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The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis Svetlana Chefer a Jurgen Seidel a Adam S Cockrell b Boyd Yount b Jeffrey Solomon c Katie R Hagen a David X Liu a Louis M Huzella a Mia R Kumar d Elena Postnikova a J Kyle Bohannon a Matthew G Lackemeyer a Kurt Cooper a Ariel Endlich Frazier d Heema Sharma d David Thomasson a Christopher Bartos a Philip J Sayre a Amy Sims b Julie Dyall a Michael R Holbrook a Peter B Jahrling a d Ralph S Baric b Reed F Johnson d a Integrated Research Facility National Institute of Allergy and Infectious Diseases National Institutes of Health Frederick Maryland USA b Department of Epidemiology University of North Carolina Chapel Hill Chapel Hill North Carolina USA c Clinical Research Directorate Clinical Monitoring Research Program Leidos Biomedical Research Inc Frederick National Laboratory for Cancer Research Frederick Maryland USA d Emerging Viral Pathogens Section Laboratory of Immunoregulation Division of Intramural Research National Institute of Allergy and Infectious Diseases National Institutes of Health Frederick Maryland USA 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 oncol ogy and cardiology Application of SPECT CT magnetic resonance imaging and pos itron 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 R ecombinant viruses expressing reporter genes such as luciferase or fluorescent proteins or viral proteins fused with a reporter protein have been used as screening tools for countermeasures and to understand pathogenesis 1 4 While the application of reporter gene technology has provided insight into viral pathogenesis these exper Received 28 September 2018 Accepted 26 October 2018 Published 12 December 2018 Citation Chefer S Seidel J Cockrell AS Yount B Solomon J Hagen KR Liu DX Huzella LM Kumar MR Postnikova E Bohannon JK Lackemeyer MG Cooper K Endlich Frazier A Sharma H Thomasson D Bartos C Sayre PJ Sims A Dyall J Holbrook MR Jahrling PB Baric RS Johnson RF 2018 The human sodium iodide symporter as a reporter gene for studying Middle East respiratory syndrome coronavirus pathogenesis mSphere 3 e00540 18 https doi org 10 1128 mSphere 00540 18 Editor Matthew B Frieman University of Maryland College Park This is a work of the U S Government and is not subject to copyright protection in the United States Foreign copyrights may apply Address correspondence to Reed F Johnson johnsonreed niaid nih gov RESEARCH ARTICLE Host Microbe Biology crossm November December 2018 Volume 3 Issue 6 e00540 18 msphere asm org 1 on March 11 2019 by guest http msphere asm org Downloaded from iments are frequently hampered by sacrificing the subject to identify the source of the reporter gene signal 5 7 Applying medical imaging technology such as single photon emission computed tomography SPECT or positron emission tomography PET to animal models of human infectious disease provides the capability to serially monitor anatomical and physiological responses to infection in the same subject that can be clinically translated Generating a virus that carries a SPECT PET compatible reporter gene furthers that capability by serial real time evaluation of virus kinetics identification of tissue tropism and determination of pathogenic mechanisms The human sodium iodide symporter hNIS gene has emerged as one of the most prom ising reporter genes in preclinical and translational research for oncology and gene therapy 8 9 The hNIS symporter is an intrinsic plasma membrane protein belonging to the sodium solute symporter family which drives negatively charged solutes into the cytoplasm using a sodium ion electrochemical gradient 10 The advantages of hNIS as an imaging reporter gene include its relatively small size H110112 kb wide availability of substrates such as radioiodines tetrafluoroborate and 99m Tc pertechnetate and well understood metabolism and clearance mechanisms of these substrates 11 Oncolytic viruses such as measles virus and replication deficient adenovirus that contain hNIS have demonstrated value as theranostics as hNIS is used both as a therapeutic platform and to track the therapeutic