The study used outbred Syrian hamsters (Mesocricetus auratus) of both genders, 18 months old. All animals were kept in the 3rd Animal Biosafety Level (ABSL-3) facility of the Central Reference Laboratory (CRL) of the Aikimbayev’s National Scientific Center for Especially Dangerous Infections (Almaty, Republic of Kazakhstan), hereinafter referred to as NSCEDI. The animals were kept in individually ventilated cages (IVC) with a HEPA filter (Allentown, USA) on a special autoclavable ssniff^® bedding (Spezialdaten GmbH, Germany). The conditions corresponded to generally accepted standards: ambient temperature 21 ± 2°C, humidity 50 ± 10%, and a day/night cycle (10:14). The diet included a standardized complete ssniff^® feed for hamsters (cat. No. V 2144-000) (Spezialdaten GmbH, Germany). Water and feed ad libitum. The animals were weighed using Adventurer RV 1502 scales (Ohaus, China). The study protocol was approved by IACUC No. 14.

The study used the strain SARS-CoV-2, isolated earlier (11, 12). The complete sequence of the protein was published in the GISAID database under the number EPI_ISL_514093. The virus was cultured on a monolayer Vero E6 cell line in Dulbecco’s Modified Eagle Medium (DMEM) with added 2% bovine fetal serum and antimycotic antibiotic solution (penicillin 10,000 U, streptomycin 10 mg. and amphotericin B 25 μg) (Gibco, USA) at a temperature 37°C in a CO₂-incubator in an atmosphere with 5% carbon dioxide for three days. One of the infected hamsters did not survive the anesthesia.

The experimental animals were randomized into 8 groups consisting of 6 individuals ( Table 1 ). Half of the animals were kept on a standard diet (RD), and the other half from 14 to 18 months received a high-fat diet (HF) with 20% coconut oil and 0.2% ssniff^® cholesterol for hamsters (cat. no. E21413-3402) (Spezialdaten GmbH, Germany). Under anesthesia, blood was taken from the gingival vein to assess biochemical changes in the blood of hamsters before and after feeding diets.

Experimental animals were infected by the intranasal route. For this, hamsters were anesthetized by intraperitoneal injection of solutions of ketamine and xylazine at doses of 100 and 10 mg/kg, respectively. Then, 10 μl of DMEM containing the hCoV-19 virus at a dose of 10^6.0 TCID₅₀ was added to each nostril. A virus-free nutrient medium was injected into the nostrils of the control animals.

The animals were weighed on days 0, 1, 2, and 3 post-infection (dpi). After that, the animals were euthanized and samples of lung and liver tissue were collected for ELISA and histological studies.

Serum was isolated from the blood of the hamsters by centrifugation at 6000 rpm for 15 minutes for clinical chemistry testing. Serum samples were examined on the A-25 biochemical analyzer (Biosystem, Spain). The following parameters were measured: alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose (Glu), cholesterol (Chol), and triglycerides (Trgly).

To study the level of cytokines in the lungs, the tissue was homogenized in a ratio of 1 to 10 PBS (Sigma, USA) with the addition of Protease Inhibitor A32965 (Thermo Scientific, USA). Cytokines were measured quantitatively by using the ELISA sandwich method with hamster kits from MyBioSource (USA) for TNF- α (MBS7606475), IL-6 (MBS700950), IFN-α (MBS010919), and IFN-γ (MBS010146)

The organs were fixed in a 10% formalin solution. After 24-hour fixation the organs were subjected to histological processing which involved four portions of isopropanol (BioVitrum), and two portions of the clearing agent Bio-Clear (Bio-Optica, Italy). Then the material was soaked in four portions of paraffin medium Histomix (BioVitrum) with the subsequent embedding of histological blocks. After that, the blocks were cut on a semi-automatic microtome MZP-01 (KB Technom), as a result of which sections were obtained with a thickness of 5 micrometers (μ). Then the sections were dewaxed in two portions of xylene and three portions of ethanol, and after that, they were stained with Mayer’s hemalum (BioVitrum) and aqueous 1% eosin (DiaPath). This was followed by a clarification in ethanol and two portions of Bio-Clear. In the end, the sections were closed with cover glasses using a synthetic medium Bio Mount (Bio-Optica, Italy). The specimens were studied using the Mshot microscope (China), MF52-N model. The photographs were taken with a Mshot MS23 camera (China) in the MShot image analysis system (China). Each slide was quantified based on the severity of histologic changes, including interstitial pneumonia, alveolitis, bronchiolitis, alveolar destruction, interstitial infiltration, pulmonary hemorrhage, and peribronchiolar inflammation. Scoring of pathological changes in the lung sections: 4 points = extremely severe; 3 points = severe; 2 points = moderate; 1 point = mild; 0 points = no changes (13).

All works were carried out in ISO 35001:2019 certified laboratories of the 3rd biosafety level (BSL3) in the CRL of NSCEDI. The works were carried out in a biosafety cabinet SG404, class II, type A2 (Baker, USA). A biosafety specialist of the CRL participated in all works.

All quantitative results are presented as the average ± SD. Statistical significance was assessed by parametric and nonparametric criteria. The body weight dynamics and cytokine level were assessed by a one-way ANOVA with Tukey’s multiple comparisons test. Biochemical analysis was assessed using the unpaired Mann-Whitney test. Histological scoring was assessed by the Kruskal-Wallis test with Dunn’s multiple comparisons tests. The differences were considered significant for the p values less than 0.05. Statistical tests were conducted using GraphPad Prism version 9.0.0 (GraphPad Software, La Jolla, CA, USA).

