Traditional pathogenesis studies of alphaviruses involves monitoring survival, viremia, and pathogen dissemination via serial necropsies; however, molecular imaging shifts this paradigm and provides a dynamic assessment of pathogen infection. Positron emission tomography (PET) with PET tracers targeted to study neuroinflammation (N,N-diethyl-2-[4-phenyl]-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide, [¹⁸F]DPA-714), apoptosis (caspase-3 substrate, [¹⁸F]CP-18), hypoxia (fluormisonidazole, [¹⁸F]FMISO), blood–brain barrier (BBB) integrity ([¹⁸F]albumin), and metabolism (fluorodeoxyglucose, [¹⁸F]FDG) was performed on C3H/HeN mice infected intranasally with 7000 plaque-forming units (PFU) of Venezuelan equine encephalitis virus (VEEV) TC-83. The main findings are as follows: (1) whole-brain [¹⁸F]DPA-714 and [¹⁸F]CP-18 uptake increased three-fold demonstrating, neuroinflammation and apoptosis, respectively; (2) [¹⁸F]albumin uptake increased by 25% across the brain demonstrating an altered BBB; (3) [¹⁸F]FMISO uptake increased by 50% across the whole brain indicating hypoxic regions; (4) whole-brain [¹⁸F]FDG uptake was unaffected; (5) [¹⁸F]DPA-714 uptake in (a) cortex, thalamus, striatum, hypothalamus, and hippocampus increased through day seven and decreased by day 10 post exposure, (b) olfactory bulb increased at day three, peaked day seven, and decreased day 10, and (c) brain stem and cerebellum increased through day 10. In conclusion, intranasal exposure of C3H/HeN mice to VEEV TC-83 results in both time-dependent and regional increases in brain inflammation, apoptosis, and hypoxia, as well as modest decreases in BBB integrity; however, it has no effect on brain glucose metabolism.

While preclinical and clinical imaging have been applied to drug discovery/development and characterization of disease pathology, few examples exist where imaging has been used to evaluate infectious agents or countermeasures to biosafety level (BSL)3/4 threat agents. Viruses engineered with reporter constructs, i.e., enzymes and receptors, which are amenable to detection by positron emission tomography (PET), single photon emission tomography (SPECT), or magnetic resonance imaging (MRI) have been used to evaluate the biodistribution of viruses containing specific therapeutic or gene transfer payloads. Bioluminescence and nuclear approaches involving engineered reporters, direct labeling of bacteria with radiotracers, or tracking bacteria through their constitutively expressed thymidine kinase have been utilized to characterize viral and bacterial pathogens post-infection. Most PET, SPECT, CT, or MRI approaches have focused on evaluating host responses to the pathogens such as inflammation, brain neurochemistry, and structural changes and on assessing the biodistribution of radiolabeled drugs. Imaging has the potential when applied preclinically to the development of countermeasures against BSL3/4 threat agents to address the following: (1) presence, biodistribution, and time course of infection in the presence or absence of drug; (2) binding of the therapeutic to the target; and (3) expression of a pharmacologic effect either related to drug mechanism, efficacy, or safety. Preclinical imaging could potentially provide real-time dynamic tools to characterize the pathogen and animal model and for developing countermeasures under the U.S. FDA Animal Rule provision with high confidence of success and clinical benefit.

The association of Zika virus (ZIKV) infection and development of neurological sequelae require a better understanding of the pathogenic mechanisms causing severe disease. The purpose of this study was to evaluate the ability and sensitivity of positron emission tomography (PET) imaging using [¹⁸F]DPA-714, a translocator protein (TSPO) 18 kDa radioligand, to detect and quantify neuroinflammation in ZIKV-infected mice.

Favipiravir (T705; 6-fluoro-3-hydroxypyrazine-2-carboxamide) is a pyrazine analog that has demonstrated potent antiviral activity against a broad spectrum of viruses in multiple in vivo disease models. To better understand the compounds anti-viral activity, assessment of the drug’s biodistribution and kinetics in vivo may lend insight into how best to evaluate the compound efficacy preclinically and to contribute to the design of clinical studies to take into account the compound’s pharmacokinetic distribution and kinetics. In the current study, a method for synthesis of [¹⁸F]favipiravir was developed and the biodistribution in mice naïve to and pre-dosed with favipiravir was assessed by PET and gamma counting of tissue samples. Fluorine-18 labeling of favipiravir was achieved in a one-pot, two-step synthesis using a commercially available precursor, methyl-5-chloroisoxazolo[4,5-b]pyrazine-3-carboxylate, with an overall radiochemical yield of 15–24%, a molar activity of 37–74 GBq/µmol in a 70 minute synthesis time. [¹⁸F]favipiravir tissue uptake and distribution was similar in naïve and pre-dosed mice; however, in the pre-dosed animals plasma clearance was more rapid and tissue clearance appeared to be prolonged. In conclusion, application of PET to the evaluation of favipiravir has demonstrated the importance of dosing regimen on the distribution and tissue uptake and clearance of the molecule. Favipiravir is cleared through the kidney as previously reported but the liver and intestinal excretion may also play an important role in compound elimination. Measurement of the tissue uptake of favipiravir as determined by PET may be a more important indicator of a compound’s potential efficacy than purely monitoring plasma parameters such as viremia and drug levels.

Critical bacterial pathogens of public health and biodefense concerns were engineered to constitutively express Escherichia coli enzyme thymidine kinase (TK) that allows for noninvasive nuclear imaging via phosphorylation and entrapment of radiolabeled nucleoside analog 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-iodouracil (FIAU). Expression of functional TK was established using a nucleoside analog Zidovudine that impeded the growth of tk-engineered bacteria. Significantly, no observable growth differences were detected for FIAU. High resolution mass spectrometry with Pseudomonas aeruginosa PAO1 and its tk variant (PAO1TK) confirmed FIAU phosphorylation and retention only in PAO1TK. In vitro gamma counting with wild-type PAO1, Acinetobacter baumannii and Burkholderia pseudomallei Bp82 and their tk derivatives with [¹⁸F]FIAU further confirmed that tk variants selectively incorporated the radiotracer, albeit with varying efficiencies. In vitro [¹⁸F]FIAU labeling coupled with in vivo Positron Emission Tomography/Computed Tomography (PET/CT) imaging of PAO1 and PAO1TK confirmed that only PAO1TK can be imaged in mice at sensitivities ≥10⁷ bacteria per infection site. This was further verified by administering [¹⁸F]FIAU to animals infected with PAO1 and PAO1TK. Utility of tk-engineered P. aeruginosa in noninvasive PET/CT imaging for bacterial therapeutic evaluation in animals was demonstrated employing antibiotic ciprofloxacin, underscoring the immediate use of PAO1TK and potentially other engineered pathogens for evaluating experimental therapeutics.