6- to 9-week-old golden Syrian hamsters were acclimatized in biosafety level-2 (BSL-2) for 1 week and then infected in the animal BSL3 (ABSL-3) institutional facility. The animals were maintained under the 12-h light and dark cycle and fed a standard pellet diet and water ad libitum. All the experimental protocols involving the handling of virus culture, and animal infection were approved by RCGM, institutional biosafety, and IAEC Animal Ethics Committee (IAEC/THSTI/105).

SARS-related coronavirus 2, isolate USA-WA1/2020 virus was grown and titrated in vero E6 cell line cultured in Dulbecco’s modified Eagle medium (DMEM) complete media containing 4.5 g/L D-glucose, 100,000 U/L penicillin–streptomycin, 100 mg/L sodium pyruvate, 25 mM HEPES, and 2% FBS. The stocks of the virus were plaque purified at the THSTI IDRF facility inside ABSL3 following institutional biosafety guidelines.

6- to 9-week-old golden Syrian hamsters were procured from CDRI and quarantined for 1 week at the small animal facility (SAF), THST before starting the experiment. The animals were then randomly divided into five groups containing five animals/group, namely, uninfected (UI), infected (I), and two infected groups receiving til tailya (TT) or Anu oil (AO) as therapeutic interventions, respectively. One group received intranasal installation of Anu oil, while the other group received intranasal installation of til tailya (50 ul/nostril/day) starting 5 days before infection and continued till 4 days postinfection (DPI). On the day of the challenge, intranasal administration of Anu oil and til tailya was carried out 30 min before infection. On the day of the challenge, the animals were shifted to ABSL3. Intranasal infection with live SARS-CoV2 10⁵PFU/100 μl or with the DMEM mock control (for uninfected control group) was established with the help of a catheter under mild anesthetized by using ketamine (150 mg/kg) and xylazine (10 mg/kg) intraperitoneal injection inside the ABSL3 facility. All the experimental protocols involving the handling of virus culture and animal infection were approved by the RCGM, Institutional Biosafety and IAEC Animal Ethics Committee.

All infected animals were euthanized after 4 days post-infection at ABSL3. Changes in body weight were observed each day post-challenge and plotted as percent change in the body weight. Post-sacrifice, the lungs and spleen of the animals were excised and imaged for gross morphological changes. The left lower lobe of the lung was fixed in 10% formalin and used for histological analysis. The remaining part of the lung’s left lobe was homogenized in 2 ml TRIzol solution for viral load estimation. The spleen was homogenized in 2 ml of TRIzol solution. The TRIzol samples were stored immediately at −80°C until further use. Blood of the animals was drawn through direct heart puncture, and serum was isolated and stored at -80°C until further use.

For viral load, estimated lungs were homogenized in TRIzol reagent (Invitrogen), and their supernatant was collected after centrifugation at 4,000 rpm for 15 min at 4°C. Thereafter, RNA was isolated by TRIzol–choloform method, and RNA yield was quantitated by nano-drop; 1 µg of total RNA was then reverse-transcribed to cDNA using the iScript cDNA synthesis kit (Biorad; #1708891) (Roche). Diluted cDNAs (1:5) were used for qPCR by using the KAPA SYBR^® FAST qPCR Master Mix (5X) Universal Kit (KK4600) on the Fast 7,500 Dx real-time PCR system (Applied Biosystems), and the results were analyzed with SDS2.1 software. In brief, 200 ng of RNA was used as a template for reverse transcription polymerase chain reaction (RT-PCR). The CDC-approved commercial kit was used for the SARS-CoV-2 N gene: 5′-GAC​CCC​AAA​ATC​AGC​GAA​AT-3′ (forward), 5′-TCT​GGT​TAC​TGC​CAG​TTG​AAT​CTG-3′ (reverse). The hypoxanthine–guanine phosphoribosyltransferase (HGPRT) gene was used as an endogenous control for normalization through quantitative RT-PCR. The relative expression of each gene was expressed as fold change and was calculated by subtracting the cycling threshold (Ct) value of hypoxanthine–guanine phosphoribosyltransferase (HGPRT-endogenous control gene) from the Ct value of the target gene (ΔCT). The fold change was then calculated according to the formula POWER(2,-ΔCT)*10,000 (Malik et al., 2017).

