real-time RT-PCR. Similar trends were seen between BALB/c and AG129 mice for all tissues except the brain. High viral loads in the brains of AG129 mice were maintained throughout the study. Tissues from mock-infected animals were used as a control. Horizontal dotted lines represent the limit of detection. Blue symbols, AG129 mice; red symbols, BALB/c mice.