A Single Mutation in the VP1 of Enterovirus 71 Is Responsible for Increased Virulence and Neurotropism in Adult Interferon-Deficient Mice

Abstract

Hand, foot, and mouth disease (HFMD) has spread throughout the Asia-Pacific region, affecting millions of young children, who develop symptoms ranging from painful blisters around their mouths and hands to neurological complications. Many members of the genus Enterovirus (family Picornaviridae) cause HFMD. Enterovirus 71 (EV71) is one of the primary causative agents and has been linked to severe disease. Vaccine efficacy and pathogenesis studies for EV71 have been limited because there is a lack of suitable animal models. Previously, we generated a mouse-adapted EV71 (mEV71) capable of infecting 12-week-old interferon receptor-deficient AG129 mice and used the model to evaluate the efficacy of candidate HFMD vaccines. Here, we present data investigating the genetic correlates of EV71 adaptation and characterize the virus's tissue tropism in mice. Using reverse genetics, a VP1 mutation (K244E) was shown to be necessary for mEV71 virulence in adult mice. Another VP1 mutation (H37R) was required for mEV71 recovery on rhabdomyosarcoma (RD) cells. Viral loads determined by real-time reverse transcription (RT)-PCR confirmed the presence of mEV71 in the sera and multiple organs of mice. Histological analysis revealed signs of meningitis and encephalitis, characteristic of severe human disease. The further description of this model has provided insight into EV71 pathogenesis and demonstrates the importance of the VP1 region in facilitating mEV71 adaptation. IMPORTANCEEV71 is a reemerging pathogen, and little is known about the genetic determinants involved in its pathogenesis. The absence of animal models has contributed to this lack of knowledge. The data presented here improve upon the existing animal models by characterizing a mouse-adapted strain of EV71. We determined that a VP1 mutation (K244E) was needed for EV71 virulence in adult AG129 mice. While this mutation was found previously for EV71 adaptation in 5-day-old BALB/c mice, neurotropic disease did not develop. Using interferon-deficient mice, we raised the age of susceptibility beyond 6 weeks and provided clear evidence that our model mimics severe human infections. The model can be exploited to identify determinants of EV71 virulence and to reveal molecular mechanisms that control the virus-host interaction, especially those associated with neurotropic disease. Furthermore, these data provide useful information regarding the importance of VP1, specifically position 244, in host adaptation and tissue dissemination. O ver the past decade, enterovirus 71 (EV71) (family Picornaviridae) was identified as one of the main causative agents responsible for large outbreaks of hand, foot, and mouth disease (HFMD) across the Asia-Pacific region. Other members of the genus Enterovirus cause HFMD, including coxsackieviruses A16, A6, A5, A7, A9, A10, B2, and B5, but EV71 has been linked to severe complications, including brainstem encephalitis, aseptic meningitis, and pulmonary edema (1-3). Picornaviruses, including the enteroviruses, have positive-sense, single-s...

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

“…10). The data also followed viral load trends similar to those seen in previous work looking at viral loads in tissues of 6-week-old AG129 mice infected with mEV71 (23). Viral loads in the brains of AG129 mice could also be detected at 42 dpi.…”

“…Viral stocks of vEV71 were prepared following previously described procedures (14). Mouse-adapted EV71 (mEV71) was created by serial passage of vEV71 in young, interferon receptor-deficient mice and extensively characterized (14,23). There were two synonymous mutations (T108A in VP4 and C51T in 2B) and two nonsynonymous mutations (H37R and K244E, both in VP1) between mEV71 and vEV71.…”

“…There were two synonymous mutations (T108A in VP4 and C51T in 2B) and two nonsynonymous mutations (H37R and K244E, both in VP1) between mEV71 and vEV71. The K244E mutation in VP1 was determined to be the critical mutation for mouse adaptation (23).…”

“…Briefly, NLuc sequences were amplified from a full-length PA-2A-NLuc fusion construct, obtained from A. Mehle (UW-Madison) (17). NLuc amplicons were placed between the 5= UTR and the VP4 gene of previously characterized infectious cDNAs for vEV71 and mEV71 (23) to create a continuous open reading frame (ORF) (Fig. 1A).…”

“…Virus recovery from full-length infectious cDNAs was completed for NLuc constructs as previously described (23). Briefly, cDNAs were linearized with SbfI-HF (New England BioLabs, Ipswich, MA), and full-length RNA transcripts were synthesized using a MEGAscript T7 transcription kit (Thermo Fisher Scientific, Waltham, MA) following the manufacturer's instructions.…”