Longitudinal analyses using 18F-Fluorodeoxyglucose positron emission tomography with computed tomography as a measure of COVID-19 severity in the aged, young, and humanized ACE2 SARS-CoV-2 hamster models.

This study compared disease progression of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in three different models of golden hamsters: aged (≈60 weeks old) wild-type (WT), young (6 weeks old) WT, and adult (14-22 weeks old) hamsters expressing the human-angiotensin-converting enzyme 2 (hACE2) receptor. After intranasal (IN) exposure to the SARS-CoV-2 Washington isolate (WA01/2020), 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography with computed tomography (18F-FDG PET/CT) was used to monitor disease progression in near real time and animals were euthanized at pre-determined time points to directly compare imaging findings with other disease parameters associated with coronavirus disease 2019 (COVID-19). Consistent with histopathology, 18F-FDG-PET/CT demonstrated that aged WT hamsters exposed to 105 plaque forming units (PFU) developed more severe and protracted pneumonia than young WT hamsters exposed to the same (or lower) dose or hACE2 hamsters exposed to a uniformly lethal dose of virus. Specifically, aged WT hamsters presented with a severe interstitial pneumonia through 8 d post-exposure (PE), while pulmonary regeneration was observed in young WT hamsters at that time. hACE2 hamsters exposed to 100 or 10 PFU virus presented with a minimal to mild hemorrhagic pneumonia but succumbed to SARS-CoV-2-related meningoencephalitis by 6 d PE, suggesting that this model might allow assessment of SARS-CoV-2 infection on the central nervous system (CNS). Our group is the first to use (18F-FDG) PET/CT to differentiate respiratory disease severity ranging from mild to severe in three COVID-19 hamster models. The non-invasive, serial measure of disease progression provided by PET/CT makes it a valuable tool for animal model characterization.

Characteristic and quantifiable COVID-19-like abnormalities in CT- and PET/CT-imaged lungs of SARS-CoV-2-infected crab-eating macaques (Macaca fascicularis).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing an exponentially increasing number of coronavirus disease 19 (COVID-19) cases globally. Prioritization of medical countermeasures for evaluation in randomized clinical trials is critically hindered by the lack of COVID-19 animal models that enable accurate, quantifiable, and reproducible measurement of COVID-19 pulmonary disease free from observer bias. We first used serial computed tomography (CT) to demonstrate that bilateral intrabronchial instillation of SARS-CoV-2 into crab-eating macaques (Macaca fascicularis) results in mild-to-moderate lung abnormalities qualitatively characteristic of subclinical or mild-to-moderate COVID-19 (e.g., ground-glass opacities with or without reticulation, paving, or alveolar consolidation, peri-bronchial thickening, linear opacities) at typical locations (peripheral>central, posterior and dependent, bilateral, multi-lobar). We then used positron emission tomography (PET) analysis to demonstrate increased FDG uptake in the CT-defined lung abnormalities and regional lymph nodes. PET/CT imaging findings appeared in all macaques as early as 2 days post-exposure, variably progressed, and subsequently resolved by 6-12 days post-exposure. Finally, we applied operator-independent, semi-automatic quantification of the volume and radiodensity of CT abnormalities as a possible primary endpoint for immediate and objective efficacy testing of candidate medical countermeasures.

Cranial Vena Cava Syndrome in Guinea Pigs with Chronic Jugular Vein Catheters.

Guinea pigs are a premier small animal model for infectious disease research, and chronic indwelling venous access ports may be used to facilitate various procedures. Here we report catheter-related lesions in 5 uninfected Dunkin-Hartley guinea pigs with chronic jugular vein catheters used for imaging studies. Three guinea pigs were found dead with no premonitory signs. At necropsy, there was severe bilateral pulmonary atelectasis due to 20 to 29 mL of pleural effusion resulting from catheter-related thrombosis and cranial vena cava syndrome. In addition, one of these 3 guinea pigs had a polymicrobial catheter infection with abscessation. A 4th clinically normal guinea pig was euthanized at the end of the study, having spontaneously lost its catheter 7 mo prior, and had 17 mL of pleural effusion. The 5th guinea pig was euthanized following pooling of contrast material around the distal catheter in the cranial vena cava on CT. By histology, affected animals had recent and remote thrombosis or fibrosis (or both) of the cranial vena cava and right atrial wall, with osseous and cartilaginous metaplasia. Cranial vena cava syndrome should be considered as a differential for dyspnea or death in chronically catheterized laboratory animals.

Animal Models of Ebolavirus Infection.