RNA from spleen samples was isolated as described earlier for the lung samples, and cDNA was prepared. Thereafter, the relative expression of each gene was expressed as fold change and was calculated by subtracting the cycling threshold (Ct) value of hypoxantine–guanine phosphoribosyltransferase (HGPRT-endogenous control gene) from the Ct value of target gene (ΔCT). Fold change was calculated according to the formula POWER(2,-ΔCT)*10,000 (Malik et al., 2017; Rizvi et al., 2021a). The list of the primers is provided as follows in Table 1

The lung of the euthanized animals was fixed in 10% formalin solution and then embedded in paraffin. Sample embedded paraffin blocks were then cut into 3-µm fine sections and mounted on silicone-coated glass slides. The slides were then stained with hematoxylin and eosin dye, as previously described (Rizvi et al., 2018). Each stained sample was then analyzed and captured at ×40 magnification. Assessment for the histological score was carried out through blind scoring for each sample by a professional histologist.

Results from the experiments were analyzed and plotted by using GraphPad Prism 7.0 software. The graph for percent change in body weight, gene expression, and lung histology scores were compared and analyzed by using the Student t-test or one-way ANOVA, with n = 5 samples per group. The p-value of less than 0.05 and was considered as statistically significant.

SARS-CoV2 infection in hamsters peaks in 4–5 days and is characterized by a reduction in body weight and appearance of pneumonitis in the lungs and splenomegaly (Imai et al., 2020; Sia et al., 2020; Rizvi et al., 2021b; Chan et al., 2020). These defined gross clinical parameters were recorded for all the groups, that is, uninfected control (UI), infected control (I), and infected hamsters receiving either Til tailya (TT) or Anu oil (AO) intranasal formulations (50 µl/nostril/day). Treatment of TT and AO was started 5 days before SARS-CoV2 live virus challenge and was continued till the end of the experiment, that is, 4 days postinfection (dpi), as presented schematically in the study design (Figure 1A). In line with the earlier published reports, a decrease in the body weight of SARS-CoV2–infected hamsters was observed with 5–8% body weight reduction on 4 days dpi. Hamsters receiving TT or AO, before SARS-CoV2 infection, did not lose body weight as observed in the SARS-CoV2–infected group (Figures 1B,C). SARS-CoV2 infection in the hamster model is characterized by lung inflammation, pneumonitis, and cytokines release (Afrin et al., 2020; Chen and Li, 2020; Moore and June 2020). To further understand the lung-associated pathologies, gross morphological changes of the excised lungs were compared between healthy, SARS-CoV2–infected, and SARS-CoV2–infected plus oil formulated groups. There was a reduction in the regions of pneumonitis in the excised lungs of the AO group, but not the TT group, as compared to infection control (Figures 1D,E). As reported earlier, splenomegaly is one of the critical parameters indicating active infection (Rizvi et al., 2021b). Thus, we tested the splenomegaly between different groups and found that AO, but not TT, showed inhibition in splenomegaly as compared to the SARS-CoV2–infected hamsters (Figures 1F,G). We also evaluated the lung viral load at four dpi and calculated the fold reduction in viral load in AO- and TT-treated groups as compared to the SARS-CoV2–infected groups. Our data indicate that compared to the SARS-CoV2–infected group, viral loads in AO- and TT-treated groups were ∼3- and ∼2-fold less, respectively (Figure 1H). Together, these data indicated that prophylactic use of intranasal instillation of TT and AO resulted in decreased lung viral load with the AO group, showing better protection in gross clinical parameters.

