The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis

Abstract

Single photon emission computed tomography (SPECT) is frequently used in oncology and cardiology to evaluate disease progression and/or treatment efficacy. Such technology allows for real-time evaluation of disease progression and when applied to studying infectious diseases may provide insight into pathogenesis. Insertion of a SPECT-compatible reporter gene into a virus may provide insight into mechanisms of pathogenesis and viral tropism. The human sodium iodide symporter (hNIS), a SPECT and positron emission tomography reporter gene, was inserted into Middle East respiratory syndrome coronavirus (MERS-CoV), a recently emerged virus that can cause severe respiratory disease and death in afflicted humans to obtain a quantifiable and sensitive marker for viral replication to further MERS-CoV animal model development. The recombinant virus was evaluated for fitness, stability, and reporter gene functionality. The recombinant and parental viruses demonstrated equal fitness in terms of peak titer and replication kinetics, were stable for up to six in vitro passages, and were functional. Further in vivo evaluation indicated variable stability, but resolution limits hampered in vivo functional evaluation. These data support the further development of hNIS for monitoring infection in animal models of viral disease.IMPORTANCE Advanced medical imaging such as single photon emission computed tomography with computed tomography (SPECT/CT) enhances fields such as oncology and cardiology. Application of SPECT/CT, magnetic resonance imaging, and positron emission tomography to infectious disease may enhance pathogenesis studies and provide alternate biomarkers of disease progression. The experiments described in this article focus on insertion of a SPECT/CT-compatible reporter gene into MERS-CoV to demonstrate that a functional SPECT/CT reporter gene can be inserted into a virus.

Keywords: MERS; coronavirus; medical imaging; reporter gene.

FIG 1

Kinetics of rMERS-CoV/hNIS and parental rMERS-CoV replication in Vero E6 cells. (a and b) Multistep (a) and one-step (b) growth curves of Vero E6 cells infected with rMERS-CoV (Parental) and rMERS-CoV/hNIS (hNIS). Quantification of the release of infectious virus at the indicated time points (hours postexposure) was determined by plaque assays. Each data point represents the mean ± standard deviation (SD) (error bar) averaged from three independent experiments. (c and d) Cytopathology of rMERS-CoV and rMERS-CoV/hNIS in Vero E6 cells. The cells were infected with either rMERS-CoV or rMERS-CoV/hNIS at an MOI of 0.01 (c) or 3 (d) and analyzed by light microscopy at the indicated time points. Photomicrographs were taken using a 40× objective.

FIG 2

Retention of hNIS transgene following viral kinetics analysis and serial passage. (a and b) Vero E6 cells were infected with rMERS-CoV/hNIS (a) or parental rMERS-CoV (b) at an MOI of 0.01 or 3 and then collected at 96 h postinfection for RT-PCR. (c) Retention of the hNIS gene following serial passage. RNA was extracted from cells 72 h postinfection followed by RT-PCR at passage 6. A positive-control virus (C+) and uninfected negative-control cells (C−) were used as controls.

FIG 3

Radio-uptake of ^99mTc-pertechnetate by planar scintigraphy. (a) Experimental overview of in vitro evaluation of the rMERS-CoV/hNIS virus. Vero E6 cells were infected with rMERS-CoV or rMERS-CoV/hNIS at an MOI of 0.01 or 0.04. At various time points postinfection, the cells were incubated with ^99mTc-pertechnetate, and images of the plates were acquired. (b) Plate layout for hNIS functional assays. (c) Representative images of the plates acquired at 24 h postinfection at an MOI of 0.01 (top plates) or 0.04 (bottom plates) after incubation with ^99mTc-pertechnetate.

FIG 4

Quantification of ^99mTc-pertechnetate uptake by rMERS-CoV/hNIS-infected cells. (a) ^99mTc-pertechnetate uptake by rMERS-CoV- or rMERS-CoV/hNIS-infected cells at an MOI of 0.01 at 24, 48, 72, and 96 h postinfection. (b) ^99mTc-pertechnetate uptake by rMERS-CoV/hNIS-infected cells at an MOI of 0.01 or 0.04 at 24 and 48 h postinfection. (c) Quantitative analysis of ^99mTc-pertechnetate uptake applied at doses ranging from 0.6 to 0.004 mCi per well.

FIG 5

Sodium perchlorate-mediated inhibition of ^99mTc-pertechnetate uptake. (a) Quantitation of ^99mTc-pertechnetate uptake in the presence of sodium perchlorate at doses ranging from 0 and 0.1 mM. ^99mTc-pertechnetate uptake was reduced in rMERS-CoV/hNIS-infected cells with increasing sodium perchlorate concentrations. (b) ^99mTc-pertechnetate uptake by rMERS-CoV- or rMERS-CoV/hNIS-infected cells at an MOI of 0.01 at 24, 48, 72, and 96 h postinfection in the presence of 0.1 mM sodium perchlorate.

FIG 6

Lung histopathology in infected mice and detection of rMERS-CoV/hNIS or rMERS-CoV in lung tissue. (a to e) Histopathology of the lungs of one representative mouse each from groups 1, 2, 3, 4, and 5 infected with rMERS-CoV or MERS-CoV/hNIS. The cells were visualized with HE stain. Magnification, ×10. Bars = 200 µm. (a) Uninfected control mouse. (b) Representative mouse infected with rMERS-CoV (group 1) with multifocal, minimal-to-mild perivascular and peribronchiolar inflammation, with congestion on day 3 postexposure (pe). (c) Representative mouse infected with rMERS-CoV/hNIS (group 2) with multifocal, minimal-to-mild perivascular and peribronchiolar inflammation, with congestion on day 3 pe. (d) Representative mouse infected with rMERS-CoV (group 4) with multifocal, minimal-to-mild perivascular and peribronchiolar inflammation with congestion at day 7 pe. (e) Representative mouse infected with rMERS-CoV/hNIS (group 5) with multifocal, minimal-to-mild perivascular and peribronchiolar inflammation with congestion at day 7 pe. (f) Viral load in lung tissue on day 3 pe in mice infected with rMERS-CoV or rMERS-CoV/hNIS as determined by plaque assay. (g) RT-PCR analysis of the lungs recovered from rMERS-CoV/hNIS- or rMERS-CoV-infected mice. The leftmost lane contains molecular size markers (in kilobases). The next three lanes contain stock controls, rMERS-CoV/hNIS (stock control, without RT step), rMERS-CoV stock, and rMERS-CoV/hNIS stock. C+ indicates positive-control virus, and C− indictes uninfected negative-control cells. Samples from groups 1 to 5 are shown in the five sets of lanes as follows: group 1, uninfected control (C−) group, lanes 1 to 5; group 2, exposed to rMERS-CoV, lanes 1 to 5; group 3, exposed to rMERS-CoV/hNIS, lanes 1 to 6; group 4, exposed to rMERS-CoV, lanes 1 to 5; group 5, exposed to rMERS-CoV/hNIS, lanes 1 to 6. The asterisk in lane 5* of the rMERS-CoV/hNIS-treated group on day 3 pe indicates altered PCR conditions (48°C annealing temperature, 3% dimethyl sulfoxide) to improve the sensitivity to detect rMERS-CoV/hNIS.