Ebola virus is a highly pathogenic member of the family Filoviridae that causes a severe hemorrhagic disease in humans and NHP. The 2013-2016 West African outbreak has increased interest in the development and refinement of animal models of Ebola virus disease. These models are used to test countermeasures and vaccines, gain scientific insights into the mechanisms of disease progression and transmission, and study key correlates of immunology. Ebola virus is classified as a BSL4 pathogen and Category A agent, for which the United States government requires preparedness in case of bioterrorism. Rodents, such as Syrian golden hamsters (Mesocricetus auratus), mice (Mus musculus), and guinea pigs (Cavia porcellus), are the most common research species. However, NHP, especially macaques, are favored for Ebola virus disease research due to similarities with humans regarding the pathogenesis, clinical presentation, laboratory findings, and causes of fatality. To satisfy the regulatory requirements for approval of countermeasures against high-consequence pathogens, the FDA instituted the Animal Rule, which permits efficacy studies in animal models in place of human clinical data when such studies are not feasible or ethical. This review provides a comprehensive summary of various animal models and their use in Ebola virus disease research.

Assurance of neuroattenuation of a live vaccine against West Nile virus: a comprehensive study of neuropathogenesis after infection with chimeric WN/DEN4Δ30 vaccine in comparison to two parental viruses and a surrogate flavivirus reference vaccine.

The upsurge of West Nile virus (WNV) human infections in 2012 suggests that the US can expect periodic WNV outbreaks in the future. Availability of safe and effective vaccines against WNV in endemic areas, particularly for aging populations that are at high risk of West Nile neuroinvasive disease (WNND), could be beneficial. WN/DEN4Δ30 is a live, attenuated chimeric vaccine against WNV produced by replacement of the genes encoding the pre-membrane and envelope protein genes of the vaccine virus against dengue virus type 4 (DEN4Δ30) with corresponding sequences derived from a wild type WNV. Following intrathalamic inoculation of nonhuman primates (NHPs), a comprehensive neuropathogenesis study was performed and neurovirulence of WN/DEN4Δ30 vaccine candidate was compared to that of two parental viruses (i.e., WNV and DEN4Δ30), as well as to that of an attenuated flavivirus surrogate reference (i.e., yellow fever YF 17D). Clinical and virological data, as well as results of a semi-quantitative histopathological analysis, demonstrated that WN/DEN4Δ30 vaccine is highly attenuated for the central nervous system (CNS) of NHPs in comparison to a wild type WNV. Importantly, based on the virus replicative ability in the CNS of NHPs and the degree of induced histopathological changes, the level of neuroattenuation of WN/DEN4Δ30 vaccine was similar to that of YF 17D, and therefore within an acceptable range. In addition, we show that the DEN4Δ30 vaccine tested in this study also has a low neurovirulence profile. In summary, our results demonstrate a high level of neuroattenuation of two vaccine candidates, WN/DEN4Δ30 and DEN4Δ30. We also show here a remarkable sensitivity of our WNV-NY99 NHP model, as well as striking resemblance of the observed neuropathology to that seen in human WNND. These results support the use of this NHP model for translational studies of WNV neuropathogenesis and/or testing the effectiveness of vaccines and therapeutic approaches.

Progressive mitochondrial compromise in brains and livers of primates exposed in utero to nucleoside reverse transcriptase inhibitors (NRTIs).

Mitochondrial compromise has been documented in infants born to women infected with the human immunodeficiency virus (HIV-1) who received nucleoside reverse transcriptase inhibitor (NRTI) therapy during pregnancy. To model these human exposures, we examined mitochondrial integrity at birth and 1 year in brain cortex and liver from offspring of retroviral-free Erythrocebus patas dams-administered human-equivalent NRTI doses for the last half (10 weeks) of gestation. Additional infants, followed for 1 year, were given the same drugs as their mothers for the first 6 weeks of life. Exposures included: no drug, Zidovudine (AZT), Lamivudine (3TC), AZT/3TC, AZT/Didanosine (ddI), and Stavudine (d4T)/3TC. In brain and liver, oxidative phosphorylation (OXPHOS) enzyme activities (complexes I, II, and IV) showed minimal differences between unexposed and NRTI-exposed offspring at both times. Brain and liver mitochondria from most NRTI-exposed patas, both at birth and 1 year of age, contained significant (p < 0.05) morphological damage observed by electron microscopy (EM), based on scoring of coded photomicrographs. Brain and liver mitochondrial DNA (mtDNA) levels in NRTI-exposed patas were depleted significantly in the 3TC and d4T/3TC groups at birth and were depleted significantly (p < 0.05) at 1 year in all NRTI-exposed groups. In 1-year-old infants exposed in utero to NRTIs, mtDNA depletion was 28.8-51.8% in brain and 37.4-56.5% in liver. These investigations suggest that some NRTI-exposed human infants may sustain similar mitochondrial compromise in brain and liver and should be followed long term for cognitive integrity and liver function.

