Compartmentalized SARS-CoV-2 Replication in the Upper vs Lower Respiratory Tract After Intranasal Inoculation or Aerosol Exposure

Abstract

The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in close to 7 million deaths as of the end of August 2023. The virus is transmitted primarily via aerosols and respiratory droplets [1]. Infected individuals can shed virus before exhibiting symptoms, with peak shedding detected at symptom onset [2]. Additionally, the virus has a half-life >1 hour as a suspended aerosol [3], which, unlike small droplets, can remain suspended for an extended period.

Animal models are instrumental in the investigation of pathogenesis caused by infectious agents, as well as in preclinical research on vaccines and antivirals [4]. In selecting an animal model, one must carefully consider the research question and weigh variables such as the inoculation route and resulting disease profile to ensure that the studies are done consistently and stringently. We previously showed that the inoculation route affects SARS-CoV-2 disease progression in Syrian hamsters [5]. SARS-CoV-2 inoculation of nonhuman primate (NHP) models has been done via a multitude of routes, including intranasal, intratracheal, and aerosol inoculation, with varying outcomes regarding disease severity, virus shedding, and tissue tropism [6–10]. In the current study, we directly compare SARS-CoV-2 tropism in rhesus macaques inoculated via the intranasal or aerosol route and compare outcomes with a previous NHP study in which we used multiroute inoculation [11].

METHODS

Biosafety and Ethics Approval

Approval for studies involving NHPs was provided by the Animal Care and Use Committee at the Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Animal studies were carried out in a facility accredited by the Association for Assessment and AAALAC International, following the basic principles and guidelines in the Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, the US Department of Agriculture, and the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. Rhesus macaques were housed in individual primate cages enabling social interactions in a climate-controlled room with a fixed light/dark cycle (12 hours/12 hours). Commercial monkey chow, treats, and fruit were provided by trained personnel. Water was available ad libitum. Environmental enrichment consisted of various human interactions, manipulanda, treats, videos, and music. Animals were observed at least twice daily. The Institutional Biosafety Committee approved work with infectious SARS-CoV-2 virus strains under biosafety level 3 conditions at minimum. Virus inactivation of all samples was performed according to committee-approved standard operating procedures for the removal of specimens from high containment areas.

Cells and Virus

SARS-CoV-2 strain SARS-CoV-2/human/USA/WA-001/2020 (MN985325.1; provided by the Centers for Disease Control and Prevention) was obtained from a patient from Washington in 2020 and propagated in Vero E6 cells in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 2% fetal bovine serum, 1mM L-glutamine, 50 U/mL of penicillin, and 50 μg/mL of streptomycin. Vero E6 cells were provided by R. Baric, University of North Carolina, and mycoplasma testing was performed monthly. The virus stock was sequenced and analyzed with Bowtie2 version 2.2.9, and no single-nucleotide polymorphisms were detected as compared with the patient sample sequence. Virus titrations were performed by end point titration in Vero E6 cells, which were inoculated with 10-fold serial dilutions of virus in 96-well plates. Cells were incubated at 37 °C and 5% CO₂. The cytopathic effect was read 5 days later.

Experimental Design

Four male rhesus macaques (aged 3 years, 4.2–5.2 kg) were inoculated with SARS-CoV-2/human/USA/WA-001/2020 via the aerosol route with an average dose of 1.5 × 10³ TCID₅₀/animal (50% tissue culture infectious dose). All animals received between 9 × 10² and 2.6 × 10³ TCID₅₀ (Supplementary Table 1). These small differences in received inoculum are not expected to make a difference in disease outcome. Aerosol exposure was performed via the AeroMP aerosol management platform (Biaera Technologies). Prior to exposure, the necessary volume of inhaled inoculum was estimated by the respiratory minute volume rates of the animals, determined by the Head-Out Buxco plethysmography unit and FinePointe software version 2.9.2.12849 (Data Sciences International). Once the necessary volume of inoculum was calculated for each macaque, it was anesthetized and exposed to the calculated inoculum volume while contained in a head-only exposure unit. Aerosol droplet nuclei were generated by a 3-jet Collison nebulizer (CH Technologies) and ranged from 1 to 5 µm. A sample of 6 L of air per minute was collected on a 47-mm gelatin filter from the head-only unit during the <10-minute exposure (Sartorius). Postexposure, the filters were dissolved in 10 mL of prewarmed (37 °C) DMEM containing 10% fetal bovine serum. Infectious virus was titrated as previously described, and the aerosol concentration was calculated. This method delivers roughly the same dose of virus to all animals and allows a close approximation of the actual virus dose received by each animal to be calculated. In the second cohort, 4 male rhesus macaques (aged 3 years, 3.6–4.8 kg) were inoculated with the same strain of SARS-CoV-2 via the intranasal route with a dose of 8 × 10⁵ TCID₅₀/animal (0.5 mL per naris; total volume, 1 mL). Hereafter, animals were observed and scored daily by the same person blinded to study group allocation using a standardized method [6]. Clinical examinations were performed on 0, 1, 3, 5, and 7 days postinoculation (DPI). Nasal and oropharyngeal swabs were collected on all days that examinations were performed. Bronchoalveolar lavage (BAL) was performed on 3 and 5 DPI.

