**1. Introduction**

Infectious bronchitis (IB) of chickens is a highly contagious disease characterized by damage to chickens' respiratory and reproductive organs [1–5]. Diseases of the respiratory system caused by the infectious bronchitis virus (IBV) [5] have common features with the pathology of the respiratory system caused by SARS-CoV-2 in humans [6]. IB and SARS-CoV-2 viruses have many similarities: both are low copy coronavirus, both have a

**Citation:** Nefedova, E.; Koptev, V.; Bobikova, A.S.; Cherepushkina, V.; Mironova, T.; Afonyushkin, V.; Shkil, N.; Donchenko, N.; Kozlova, Y.; Sigareva, N.; et al. The Infectious Bronchitis Coronavirus Pneumonia Model Presenting a Novel Insight for the SARS-CoV-2 Dissemination Route. *Vet. Sci.* **2021**, *8*, 239. https:// doi.org/10.3390/vetsci8100239

Academic Editors: Ana Faustino and Paula A. Oliveira

Received: 7 September 2021 Accepted: 12 October 2021 Published: 18 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

lipid envelope, and for both, the immune system reaction contributes significantly to the damage. These give the basis to believe that the mechanisms of action of these two viruses are similar.

Bacterial viruses (bacteriophages) are a very convenient model for studying various drug virucidal activities. Specifically, bacteriophage ϕ6 belonging to the Cystoviridae family is a promising model. These phage particles have a spherical or icosahedral shape and dsRNA type of nucleic acid. The presence of capsids in its composition makes it an effective model for testing the activity of virucidals and disinfectants against enveloped viruses, including coronaviruses. The virucidal activity of substances determined with the ϕ6 bacteriophage model allows the estimation of their activity against various viruses containing lipids in their capsid, such as herpesviruses 1 and 2 and coronavirus SARS-CoV-2, which infect eukaryotic cells [7–9]. The ϕ6 bacteriophage infects *P. phaseolicola* plant pathogen bacteria. Consequently, the advantage of working experimentally with a ϕ6 bacteriophage and plant pathogenic bacteria is that neither the phage nor the bacteria are pathogenic to humans, which minimizes the requirements for the laboratory biosafety level. Some research groups successfully applied the ϕ6 bacteriophage as a model for the SARS-CoV-2 virus [10,11].

Drugs possessing virucidal activity in the small intestine can be effective against viral infections in other tissues. Clinical observations showed the effectiveness of Ecocid, Triviron, Argovit, and lauric acid monoglyceride (C12) against viral mal-absorption in poultry farms (flavivirus, astroviral etiology). Ecocid shows high antibacterial and virucidal activity combined with low toxicity and stability in a living organism, making it a promising alternative as a topical virucidal agent [12] and a disinfectant that prevents the horizontal transfer of antibiotic resistance genes [13]. Triviron virucidal formulation began to be used in veterinary medicine relatively recently [14,15]. The mechanism of action has no analogs and belongs to a new pharmacological group of synthetic ribonucleases [2,16]. Silver nanoparticles [8,17–20] including Argovit AgNPs [21–24] have recently shown virucidal effects against some viruses, and are therefore also potentially effective against the SARS-CoV-2 virus. Lauric acid monoglyceride (Monolaurin) is known to inactivate lipid-coated viruses by binding to the lipid–protein envelope of the virus, thereby preventing it from attaching and entering host cells, making infection and replication impossible [25]. Other studies show that Monolaurin disintegrates the protective viral envelope, killing the virus [26,27]. Monolaurin has been studied to inactivate many pathogens, including the Herpes simplex virus [25–28].

The present study aimed to screen the virucidal activity of the four formulations mentioned above on in vitro (bacteriophage ϕ6) and in vivo (IB of chickens) models for SARS-CoV-2 infection.

#### **2. Materials and Methods**

#### *2.1. Formulations*

Argovit 1% is an aqueous suspension of highly dispersed silver nanoparticles (0.6 mg/mL of metallic silver) stabilized with polyvinylpyrrolidone, produced by the Center of Investigation and Production "Vector-Vita", Novosibirsk, Russia. Argovit™ is a stable suspension in water with an AgNP concentration of 200 mg/mL (20% *w*/*w*). The metallic Ag (content 1.2% *w*/*w*) is stabilized with polyvinylpyrrolidone (PVP 12.6 ± 2.7 kDa, content 18.8%) of AgNP total weight. The remaining 80% of the weight is distilled water. AgNPs have a spheroidal shape with a size varying from 1 to 90 nm and an average diameter of 35 ± 12 nm. The hydrodynamic diameter is 70 nm, the ζ potential is −15 mV, and a plasmonic resonance peak is registered at 420 nm [29]. Triviron (0.03% synthetic ribonuclease (1,5-bis-[N, N-1- (4-tetradecyl) diazoniabicyclo [2.2.2] octyl] pentane tetrobromide) was produced by «Trionisvet» Ltd., Korolev, Russia. Ecocid (0.05%), produced by Krka, d. d., Novo Mesto, Slovenia, contains a triple salt of potassium peroxomonosulfate (50%), as well as auxiliary substances: surfactants (sodium dodecylbenzene sulfonate), organic acids (malic, sulfamic), inorganic buffer systems (sodium chloride and sodium polyphosphate),