Metabolism and pharmacokinetics of the combination Zidovudine plus Lamivudine in the adult Erythrocebus patas monkey determined by liquid chromatography-tandem mass spectrometric analysis.

Because of their similarity to humans, non-human primates constitute useful preclinical models in which to examine potential human drug toxicities. Antiretroviral nucleoside reverse transcriptase inhibitor (NRTI) toxicity is currently under investigation in Erythrocebus patas monkeys, and whereas NRTI pharmacokinetics have been studied in other monkey species, pharmacokinetics for Zidovudine plus Lamivudine (AZT/3TC) dosing have not been reported in the patas. Here we present 24 h serum pharmacokinetic parameters after a single oral exposure to the combination of AZT (40 mg) and 3TC (24 mg), doses equivalent to a human daily dose of Combivir. The patas (n=3) AZT/3TC pharmacokinetic profiles were similar to those seen in other primate species. Average maximum serum concentrations (Cmax) for AZT and 3TC were 2.35 and 2.65 microg/ml, respectively, and were observed at 0.83 h (Tmax). Cmax was 13.34 microg/ml for the AZT-glucuronide (AZT-G) and was 0.023 microg/ml for the potentially toxic minor metabolite 3'-amino-3'-deoxythymidine (AMT), both occurring at about 1 h after dosing. Similar elimination half-times, 0.70 and 0.68 h(-1), were found for AZT and AZT-G, respectively, while 3TC was eliminated about half as fast (0.33 h(-1)) resulting in AUC(0-infinity) values of 6.97 microg/ml h for 3TC, 2.99 microg/ml h for AZT, 20.5 microg/ml h for AZT-G and 0.002 for AMT 6.97 microg/ml h. This study shows similar metabolism and pharmacokinetics for oral administration of AZT/3TC in the adult patas monkey, other primate species and humans. The data validate the use of the patas monkey for studies of NRTI toxicity.

Erythrocebus patas monkey offspring exposed perinatally to NRTIs sustain skeletal muscle mitochondrial compromise at birth and at 1 year of age.

Antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs), given to human immunodeficiency virus-1-infected pregnant women to prevent vertical viral transmission, have caused mitochondrial dysfunction in some human infants. Here, we examined mitochondrial integrity in skeletal muscle from offspring of pregnant retroviral-free Erythrocebus patas dams administered human-equivalent NRTI doses for the last 10 weeks of gestation or for 10 weeks of gestation and 6 weeks after birth. Exposures included no drug, Zidovudine (AZT), Lamivudine (3TC), AZT/3TC, AZT/Didanosine (ddI), and Stavudine (d4T)/3TC. Offspring were examined at birth (n=3 per group) and 1 year (n=4 per group, not including 3TC alone). Circulating levels of creatine kinase were elevated at 1 year in the d4T/3TC-exposed group. Measurement of oxidative phosphorylation enzyme activities (complexes I, II, and IV) revealed minimal NRTI-induced changes at birth and at 1 year. Histochemistry for complex IV activity showed abnormal staining with activity depletion at birth and 1 year in groups exposed to AZT alone and to the 2-NRTI combinations. Electron microscopy of skeletal muscle at birth and 1 year of age showed mild to severe mitochondrial damage in all the NRTI-exposed groups, with 3TC inducing mild damage and the 2-NRTI combinations inducing extensive damage. At birth, mitochondrial DNA (mtDNA) was depleted by approximately 50% in groups exposed to AZT alone and the 2-NRTI combinations. At 1 year, the mtDNA levels had increased but remained significantly below normal. Therefore, skeletal muscle mitochondrial compromise occurs at birth and persists at 1 year of age (46 weeks after the last NRTI exposure) in perinatally exposed young monkeys, suggesting that similar events may occur in NRTI-exposed human infants.

Transplacentally exposed human and monkey newborn infants show similar evidence of nucleoside reverse transcriptase inhibitor-induced mitochondrial toxicity.