effect 8 9 In addition hNIS is unlikely to perturb the underlying cell biochemistry and no negative effects of resultant sodium influx have been observed 12 Finally once incorporated into the viral genome the relatively small size of the reporter gene is less likely than larger reporter genes to alter viral pathogenic properties 13 Middle East respiratory syndrome CoV MERS CoV recently emerged and is associ ated with Middle East respiratory syndrome MERS a severe frequently lethal pneu monia in humans 14 16 Viral pathogenesis is not well understood in part because of limited autopsy information and a lack of animal models that fully recapitulate human disease As with most lethal infectious diseases animal models are the corner stone for preclinical countermeasure evaluation and understanding pathogenesis MERS CoV provides a unique opportunity to incorporate reporter gene technology to better understand viral pathogenesis because its larger genome size may be more amenable to reporter gene insertion than other viruses Animal models for MERS are under development with no single model identified as the standard New World and Old World nonhuman primates infected with MERS CoV develop transient respiratory disease with little or no virus replication and varying disease outcome 17 19 MERS CoV exposed New Zealand White rabbits develop limited lung pathology with evidence of viral replication but did not show overt clinical signs of disease 20 21 Transgenic mice globally expressing the human CD26 dipeptidyl peptidase 4 DPP4 receptor 22 expressing the human receptor under the murine promoter 23 or transduced with DPP4 receptor 24 become permissive to the virus but do not develop fulminant lethal respiratory disease Therefore changes in reporter gene signal may serve as a biomarker for countermeasure evaluation The objective of this study was to incorporate hNIS into MERS CoV to improve the MERS animal models Incorporation of a SPECT PET compatible reporter gene with an emerging virus such as MERS CoV requires functional evaluation of the recombinant virus to ensure similar fitness to the parental pathogen We hypothesized that insertion of hNIS would result in stable expression of a SPECT PET compatible reporter gene A recombinant MERS CoV carrying hNIS rMERS CoV hNIS was generated We assessed the stability fitness and functionality of this recombinant pathogen in vitro and in CRISPR generated transgenic mice that support replication of wild type MERS CoV 25 RESULTS rMERS CoV hNIS genetic stability kinetics and fitness Recombinant virus was evaluated by one step and multistep kinetics and by serial passaging of the virus rMERS CoV hNIS replicated similarly to rMERS CoV in Vero E6 cells infected at a multi Chefer et al November December 2018 Volume 3 Issue 6 e00540 18 msphere asm org 2 on March 11 2019 by guest http msphere asm org Downloaded from plicity of infection MOI of 0 01 with a peak in virus yield of 4 log 10 plaque forming units PFU ml at 24 h At an MOI of 3 viral yields peaked at 7 log 10 PFU ml at 48 h and plateaued at 72 h postinfection Fig 1a and b The correlations between the multistep growth curves for cells infected at an MOI of 0 01 g rH110050 96 Pearson correlation PH110050 0082 Fig 1a and one step growth curves of cells infected at an MOI of 3 rH110050 90 PH110050 03 Fig 1b for both viruses were high Both viruses had comparable cytopathic effects Fig 1c and d Expression of the hNIS transgene was evaluated by reverse transcriptase PCR RT PCR using primers to specifically detect hNIS expression by MERS CoV The result ing 635 bp PCR product includes the MERS CoV leader sequence and a portion of the hNIS transgene RT PCR confirmed hNIS expression in rMERS CoV hNIS infected Vero E6 cells up to 96 h Fig 2a The PCR product was not detected in parental rMERS CoV infected cells Fig 2b The RT PCR assay was also performed to determine the stability of the hNIS transgene following serial passage The hNIS transgene was stable in rMERS CoV hNIS for up to six cell culture passages Fig 2c hNIS functionality in MERS CoV hNIS infected cells To demonstrate in vitro hNIS functionality a series of