The animal experiment was carried out at the Pomeranian Medical University in Szczecin under the approval of the Local Ethical Committee for Animal Experiments in Poznań No. 35/2022.

Non- vaccinated against Lagovirus europaeus/GI.1 and GI.2 European rabbits Oryctolagus cuniculus/Crl:KBL (NZW)/0052 were used in the experiment. The animals were purchased from a licensed breeder (AnimaLab Sp. z o.o., Poznań, Poland). Twenty animals (6 months old) with body weights in the range of 4.5 kg (±10%) were used in the study. The animals were randomly divided into two groups: a control group (n = 10; 50% males and 50% females) and an experimental group infected with the Lagovirus europaeus/GI.2 strain (n = 10; 50% males and 50% females). After delivery to the laboratory, the animals underwent adaptation (3-week period). In their cages, they had access to hay balls and wooden blocks as environmental enrichment. The animals were maintained under standard laboratory conditions.

The virus strain used in the study was isolated from a rabbit that died because of naturally acquired infection in Italy (RHDV2_Ri2017 strain; GenBank: OQ680671). The inoculum (1 mL) intramuscular administered to animals from the experimental group was prepared according to the method described by us earlier (Niedźwiedzka-Rystwej and Deptuła, 2023). Infection was provoked by the intramuscular injection of 2 × 10⁴ haemagglutination units of the virus (Vallejo et al., 2014; Tunon et al., 2003) [assuming that 1 HA unit corresponds to 10⁴ particles per mL (Ryu, 2017)], which was determined by the haemagglutination assay as described earlier (Bębnowska et al., 2023). The hemagglutination titer was obtained at a 1:16 dilution. The animals from the control group received the same amount of placebo (1 mL) in the form of PBS (phosphate-buffered saline).

The experiment started with the administration of the inoculum, and this moment was marked as 0 h of the experiment. The health status of all rabbits, including rectal measurement of body temperature, was monitored at least twice a day from the time of inoculate injection. During the experiment blood was collected from each animal into an anticoagulant (heparin) tube (1 mL) at selected timepoints: 0, 12, 24, 36 and 48 h post inoculation (h p.i.). The onset of severe symptoms of the disease was considered the terminal moment of the experiment. Animals qualified for euthanasia were anesthetized by intravenous injection of sodium pentobarbital at a dose of 90 mg/kg, and then cardiac arrest was induced by administration of sodium pentobarbital at a dose of 250 mg/kg. Tissue samples (from liver, spleen, and kidney) were collected from rabbits immediately after death (both from animals that died suddenly during the experiment and those euthanized) and were preserved in RNAlater stabilizing solution (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and then stored at −80°C until analysis.

Immediately after blood collection at selected timepoints erythrocytes in peripheral blood were lysed using Red Blood Cell Lysis Buffer (A&A Biotechnology, Gdańsk, Poland), using the protocol provided by the producer. Total RNA was isolated from PBL pellet and tissue samples using RNeasy Mini Kit (A&A Biotechnology, Poland) according to the manufacturer’s instructions. The quantity and quality of the isolate were measured using NanoDrop ™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, United States). To obtain cDNA the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, Massachusetts, United States) was used as recommended by the manufacturer.

To analyze relative gene expression quantitative real-time PCR was performed using the LightCycler® 480 Instrument II (Roche Diagnostics GmbH, Mannheim, Germany) and the PowerUp™ SYBR™ Green Master Mix Thermo Fisher Scientific, Waltham, Massachusetts, United States according to manufacturer’s recommendations. In the study, specific primers were used (Table 1). Obtained threshold cycle (Ct) values were normalized by β-actin gene expression, and the relative gene expression was calculated using the Pfaffl method (Pfaffl, 2001). To determine viral genome copies a dilution series of full-length L. europaeus GI.2 transcript standards was prepared (ranging from 1 × 10⁶ copies/μl to 1 × 10¹ copies/μl). A standard curve and absolute quantification were performed using a LightCycler 480 II instrument.