RNA Extraction and Quantitative Reverse-Transcription Polymerase Chain Reaction

Up to 30 mg of tissue was homogenized in RLT buffer and RNA was extracted via the RNeasy Kit (Qiagen), whereas RNA was extracted from BAL fluid and swabs via the QiaAmp Viral RNA Kit (Qiagen), both according to the manufacturer's instructions. A viral genomic RNA-specific assay [12] was used for the detection of viral RNA.

Histology

Collected tissues were fixed for a minimum of 7 days in 10% neutral-buffered formalin and subsequently embedded in paraffin, processed with a VIP-6 Tissue Tek tissue processor (Sakura Finetek), and embedded in Ultraffin paraffin polymer (Cancer Diagnostics). Samples were sectioned at 5 μm, stained with hematoxylin and eosin, and evaluated by a board-certified veterinary pathologist blinded to study groups.

Statistical Analyses

To investigate significant differences among the 3 study groups, 2-way analysis of variance was conducted with Prism version 9.3.1 (GraphPad). Analysis was performed with either a Geisser-Greenhouse correction followed by a Tukey multiple-comparisons test or a Kruskal-Wallis test followed by a Dunn multiple-comparisons test.

Data Availability

All data can be accessed on https://figshare.com/articles/journal_contribution/_b_Compartmentalized_SARS-CoV-2_replication_in_upper_versus_lower_respiratory_tract_after_intranasal_inoculation_or_aerosol_exposure_b_/24036612.

RESULTS

An increase in clinical score was observed in all SARS-CoV-2–inoculated animals and was defined as mild disease (Figure 1A; euthanasia is mandated at a clinical score ≥35). The clinical score was driven by respiratory signs and decreased appetite (Supplementary Table 2). No significant difference was observed among the clinical scores of the study groups at any time point or when the area under the curve for the clinical score was determined (Figure 1B). We then compared clinical scores with those obtained from the NHP study previously performed in our laboratory [11], in which animals were inoculated via a combination of an intranasal, intratracheal, oral, and ocular route and received a total dose of 2.6 × 10⁶ TCID₅₀/animal. These animals displayed higher clinical scores at 3, 4, 5, and 6 DPI than animals exposed to aerosols (Figure 1A), which was reflected in the area under the curve analysis (Figure 1B). In nasal swabs, the highest amount of viral RNA was detected at 1 DPI in animals inoculated via the intranasal and multiroute method and at 3 DPI in animals exposed to aerosols. A significant difference was noted at 1 DPI between the multiroute and aerosol-inoculated animals (Figure 1C). All animals had evidence of virus replication in nasal swabs, and no significant difference in the total amount of virus shed was detected among the groups (Figure 1D). BAL was performed on 3 and 5 DPI. Genomic viral RNA levels were low in BAL fluid in animals inoculated via the intranasal or aerosol route and significantly higher in animals inoculated via the multiroute (Figure 1E). Genomic viral RNA was detected in the BAL fluid of all animals inoculated via the aerosol route but in only 1 of 4 animals inoculated via the intranasal route (animal IN1, Figure 1F). Interestingly, animal IN1 also had a consistently higher clinical score throughout the study (Figure 1B). Genomic viral RNA detected in tissues isolated at 7 DPI was low and limited to the upper respiratory tract in animals inoculated via the intranasal route but in the lower respiratory tract of 3 of 4 animals exposed to aerosols and 6 of 6 animals exposed via the multiroute (Figure 1G). Thus, although no differences among groups were detected in virus replication in the upper respiratory tract, virus replication in the lower respiratory tract was observed just in animals exposed to aerosols as compared with those inoculated via the intranasal route.