assays were performed to characterize the kinetics of 99m Tc pertechnetate cellular uptake the relationship between virus concentration and probe cellular uptake and between probe dose and its detectability by the gamma camera The in vitro hNIS functionality assay is outlined in Fig 3a and b The 99m Tc pertechnetate uptake by rMERS CoV hNIS infected cells in six well plates was visible on the tissue culture plate images and distinguishable from the background radioactivity in the wells with uninfected and rMERS CoV infected cells Fig 3c In rMERS CoV hNIS infected cells uptake increased with time after infection and reached a maximum 20 of the dose applied to the well at the final 96 h postinfection time point Fig 4a The mean radioactivity values measured in the wells were 89 8H110065 6 0 77H110060 15 and 0 57H110060 04 H9262Ci for rMERS CoV hNIS infected rMERS CoV infected and uninfected cells respectively 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 meanH11006standard 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 40H11003 objective rMERS CoV Expressing the Sodium Iodide Symporter November December 2018 Volume 3 Issue 6 e00540 18 msphere asm org 3 on March 11 2019 by guest http msphere asm org Downloaded from 99m Tc pertechnetate uptake was dependent on the virus MOI Fig 4b Higher MOI was associated with greater accumulation of 99m Tc pertechnetate by rMERS CoV hNIS infected cells Uptake by rMERS CoV hNIS infected cells was also dependent on the dose of 99m Tc pertechnetate Fig 4c The relationship between radioactivity measured by the gamma camera on plate images and the dose of 99m Tc pertechnetate added to each well was linear with high correlation R 2 H11005 0 99 linear regression At 96 hours postinfection with an MOI of 0 01 uptake was slightly above the background level when the 99m Tc pertechnetate dose of 0 02 mCi per well was applied and no uptake was detected when 0 004 mCi of 99m Tc pertechnetate was added to each well Fig 4c Therefore the 99m Tc pertechnetate dose should be considered a limiting factor for detection of hNIS expression in rMERS CoV hNIS infected cells Importantly no changes 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 CH11001 and uninfected negative control cells CH11002 were used as controls FIG 3 Radio uptake of 99m Tc 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 99m Tc 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 99m Tc pertechnetate Chefer et al November December 2018 Volume 3 Issue 6 e00540 18 msphere asm org 4 on March 11 2019 by guest http msphere asm org Downloaded from in 99m Tc pertechnetate accumulation were detected in the wells infected with the parental virus rMERS CoV regardless of the dose The specificity of 99m Tc pertechnetate uptake by rMERS CoV hNIS infected cells was confirmed by adding sodium perchlorate a specific and competitive inhibitor of hNIS protein function and assessing uptake at 24 h postinfection Fig 5a At a concentration of 0 1 mM sodium perchlorate blocked 99 5 of the cellular uptake of 99m Tc pertechnetate in rMERS CoV hNIS infected cells at an MOI of 0 01 Sodium perchlorate concentrations below 0 1 mM reduced the inhibitory effect For example radiotracer uptake was inhibited by 50 at sodium perchlorate concentrations of 0 001 mM at 24 h postinfection Fig 5a The effective inhibition of 99m Tc pertechnetate uptake with 0 1 mM sodium perchlorate declined between 48 and 96 h postinfection Fig 5b sug gesting that viral transcription can overcome the inhibitory effect By 96 h postinfec FIG 4 Quantification of 99m Tc pertechnetate uptake by rMERS CoV hNIS infected cells a 99m Tc 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 99m Tc 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 99m Tc pertechnetate uptake applied at doses ranging from 0 6 to 0 004 mCi per well FIG 5 Sodium perchlorate mediated inhibition of 99m Tc pertechnetate uptake a Quantitation of 99m Tc pertechnetate uptake in the presence of sodium perchlorate at doses ranging from 0 and 0 1 mM 99m Tc pertechnetate uptake was reduced in rMERS CoV hNIS infected cells with increasing sodium per chlorate concentrations b 