Since gross clinical parameters and lung viral load data suggested protection from SARS-CoV2 infection in AO and TT groups, we set out to study the mitigation of pulmonary pathologies such as lung injury, alveolar epithelial injury, bronchitis, pneumonitis, and inflammation using histological analysis (Bao et al., 2020; Lee et al., 2020; Leng et al., 2020; Rizvi et al., 2021b). Hematoxylin and eosin (H and E)–stained lung data showed a reduction in alveolar epithelial injury, inflammation, and pneumonitis in SARS-CoV2–infected AO-treated hamsters as compared to SARS-CoV2–infected hamsters. There was no sign of bronchitis in SARS-CoV2–infected AO-treated hamsters with overall significant mitigation in the disease score as compared to SARS-CoV2–infected hamsters (Figures 2A,B). Hamsters treated with TT however showed little or no improvement in lung injury and overall disease score as compared to the infected control (Figures 2A,B). Together, lung pathology associated with SARS-CoV2 was found to be resolved in the AO group but not in the TT group, with around 1.5-fold rescue in disease index in the AO group as compared to the infected control group.

Lung histology data indicated overall improvment in histological changes of SARS-CoV2–infected AO-treated groups as compared to SARS-CoV2–infected group. These data compelled us to explore the mechanism involved in the rescue of lung pathologies. Injury to the pulmonary region is characterized by elevated expression of surfactant D (sftp-D), increased mucus (muc-1) secretion, and increased expression of eotaxin, which promotes infiltration of granulocytes and mast cells and an increased risk of pulmonary thrombosis as seen in COVID-19 patients (Crouch, 2000; Guo et al., 2001; Prabhakaran et al., 2003; Xie et al., 2017; Chatterjee et al., 2020). We observed elevated levels of sftp-D, muc-1, eotaxin, muc-1, chymase, tryptase, and plasmonigen activator inhibitor-I (PAI-1: a key factor for lung fibrosis) in the lungs of infected hamsters (Figures 3A–C). However, prophylactic intranasal use of AO in SARS-CoV2–infected hamster significantly reduced the mRNA expression of these lung injury genes and genes that are required for chemotaxis of granulocytes and function of mast cells (Figures 3A,B). Prophylactic use of AO in SARS-CoV2–infected hamsters did not reduce PAI-1 expression (Figure 3C). In contrast to AO, TT data showed no reduction in lung injury as compared to infected control (Figure 3A). Surprisingly, there was a marked increase in mast cell markers in the TT group as compared to the infected control (Figure 3B). Overall, consistent with our lung histology data, a profound reduction in the expression of lung injury genes and mast cell markers as compared to the infected control was observed in AO-treated groups.

COVID-19–related respiratory distress is associated with inflammation in the lungs. The increase in lung inflammation is characterized by the secretion of pro-inflammatory cytokines in COVID-19 patients (Iwasaki and Yang, 2020; Mathew et al., 2020; Moore and June 2020; Verity et al., 2020). Cytokine expression data from splenocytes indicate elevated expression of Th1 cytokines (IFNγ and TNFα), Th2 cytokine (IL-4, IL-13), Th17 cytokine (IL-17 A), and various other pro-inflammatory cytokine-like IL-6 and IL-13 in SARS-CoV2–infected group as compared to uninfected hamsters (Figures 4A–D). However, there was not much change observed in anti-inflammatory cytokine IL-10 expression as compared to the challenge control group. Prophylactic intranasal installation of AO and TT in SARS-CoV2–infected hamsters resulted in the reduction in Th1 and Th17 cell cytokines, together with pro-inflammatory cytokine expression (Figures 4A,B,D). However, surprisingly, only TT but not AO was able to reduce the Th2 cytokine gene expression (Figures 4A,C). Furthermore, we found an elevated IFN-gamma secretion in both AO- and TT-treated animals (Figures 4A,C). Since C-X-C motif chemokine ligand 10 (CXCL10), a chemoattractant, is an important mediator of IFN-gamma response and is secreted by various immune cells, we evaluated the mRNA expression of CXCL10 from treated (AO/TT) vs challenge (I) samples (Zhang et al., 2020). As compared to the challenge control group, neither the AO nor TT group did not show significant changes (Figures 4B,D). Together, we show that AO and TT reduced the pro-inflammatory cytokines.