The dissected intestinal tissues were fixed with 10% formalin for at least 24 h, but for no more than 36 h, and then treated as described previously (Bębnowska et al., 2024). For IHC reactions we used ImmPRESS HRP Universal (Horse Anti-Mouse/Rabbit IgG), PLUS Polymer Kit, Peroxidase (Vector Laboratories, Newark, CA, United States; cat. no. 7800). To expose the epitopes to IHC procedure, deparaffinized and rehydrated sections were boiled in Target Retrieval Solution (DakoCytomation, Glostrup, Denmark, cat. no. S2369) in a water bath (99°C for 8 min.). Once cooled and washed with PBS, the endogenous peroxidase activity was blocked using peroxidase blocking reagent. Antibodies were diluted in Antibody Diluent with Background Reducing Components (Dako, Dako, Santa Clara, CA, United States, S302 S3022). Slides were incubated for 1 h at room temperature (RT), with primary antibodies against LC3b (anti-LC3b- antibody, rabbit polyclonal, cat# NB100-2220, Novus Biologicals, Centennial, CO, United States); against Atg12 (anti-Atg12 antibody, rabbit polyclonal, cat# orb153327, Biorbyt, Cambridge, United Kingdom) and against Beclin-1 (anti-Beclin 1 antibody, cat# orb625103, rabbit polyclonal, Biorbyt, Cambridge, United Kingdom). Antibodies were diluted in Antibody Diluent (cat# ab64211, Abcam, Cambridge, United Kingdom). To visualize the antigen–antibody complex, sets of reagent HRP-related system were used, based on the reaction of avidin-biotin-horseradish peroxidase with DAB as a chromogen, according to included staining procedure instructions. Sections were washed in distilled H₂O and counterstained with hematoxylin (Mar-Four, Konstantynów Łódzki, Poland). For a negative control, specimens were processed in the absence of primary antibodies. Positive staining was determined microscopically (Leica DM5000B, Wetzlar, Germany) by visual identification of brown pigmentation. The experiments were repeated independently twice.

Micrographs of tissues (liver, kidney, and spleen) stained with antibodies against LC3b, Atg12, and Beclin-1 were analyzed with the use of ImageJ Fiji software.¹ First, the downloaded image was opened in Fiji software and color deconvolution was performed (Image>Color>Color Deconvolution), and the hematoxyline + DAB option was selected. Next, the threshold was selected (Image>Adjust>Treshold); the minimum threshold value was set at zero and the maximum threshold value was adjusted so that the background signal was removed, without removing the true signal from the hematoxyline + DAB-stained tissues. The percentage of area covered by the pigmentation was measured (Analyze > Measure). Three independent analyses (with a slightly changed maximum threshold) were performed and the results were averaged. The area of tissue where preparation led to bending of the specimen or tissue damage was not included in the analysis.

Levels of selected apoptotic proteins were estimated from tissue sample extracts treated with T-PER™ Tissue Protein Extraction Reagent (Cat. No. 78510, ThermoScientific™) and a mixture of protease and phosphatase inhibitors (Roche Applied Science, Penzberg, Germany; #05892791001 and #11873580001), and Bead Ruptor Elite (Cat. No. SKU 19-040E, Omni international). The samples were centrifuged for 10 min (12,000 rpm, 4°C). Protein lysates were transferred to new Eppendorf tubes. Analysis of protein levels in lysates was conducted using a Bradford reagent. Proteins were separated using the WES system (WES - Automated Western Blots with Simple Western; ProteinSimple, San Jose, California, United States), with a 12–230 kDa separation module (#SM-W003), and detected using an Anti-Mouse (#DM-002) or Anti-Rabbit (#DM-001) detection module, according to the manufacturer’s instructions. The Total protein module (#DM-TP01, ProteinSimple, San Jose, CA, United States) was used as a loading control. The following primary antibodies were used in the study: cleaved caspase-3 (Asp175) (5A1E) rabbit mAb (#9664, Cell Signaling Technology), cleaved caspase-6 (Asp162), rabbit mAb (#9761, Cell Signaling Technology), and cleaved PARP (Asp214) (D64E10) Rabbit mAb (no. 5625, Cell Signaling Technology), LAMP1 (#9091, Cell Signaling), LAMP2 (#49067, Cell Signaling), Beclin-1 (#3495S, Cell Signaling), p62/SQSTM1 (sc-48402, Santa Cruz Biotechnology) 1:50 and LC3 LC3A/LC3B (sc-398822, Santa Cruz Biotechnology) 1:250.

Caspase-3 Activity Assay Kit (#5723, Cell Signaling Technology) was used to determine caspase-3 activity according to the recommendation of the manufacturer. Samples with a protein content of 0.5 mg were prepared for the experiment. EnSpire (PerkinElmer, Waltham, Massachusetts, United States) device was used for fluorescence measurement using excitation at 380 nm and emission at 440 nm wavelength and with a gain value of 60.

To perform statistical analysis Tibco Statistica 13.3 (StatSoft, Palo Alto, CA, USA) was used. The values of the parameters were presented as arithmetic means, standard deviations (SD), and standard error (SE). The Shapiro–Wilk test was used to test the normal distribution of continuous variables. Student’s t-test was used to compare data of 2 groups data with a normal distribution and the Mann–Whitney U test was used for data with a non-normal distribution.