99m Tc 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 rMERS CoV Expressing the Sodium Iodide Symporter November December 2018 Volume 3 Issue 6 e00540 18 msphere asm org 5 on March 11 2019 by guest http msphere asm org Downloaded from tion 99m Tc pertechnetate uptake in rMERS CoV hNIS infected cells was nearly double the uptake at 24 h postinfection in the presence of sodium perchlorate Fig 5b In vivo evaluation of rMERS CoV hNIS CRISPR generated 288 330 H11001 H11001 DPP4 hu manized mice were challenged with parental rMERS CoV or rMERS CoV hNIS by the intranasal route and monitored by SPECT imaging One group each of mice infected with rMERS CoV hNIS and parental rMERS CoV were euthanized on day 3 postexposure pe while another set was monitored to day 7 pe and euthanized SPECT CT imaging was performed to determine whether sites of virus replication were evident in these animals Unfortunately the low resolution of the clinical scanner used in our biocon tainment facility prevented clear discrimination of hNIS expressing tissues and limited the utility of the imaging Details of the SPECT CT data can be found in Fig S1 and the methodology is described in Text S1 in the supplemental material Macroscopic evaluation of the lungs from virus and sham exposed animals did not reveal any significant pathological changes except in one of the five mice in group 2 rMERS CoV The right caudodorsal lung lobe of this animal was congested Micro scopically minimal to mild perivascular and peribronchiolar inflammation was noted multifocally with variable congestion in rMERS CoV and rMERS CoV hNIS infected mice Fig 6a to e Kidney congestion was also noticeable in 40 and 50 of mice in groups 2 and 3 respectively on day 3 pe and 33 and 50 on day 7 pe in mice in groups 4 and 5 respectively infected with rMERS CoV or rMERS CoV hNIS Table 1 The percentage of mice with alterations in the lung was higher on day 7 pe compared to that observed on day 3 pe 100 vs H1102183 respectively Extensive focal congestion in the lungs and multifocal congestion in the kidneys were observed in one of five mice from group 1 receiving sham infection but inflammation was not manifested in this group Table 1 Infectious virus could be detected only in the lungs from 3 6 mice from group 3 5 01 H11006 0 45 log 10 PFU mg but was detected in the lung tissue by plaque assay in 4 of 5 mice from group 2 6 7H110060 43 log 10 mean H11006 SD PFU mg Fig 6f The differences in viral loads between groups 2 and 3 were not statistically significant two tailed t test PH110050 214 RT PCR indicated maintenance of the hNIS transgene in 3 of 6 mice in group 3 Fig 6g The inability to detect virus in 3 of 6 mice and the nearly 2 log 10 decrease in PFU mg observed for rMERS CoV hNIS at day 3 supports a loss of viral fitness in vivo Virus could not be detected by plaque assay or RT PCR at 7 days postinfection DISCUSSION This is the first report describing the application of a recombinant RNA virus expressing a SPECT PET reporter gene to study viral pathogenesis Our data demon strate the feasibility of using hNIS as a reporter protein in animal models of human disease provided a higher resolution SPECT CT or PET CT is available Despite the presence of a robust signal the clinical grade SPECT CT in our facility did not provide the necessary resolution to demonstrate in vivo functionality of the rMERS CoV hNIS in mice If available a micro PET CT and the use of tetrafluorborate an alternate hNIS ligand could be used to increase sensitivity and improve signal to noise 26 The recombinant MERS CoV hNIS has similar fitness to the parental virus rMERS CoV as evidenced by similar kinetics fitness and cytopathic effect in vitro The results of in vivo evaluation also support similar fitness however further development would require insertion of the hNIS transgene into other locations within the virus and insertion of the transgene into the MERS 15 virus which can cause lethal respiratory disease in the 288 300 H11001H11001 mice Genetic stability of the hNIS transgene cloned into MERS CoV was confirmed over six in vitro passages hNIS maintained its function in rMERS Co