SARS-CoV-2 Aerosol and Intranasal Exposure Models in Ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the worldwide COVID-19 pandemic. Animal models are extremely helpful for testing vaccines and therapeutics and for dissecting the viral and host factors that contribute to disease severity and transmissibility. Here, we report the assessment and comparison of intranasal and small particle (~3 µm) aerosol SARS-CoV-2 exposure in ferrets. The primary endpoints for analysis were clinical signs of disease, recovery of the virus in the upper respiratory tract, and the severity of damage within the respiratory tract. This work demonstrated that ferrets were productively infected with SARS-CoV-2 following either intranasal or small particle aerosol exposure. SARS-CoV-2 infection of ferrets resulted in an asymptomatic disease course following either intranasal or small particle aerosol exposure, with no clinical signs, significant weight loss, or fever. In both aerosol and intranasal ferret models, SARS-CoV-2 replication, viral genomes, and viral antigens were detected within the upper respiratory tract, with little to no viral material detected in the lungs. The ferrets exhibited a specific IgG immune response to the SARS-CoV-2 full spike protein. Mild pathological findings included inflammation, necrosis, and edema within nasal turbinates, which correlated to positive immunohistochemical staining for the SARS-CoV-2 virus. Environmental sampling was performed following intranasal exposure of ferrets, and SARS-CoV-2 genomic material was detected on the feeders and nesting areas from days 2-10 post-exposure. We conclude that both intranasal and small particle aerosol ferret models displayed measurable parameters that could be utilized for future studies, including transmission studies and testing SARS-CoV-2 vaccines and therapeutics.

Comparison of Alfaxalone-Midazolam, Tiletamine-Zolazepam, and KetamineAcepromazine Anesthesia during Plethysmography in Cynomolgus Macaques (Macaca fascicularis) and Rhesus Macaques (Macaca mulatta).

Plethysmography is used in nonhuman primates (NHPs) to measure minute volume before aerosol exposure to an agent to calculate total time necessary in the exposure chamber. The consistency of respiratory parameters during the entire exposure time is paramount to ensuring dosing accuracy. Our study sought to validate an alfaxalone-midazolam (AM) anesthetic combination for use in aerosol studies. We hypothesized that AM would provide an adequate duration of anesthesia, achieve and maintain steady state minute volume (SSMV) for 20 min, and have anesthetic quality and side effects comparable to or better than either tiletamine-zolazepam (TZ) and ketamine-acepromazine (KA), the most common anesthetics used for this purpose currently. Two groups of NHPs, one consisting of 15 cynomolgus macaques and one of 15 rhesus macaques, received 3 intramuscular anesthetic combinations (AM, TZ, and KA), no less than one week apart. Anesthetized NHPs were placed in a plethysmograph chamber and their minute volumes were measured every 10 s to determine whether they had achieved SSMV and maintained it for at least 20 consecutive min. Achieving and reliably maintaining an SSMV for at least 20 min facilitates precise aerosol dosing of a challenge agent. Quality of anesthesia, based on the NHP's ability to achieve and maintain SSMV, was higher with AM compared with TZ and KA in both species, and AM had a longer duration of SSMV as compared with TZ and KA in cynomolgus macaques. Average SSMV was larger with AM compared with TZ in cynomolgus macaques, but larger with KA compared with AM in rhesus macaques. Duration of anesthesia was sufficient with all combinations but was longer for TZ than both AM and KA in both species. These results suggest that the AM anesthetic combination would produce the most accurate dosing for an aerosol challenge.

Effects of a Heated Anesthesia Breathing Circuit on Body Temperature in Anesthetized Rhesus Macaques (Macaca mulatta).

This study evaluated the effects of using a heated anesthesia breathing circuit in addition to forced-air warming on body temperature in anesthetized rhesus macaques as compared with forced-air warming alone. Hypothermia is a common perianesthetic and intraoperative complication that can increase the risk of negative outcomes. Body heat is lost through 4 mechanisms during anesthesia: radiation, conduction, convection, and evaporation. Typical warming methods such as forced-air warming devices, conductive heating pads, and heated surgical tables only influence radiative and conductive mechanisms of heat loss. A commercially available heated breathing circuit that delivers gas warmed to 104 °F can easily be integrated into an anesthesia machine. We hypothesized that heating the inspired anesthetic gas to address the evaporative mechanism of heat loss would result in higher body temperature during anesthesia in rhesus macaques. Body temperatures were measured at 5-min intervals in a group of 10 adult male rhesus macaques during 2 anesthetic events: one with a heated anesthesia breathing circuit in addition to forced-air warming, and one with forced-air warming alone. The addition of a heated breathing circuit had a significant positive effect on perianesthetic body temperature, with a faster return to baseline temperature, earlier nadir of initial drop in body temperature, and higher body temperatures during a 2-h anesthetic procedure. Use of a heated anesthesia breathing circuit should be considered as a significant refinement to thermal support during macaque anesthesia, especially for procedures lasting longer than one hour.

Human convalescent plasma protects K18-hACE2 mice against severe respiratory disease.

SARS-CoV-2 is the causative agent of COVID-19 and human infections have resulted in a global health emergency. Small animal models that reproduce key elements of SARS-CoV-2 human infections are needed to rigorously screen candidate drugs to mitigate severe disease and prevent the spread of SARS-CoV-2. We and others have reported that transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the Keratin 18 (K18) promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge. Here we report that some infected mice that survive challenge have residual pulmonary damages and persistent brain infection on day 28 post-infection despite the presence of anti-SARS-COV-2 neutralizing antibodies. Because of the hypersensitivity of K18-hACE2 mice to SARS-CoV-2 and the propensity of virus to infect the brain, we sought to determine if anti-infective biologics could protect against disease in this model system. We demonstrate that anti-SARS-CoV-2 human convalescent plasma protects K18-hACE2 against severe disease. All control mice succumbed to disease by day 7; however, all treated mice survived infection without observable signs of disease. In marked contrast to control mice, viral antigen and lesions were reduced or absent from lungs and absent in brains of antibody-treated mice. Our findings support the use of K18-hACE2 mice for protective efficacy studies of anti-SARS-CoV-2 medical countermeasures (MCMs). They also support the use of this system to study SARS-CoV-2 persistence and host recovery.

Investigation of Various Intramuscular Volumes Delivered to the Semimembranosus Muscle of Cavia porcellus.

The goal of this study is to provide quantitative data on the ideal volume for intramuscular (IM) injections into the semimembranosus muscle of guinea pigs weighing between 320 to 410 grams. This evaluation comprised 2 experiments. The first was to assess dispersion leakage of intramuscularly injected iohexol, a radiocontrast agent commonly used in Computed Tomography (CT), based on analysis of in vivo imaging. The second used varying volumes of intramuscularly injected sodium chloride (0.9% NaCl) to assess pain and pathology associated with IM injection. Hartley guinea pigs were injected IM with varying volumes of either iohexol or sodium chloride (150, 300, 500, 1000 and 1500 µL). In the iohexol experiment, results suggest IM volumes of 150 and 300 µL remain within the target muscle. In the experiment using sodium chloride, pain and pathology did not increase as IM volume increased. The pathology noted was related to needle tract through the musculature rather than the volume size of the injectate. The results did not reveal a correlation between volume of IM 0.9% NaCl and pain levels. We conclude that volume size correlates more with precision and accuracy of delivery into the intended muscle tissue. Regarding tissue distribution, our findings also suggest that the optimal capacity for IM injection in the semimembranosus muscle should be less than 500 µL.