Clinical signs observation showed that some animals died suddenly at 12–36 h p.i. without any symptoms of disease (n = 6). From 24 h p.i. remaining animals (n = 4) showed apathy, conjunctival congestion, dysponoea, fever >41°, anorexia, and the human endpoints of the experiment reached 36–48 h p.i. Post-mortem analysis confirmed the characteristic signs of RHD in all animals.

To confirm the effectiveness of the infection protocol we determined the level of viral load in blood samples at each timepoint (Figure 1).

Insights into apoptotic death in selected organs of rabbits infected with Lagovirus europaeus/GI.2 have been the subject of our previous studies (Bębnowska et al., 2023; Bębnowska et al., 2024). However, given that the liver is a central organ targeted by the virus, we decided to further explore the characteristics of this process. In this study, we demonstrated an increased amount of cleaved caspase-3 and caspase-6 (executioner caspases) in the experimental group compared to the control (p ≤ 0.03; Figure 2). In addition, we also determined the level of PARP protein, whose cleavage leads to activation of the apoptotic pathway via the intrinsic pathway (Chaitanya et al., 2010). Our results showed that there was an increased amount of cleaved PARP in the liver of infected rabbits (p ≤ 0.01; Figure 2). Since caspase-3 is a key performer of apoptosis, we also decided to determine its activity. As a result, our results indicated that caspase-3 activity in the liver is increased in infected rabbits compared to control (p ≤ 0.01; Figure 2).

Executor caspase-3 is one of the main effectors of apoptosis, which is involved in both apoptosis induced by the extrinsic death receptor-mediated pathway and the mitochondrial pathway (Thomson, 2001). The Bax and Bcl-2 proteins belong to the regulatory proteins and have anti-apoptotic and pro-apoptotic effects. Bcl-2-family proteins regulate mitochondrial membrane permeability, enabling cytochrome c release from mitochondria. Therefore, a high Bax/Bcl-2 mRNA expression level ratio indicates apoptotic activity (Guicciardi and Gores, 2005).

We assessed selected apoptotic markers to determine whether apoptotic gene expression changes in peripheral blood cells during Lagovirus europaeus/GI.2 infection (Table 2; Figure 3). We recorded at 12 h p.i. increased expression of caspase-3 (p ≤ 0.01), as well as Bax (p ≤ 0.01) and Bcl-2 (p ≤ 0.01), in infected rabbits compared to the control group. At 24 h p.i. there was an increase in the expression of anti-apoptotic Bcl-2 (p ≤ 0.01), and reduced levels of Bax/Bcl-2 mRNA expression ratio (p ≤ 0.01). Subsequently, we obtained increases in proapoptotic Bax at 36 h p.i. (p ≤ 0.05) and 48 h p.i. (p ≤ 0.01), and higher levels of Bax/Bcl-2 mRNA expression ratio – 36 h p.i. (p ≤ 0.01) and 48 h p.i. (p ≤ 0.01), in addition to an increase in caspase-3 gene expression only at 36 h p.i. (p ≤ 0.03).

The first stage of autophagy is the nucleation of the vesicles, and at this stage, the elements targeted for degradation are surrounded by an insulating membrane - phagophore. A key role during this phase plays a protein complex containing class III phosphatidylinositol 3-kinase (PI3K3 or Vps34), serine–threonine kinase (p150 or Vps15), and Beclin-1 (Atg6), which is directed to the site of autophagosome formation by Atg14 (Parzych and Klionsky, 2014). The activity of the PI3K3–Beclin-1–p150 complex can be regulated by a variety of proteins. The positive regulators of this complex include, among others UVRAG, which increases the activity of PI3K3 kinase and is involved in the maturation of the autophagosome (Bębnowska and Niedźwiedzka-Rystwej, 2022). The formation of the autophagosome is dependent on two ubiquitin-like protein systems. The first complex is formed by the conjugated Atg12 and Atg5 which then interact with Atg16L to form the Atg12–Atg5–Atg16L complex. The formation of this complex enables the elongation of the insulating membrane and thus the formation of an autophagosome (Parzych and Klionsky, 2014). The second complex necessary for autophagosome formation is LC3-II–PE. The light chain of microtubule-associated protein (LC3) is formed as a pro-LC3 precursor and transformed into the cytosolic form of LC3-I by proteolytic cleavage by Atg4. Through the activity of Atg7 and Atg3, the LC3-I form is linked with phosphatidylethanolamine (PE) and transforms into the mature LC3-II form, which is incorporated into the isolating membranes of autophagosomes. In the next step, LC3-II binds to the p62/SQSTM1 protein (Sequestosome 1), which recognizes ubiquitin located on the material to be degraded, creating a bridge between the autophagosome and the cargo to be removed (Parzych and Klionsky, 2014). The autophagosome is combined with the lysosome to form autolysosomes inside which the load is degraded (Parzych and Klionsky, 2014). The process of fusion of autophagosomes with lysosomes involves, among others, lysosomal associated membrane protein 1 (LAMP-1) and lysosomal associated membrane protein 2 (LAMP-2), which is why they serve as markers of late autophagic organelles (Klionsky et al., 2021).