Hamsters are a suitable model for diet-induced hypercholesterolemia, hypertriglyceridemia, and obesity (14). For the development of hypercholesterolemia and hypertriglyceridemia, the animals received a high-fat diet with added cholesterol for four months. There was a decrease in weight in males on the HF diet (p<0.001). At the same time, a slight increase in weight was noted in females from the HF group (p<0.001) ( Figure 1A ). Histological examination of the liver showed the presence of fatty changes, with inflammatory infiltrates ( Figures 1C–E ), compared with the standard diet group ( Figure 1B ).

An increase in cholesterol levels was observed in 18 females (95% confidence interval (CI), 7,6-10,0) compared to 14-month-old females (95% CI, 2,0-3,0; p<0.01) and in 18-month-old males (95% CI, 8,2-9,4) compared to 14-month-old males (95% CI, 2,4-4,4; p<0.05) ( Figures 2A, B ). An increase in triglyceride levels was observed in 18 females (95% CI, 8,9-11,3) compared to 14-month-old females (95% CI, 1,5-4,6) p<0.05) and in 18-month-old males (95% CI, 9,1-11,3) compared to 14-month-old males (95% CI, 0,7-2,6; p<0.01) ( Figures 2A, B ). At the same time, glucose levels, aspartate aminotransferase, and alanine aminotransferase did not change ( Figures 2C–E ).

The old age of hamsters begins at 18 months and is characterized by a number of significant changes in the body. These include degenerative processes in the gonads (15), polycystic liver disease, and other characteristic signs (16). Signs of aging in experimental animals were established during the external examination, after the autopsy, and macroscopic and histological examination of organs. Motor activity of hamsters decreased and foci of alopecia appeared, which became larger over time. At autopsy, most of the individuals were found to have characteristic multi-lobular mono- and multi-locular cysts of various sizes ( Figure 3 ).

After infection, animals fed both standard RD and high-fat HF diets showed daily clinical signs of COVID-19, such as behavior and body weight dynamics. Weight loss was noted on day 3 after infection ( Figure 4 ).

Next, the animals were euthanized and lungs were collected to determine the levels of TNF-α, IL-6, IFN-α, and IFN-γ. The lungs and liver were also histologically examined. Changes in the levels of TNF-α in the lungs of all groups were not observed ( Figures 5A, B ). There was also a significant increase in IL-6 levels in males from the infected groups, both on the RD diet (95% CI, 1867,0-5351,0) and the HF diet (95% CI, 1705,0-5247,0), compared with healthy animals on the RD diet (95% CI, 45,3-682,5; p<0.001 and p<0.01 respectively), and the HF diet (95% CI, 289,9-1369,0; p<0.01, both) ( Figure 5С ). Infected females on the RD (95% CI, 721,1-4647,0) and HF diets (95% CI, 2447,0-3945,0) showed an increase in IL-6 compared to healthy females on the RD diet (95% CI, 41,3-1519,0; p<0.05 and p<0.01, respectively) ( Figure 5D ).

The study of the effect of the SARS-CoV-2 virus on type I and II interferons showed the following results. IFN-α levels significantly increased in SARS-CoV-2-infected males on RD (95% CI, 135,9-244,7) and HF diets (95% CI, 153,1-223,7) compared to healthy animals on RD diet (95% CI, 70,3-105,6; p<0.001 and p<0.01 respectively) ( Figure 6A ). At the same time, an increase in the production of this cytokine was observed in the group of healthy males on the HF diet (95% CI, 106,9-204,5) in comparison with healthy males on the RD diet (95% CI, 70,27-105,6; p<0.05). However, changes in the levels of IFN-α in the lungs of females were not observed ( Figure 6B ). Another key cytokine of inflammation, IFN-γ, did not respond in either males or females ( Figures 6C, D ). Only a difference was observed among healthy males between HF (95% CI, 161,1-222,8) and RD diets (95% CI, 105,2-162,5; p<0.05).

Histological examination of the liver revealed the presence of pathological changes of varying degrees within the same group. A common characteristic for all animals, regardless of the group, was the presence of dilated sinusoidal spaces and multifocal foci of low-grade mononuclear-macrophage infiltration in the parenchyma ( Figure 7A ). Single foci were encountered with a group of pigment-filled hepatocytes ( Figure 7B ). In the groups infected with SARS-CoV-2, there were marked foci of granulocyte infiltration ( Figure 7C ), as well as multifocal lymphocytic foci of inflammation of the liver triad region ( Figures 7D, E ). Some of them had pigment-filled macrophages ( Figure 7E ).

Morphological studies of the lungs of hamsters infected with the SARS-CoV-2 virus showed the presence of a pathological process characteristic of ARDS in the form of bronchointerstitial pneumonia and diffuse alveolar damages ( Figure 8 ). Extensive foci of changes coexisted with healthy sites within the same group, and varied in severity from group to group, but were similar ( Figure 9 ). Pathological changes represented significant peribronchial infiltrates and desquamation of the epithelium in the bronchi lumen. Edema, interstitial and intra-alveolar infiltrates were observed in the lung parenchyma, in which lymphocytes and single neutrophils predominated. Part of the alveolar-capillary units in the foci of edema, hemorrhage, and cell debris was also disturbed.