Effective reduction in maternal-fetal human immunodeficiency virus-1 (HIV-1) transmission has been achieved by administration of nucleoside reverse transcriptase inhibitors (NRTIs) during pregnancy, and although most exposed children are clinically normal at birth, mitochondrial dysfunction has been reported. To examine mitochondrial integrity on a molecular level, we evaluated mitochondrial morphology by electron microscopy (EM) and mitochondrial DNA (mtDNA) quantity in umbilical cords and cord blood from NRTI-exposed and unexposed human and monkey newborns. Human subjects included infants born to HIV-1-infected mothers who received Combivir (Zidovudine [AZT] plus Lamivudine [3TC]) (n = 9) or AZT plus Didanosine [ddI] (n = 2) during pregnancy, and infants born to HIV-1-uninfected mothers (n = 7). NRTI-exposed Erythrocebus patas monkey dams (n = 3 per treatment group) were given human-equivalent dosing regimens containing 3TC, AZT/3TC, AZT/ddI, or Stavudine (d4T)/3TC during gestation. Four infants born to unexposed patas dams served as controls. Mitochondria in umbilical cord endothelial cells from NRTI-exposed monkey and human infants showed substantial abnormal pathology by EM, the extent of which was quantified from coded photomicrographs and shown to be different (P < 0.05) from the unexposed monkey and human newborns. Significant (P < 0.05) mtDNA depletion was found in umbilical cords from both human and monkey NRTI-exposed infants and in human, but not in monkey, cord blood leukocytes. For umbilical cords, an increase in mitochondrial morphological damage correlated with reduction in mtDNA quantity in fetal monkeys (r = 0.94). The treatment-induced mitochondrial compromise in infant monkeys ranked as follows: d4T/3TC > AZT/ddI > AZT/3TC > 3TC. The study demonstrates that transplacental NRTI exposures induce similar mitochondrial damage in cord blood and umbilical cords taken from retroviral-uninfected monkey infants and from human infants born to HIV-1-infected women.

Cardiac mitochondrial compromise in 1-yr-old Erythrocebus patas monkeys perinatally-exposed to nucleoside reverse transcriptase inhibitors.

Hearts from 1-yr-old Erythrocebus patas monkeys were examined after in utero and 6-wk-postbirth exposure to antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs). Protocols were modeled on those given to human immunodeficiency virus (HIV)-1-infected pregnant women. NRTIs were administered daily to the dams for the last 20% or 50% of gestation, and to the infants for 6 wk after birth. Exposures included: no drug (n = 4); Zidovudine, 3'-azido-3'-deoxythymidine (AZT; n = 4); AZT/Lamivudine, (-)-beta-L-2', 3'-Dideoxy-3'-thiacytidine (Epivir, 3TC) (n = 4); AZT/Didanosine (Videx, ddI) (n = 4); and Stavudine (Zerit, d4T)/3TC (n = 4). Echocardiograms and clinical chemistry showed no drug-related changes, but the d4T/3TC-exposed fetuses at 6 and 12 mo had increased white cell counts (p < 0.05). At 1 yr of age, oxidative phosphorylation (OXPHOS) enzyme activities were similar in heart mitochondria from all groups. Mitochondrial pathology, that included clones of damaged mitochondria (p < 0.05), was found in hearts of all 1-yr drug-exposed infants. Levels of mtDNA were elevated (p < 0.05) in hearts of all NRTI-exposed monkeys in the following order: control < d4T/3TC < AZT < AZT/3TC < AZT/ddI. The clinical status of NRTI-exposed infants, as evidenced by behavior, clinical chemistry, OXPHOS activity and echocardiogram, was normal. However, extensive mitochondrial damage with clusters of similar-appearing damaged heart mitochondria observed by electron microscopy, and an increase in mtDNA quantity, that persisted at 1 yr of age, suggest the potential for cardiotoxicity later in life.

Mitochondrial toxicity in fetal Erythrocebus patas monkeys exposed transplacentally to zidovudine plus lamivudine.

This study was designed to investigate fetal mitochondrial toxicity in Erythrocebus patas monkeys exposed in utero to zidovudine (AZT) and lamivudine (3TC), and taken at term. Pregnant patas monkeys were given a daily dose of 40 mg AZT (86% of the human daily dose, based on body weight), for the last 10 weeks (50%) of gestation, and a daily dose of 24 mg 3TC (84% of the human daily dose, based on body weight) for the last 4 weeks of gestation. At term, AZT was found to be incorporated into fetal mitochondrial DNA from skeletal muscle, liver, kidney, and placenta. By transmission electron microscopy (EM) drug-exposed fetal cardiac and skeletal muscle cells showed mitochondrial membrane compromise, mitochondrial proliferation, and damaged sarcomeres, while mitochondria in brain cerebrum and cerebellum were morphologically normal. Substantial depletion of oxidative phosphorylation (OXPHOS) Complex I specific activities was observed in heart (87% reduction in mean, p = 0.02) and skeletal muscle (98% reduction in mean, p = 0.002) from drug-exposed fetuses, compared to unexposed fetuses. In addition Complex IV activity was highly depleted (85% reduction in mean, p = 0.004) in skeletal muscle from the drug-exposed fetuses (p = 0.004). Brain cerebrum and cerebellum showed no statistically significant OXPHOS changes with drug exposure. Mitochondrial DNA quantity was substantially depleted (>50%) in heart, skeletal muscle, cerebellum, and cerebrum from drug-exposed fetuses compared to unexposed controls. Overall, the data indicate that significant mitochondrial damage was observed at birth in monkey fetuses exposed in utero to AZT plus 3TC in a human-equivalent dosing protocol.