As the liver is the main target of viral replication, we decided to analyze autophagic flux in this organ. Our results indicate that after the infection autophagic genes [Beclin-1 (p ≤ 0.03), UVRAG (p ≤ 0.03), Atg5 (p ≤ 0.01), Atg12 (p ≤ 0.01) and Atg16L (p ≤ 0.03)] are upregulated in contrast to the control group (Table 3; Figure 4). We performed also immunohistochemical detection of three autophagic markers: LC3B, Beclin-1, and Atg12. Our results indicate that the expression of these three markers was higher in infected animals in contrast to the control group [LC3B (p ≤ 0.01), Beclin-1 (p ≤ 0.01), and Atg12 (p ≤ 0.01)] (Figure 4). Figure 5 shows the representative pictures of a comparison between the experimental and control groups. We also assessed the levels of key proteins involved in autophagy. Our results indicate that in the liver of infected rabbits compared to the control group there is an increased level of Beclin-1 (p ≤ 0.03), LAMP-2 (p ≤ 0.05), LC3-II (p ≤ 0.03), but also a decreased level of p62 protein (p ≤ 0.01), indicating an autophagic flux in this organ (Figure 4).

Our investigation also included two additional organs because we have lately shown a strong apoptotic response in kidneys and spleen tissue during Lagovirus europaeus/GI.2 infection (Bębnowska et al., 2023). We looked at whether the autophagic response is equally noticeable in these organs. Results showed that in the kidney tissue of infected rabbits, some autophagic genes are upregulated [UVRAG (p ≤ 0.03), Atg5 (p ≤ 0.01), Atg12 (p ≤ 0.01), Atg16L (p ≤ 0.05) and MAP1LC3B (p ≤ 0.01)] compared to the control group (Table 3, Figure 6). Immunohistochemical assay indicated high expression of LC3B (p ≤ 0.03), Atg12 (p ≤ 0.03), and Beclin-1 (p ≤ 0.01) proteins and changes were detected in the renal cortex and renal medulla (Figures 6–8). Additional analysis of relative protein levels indicated elevated values of Beclin-1 (p ≤ 0.03), LAMP-1 (p ≤ 0.03), LAMP-2 (p ≤ 0.01), and LC3-II (p ≤ 0.03), but also decreased level of p62 (p ≤ 0.03) compared to the control group (Figure 6).

Analysis of spleen tissue showed that autophagic genes [Beclin-1 (p ≤ 0.01), UVRAG (p ≤ 0.05), Atg5 (p ≤ 0.01), Atg12 (p ≤ 0.01), Atg16L (p ≤ 0.01) and MAP1LC3B (p ≤ 0.01)] were upregulated in the experimental group, and also a high expression of Beclin-1 (p ≤ 0.01) and Atg12 (p ≤ 0.03) proteins (Table 3; Figure 9) was detected. Figure 10 shows representative pictures of the result from the immunohistochemical assay. Results of relative protein levels analysis of autophagic markers determined by the Western Blot method showed in the spleen tissue higher values of Beclin-1 (p ≤ 0.05), LAMP-1 (p ≤ 0.03), LAMP-2 (p ≤ 0.03), and LC3-II (p ≤ 0.05), but also a lower level of p62 (p ≤ 0.05) compared to the control group.

To determine whether autophagic genes are upregulated in peripheral blood cells during Lagovirus europaeus/GI.2 infection, we assessed the expression of several key markers (Table 4; Figure 2). Our results indicate that in the experimental group, compared to the healthy control, there is a decrease in the expression of autophagy-related genes during the initial stage of infection, which increases over time. We registered that the mRNA expression of Beclin-1 (p ≤ 0.01), MAP1LC3B (p ≤ 0.03), Atg5 (p ≤ 0.05), Atg12 (p ≤ 0.01) and Atg16L (p ≤ 0.03) was decreased in the experimental group compared to the control group at 12 h p.i. At 24 h p.i. decrease in autophagy gene expression was ongoing and significant differences were registered in Beclin-1 (p ≤ 0.03) and MAP1LC3B (p ≤ 0.03). From 36 h p.i. there was an increase in UVRAG (p ≤ 0.05), Atg5 (p ≤ 0.03), and Atg16L (p ≤ 0.03) mRNA expression. At 48 h p.i., higher levels of Atg5(p ≤ 0.01), Atg12 (p ≤ 0.01), and Atg16L (p ≤ 0.01) were obtained in the experimental group compared to the control.