Azo Diestaff dye, and Citron as an aromatic additive with a lemon scent. Used in our work, Ecocid, Triviron, and Argovit are certified and are commercially produced. Lauric acid monoglyceride (0.005%) was synthesized and kindly provided by Dr. Fomenko Vladislav from institute N.N. Vorozhtsov, Novosibirsk Institute of Organic Chemistry of SB RAS.

#### *2.2. Bacteriophage ϕ6 In Vitro Model*

Bacteriophage ϕ6 (belonging to the *Cystoviridae* family) was used as an in vitro model to assess the formulations' virucidal activity. A *Pseudomonas phaseolicola* cell culture was used to determine phage particle activity. The tested preparations were incubated with a bacteriophage culture at room temperature at various concentrations. After 1, 5, 15, 30, and 60 min of incubation, inoculation of samples containing bacteriophages and formulations and only bacteriophage (control group) was carried out. The residual concentration of bacteriophage inoculations was determined in 10-fold dilutions. The survival rate of phage particles was determined by the method of two-layer agar (Grazia method). The studies were carried out at room temperature (26 ± 2 ◦C). All experiments were performed in duplicate.

## *2.3. Poultry*

Cross Shaver male chickens aged 14 days and 198–210 g each were kept indoors and fed with standard granulated chicken feed. The experimental protocol involving animals was reviewed and approved by the Ethical Committee of Novosibirsk State Agrarian University of Siberian Federal Scientific Center of Agro-BioTechnologies of the Russian Academy of Sciences CM K PO 15-01-2019/No.3 of 10.03.2021.

#### *2.4. Inoculum Administration*

Chickens received a tenfold dose of a vaccine against IBV (from the H120 strain, live, dry) consisting of 5 lg Embryo Infectious Dose (40 per head). It was administered orally to each chicken. Experimental groups and a control group of 13 animals were formed.

#### *2.5. Dosage Administration*

The dosages of the preparations were as follows: Argovit and Triviron, 250 and 285 µL/animal, respectively. They were administered two times a day (in the morning and the evening) to each animal. The lauric acid monoglyceride dose was 0.1 mg/animal. Ecocide C (0.05%) dose chickens drank freely when they wanted (57 mL per day per head). For drinking water sanitation for animals, including birds, it is recommended to use 0.1% Ecocide C in water. Here, half of the recommended concentration was applied. All formulations were administered for 5 days. The animals were slaughtered on the 6th day. In Table 1, consumption per head per day for the studied formulations is summarized.


**Table 1.** Consumption per head per day for studied formulations.

#### *2.6. Histopathological Evaluation*

The lung histopathological analysis was performed under an Imager D1 luminescence microscope (Zeiss) using AxioVision v 4.6.3.0 software (Zeiss, Jena, Germany). Briefly, the lung pieces (~1 mm thick) were placed in a 96-well plate filled with distilled water (150 µL per well), and 20 µL SYBRTM Green (1:1000) and 5 µL of SyproTM Ruby dye

(BioRad Laboratory) were added per well. Then, they were kept for 20 min to counterstain the preparations.

Luminescence microscopic analysis of lung pieces was carried out according to an original technique of a short period staining mode, whose principle consisted of staining a formalin-fixed biomaterial with two dyes. SYPRO Ruby intercalates proteins and glows, while SYBR Green I binds to DNA and fluoresces in orange-red and green ranges, respectively. The staining is carried out within a short period (20 min), during which dye diffusion occurs in a thin layer of cells, which avoids the intense background fluorescence of entire pieces of tissue. Thus, in contrast to histological examination, a short period staining mode is suitable for studying thin tissue layers. This mode makes it possible to visualize better epithelial and endothelial cells (for example, the inner surface of the bronchi and blood vessels and intestinal epithelium). Additionally, this mode provides a good visualization of blood capillaries, hemorrhages, and tissue saturation with hemoglobin, which is accompanied by a sharp decrease in fluorescence intensity. The difference of this luminescence microscopic mode from confocal microscopy lies in the lower price of the used equipment, faster staining process, more detailed observations of epithelium surfaces, and the influence of the diffusion process microscopic picture.