Figure 1

Clinical score and viral load in rhesus macaques after inoculation with SARS-CoV-2. Animals were inoculated via the intranasal route (blue); exposed to aerosols (orange); or inoculated via the intranasal, intratracheal, oral, and ocular route (gray; historical data from van Doremalen et al [11]). A, Clinical signs were scored daily. Data are presented as median (symbols) with 95% CI (shaded areas). B, Total clinical score for experiment. C, E, Genomic viral RNA detected in nasal swabs and BALF. Data are presented as median (symbols) with 95% CI (shaded areas). Line: qualitative limit of detection. D, F, Total amount of genomic viral RNA shed per animal as measured in nasal swabs and BALF. Bar: median. G, Genomic viral RNA detected in tissue samples collected at 7 days postinoculation. Bar: median. B, D, F, Circle, animal 1; square, animal 2; upward triangle, animal 3; downward triangle, animal 4; diamond, animal 5; hexagon, animal 6. Statistical significance was determined via 2-way analysis of variance with (A, C, E) a Geisser-Greenhouse correction followed by a Tukey multiple-comparisons test or (B, D, F) a Kruskal-Wallis test followed by a Dunn multiple-comparisons test. Asterisks display group-specific differences by color. ***P < .001. **P < .01. *P < .05. BALF, bronchoalveolar lavage fluid; IN, intranasal.

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Histologic examination of lung tissue showed evidence of interstitial pneumonia in 3 of 4 animals in both groups, defined as minimal lesions. The lesions in the lungs of 2 of 4 animals exposed to aerosols included type 2 pneumocyte hyperplasia, which is typical for SARS-CoV-2 infection as described previously [6], whereas in accordance with the lack of SARS-CoV-2 replication in the lower respiratory tract, type 2 pneumocyte hyperplasia was absent in the lung tissue of animals inoculated via the intranasal route (Table 1). When we used the multiroute to inoculate animals, type 2 pneumocyte hyperplasia was found in 3 of 6 animals [11], suggesting a phenotype more like the aerosol-exposed animals than the intranasally inoculated animals. Common extrapulmonary background pathology not necessarily linked to SARS-CoV-2 infection was present in both groups with no differences between groups.

DISCUSSION

Both infection routes resulted in mild and transient respiratory disease as defined by the clinical signs observed during the experiment. No differences were observed in virus replication in the upper respiratory tract, but replication in the lower respiratory tract was more likely to occur in animals inoculated with aerosols. This is similar to previous work from our group, in which we showed differences in disease progression in Syrian hamsters driven by the inoculation route: intranasal and aerosol inoculation was associated with more severe disease than fomite exposure [5]. Differences observed in this study were correlated with where the virus was deposited during inoculation: our data suggested that intranasal inoculation primarily deposited infectious virus in the upper respiratory tract, whereas aerosol and multiroute inoculation deposited virus in the entire respiratory tract, including the lower respiratory tract. This is in accordance with previous studies showing that the inoculation route greatly affects deposition in the respiratory tract [13, 14].

Although aerosol inoculation occurred with a much lower inoculum than intranasal inoculation (1.5 × 10³ vs 8 × 10⁵ TCID₅₀), no significant differences were found in upper respiratory tract replication. Likewise, in comparison with the multiroute inoculation, SARS-CoV-2 replication in the respiratory tract was reduced only in the lower respiratory tract, not the upper. Analysis of 12 influenza challenge studies in humans concluded that intranasal inoculation leads to 20-times lower infectivity than aerosol exposure [15]. It is possible that the bioavailability of aerosolized virus is much higher than that delivered in a mass volume. It should be noted that at 1 DPI, virus replication in the upper respiratory tract trended lower in the aerosol-exposed group vs the other 2 groups. This suggests that in animals exposed to aerosols, virus kinetics differed from the other 2 groups, but this did not affect the total amount of virus detected in nasal swabs. We previously observed exposure-dependent SARS-CoV-2 shedding in Syrian hamsters [5].

In conclusion, we show that the SARS-CoV-2 inoculation route utilized in rhesus macaque models affects the resulting virus tropism. Our findings support SARS-CoV-2 inoculation via the multiroute for the preclinical evaluation of vaccines and antivirals since this results in more virus replication in lung tissue and a robust phenotype. We recommend carefully considering these variables when selecting an animal model for subsequent studies.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Acknowledgments. We thank the animal caretakers of the Rocky Mountain Veterinary Branch, and Myndi Holbrook, Laboratory of Virology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for their assistance during the study.

Author contributions. H. F., V. J. M., E. d. W., and N. v. D. designed the study. R. J. F., T. B., B. N. W., L. P.-P., F. F., J. L., G. S., D. S., V. J. M., E. d. W., and N. v. D. acquired, analyzed, and interpreted the data. R. J. F. and N. v. D. wrote the manuscript. All authors have approved the submitted version.

Financial support. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

References

Author notes