Antiviral effect of the viroporin inhibitors against Taiwan isolates of infectious bronchitis virus (IBV)

Highlights

• •The envelope (E) proteins of infectious bronchitis virus (IBV) resulted in inhibited bacteria growth, increased permeability of bacteria, and blocked protein synthesis of bacteria.
• •Hygromycin B impeded protein translation in DF-1 cells and damaged their membrane integrity in the presence of E proteins.
• •The viroporin activity of the E proteins from IBV H-120, IBV serotype TW-I, and IBV serotype TW-II was confirmed.
• •Treatments with the viroporin inhibitors increased the survival rates in IBV-infected chicken embryos and chickens.
• •Viroporin inhibitors bound to the lipid-facing surface within the transmembrane domain of the E protein, inhibiting ion conduction.

Abstract

Coronaviruses (CoVs) are significant animal and human pathogens, characterized by being enveloped RNA viruses with positive-sense single-stranded RNA. The Coronaviridae family encompasses four genera, among which gammacoronaviruses pose a major threat to the poultry industry, which infectious bronchitis virus (IBV) being the most prominent of these threats. Particularly, IBV adversely affects broiler growth and egg production, causing substantial losses. The IBV strains currently circulating in Taiwan include the IBV Taiwan-I (TW-I) serotype, IBV Taiwan-II (TW-II) serotype, and vaccine strains. Therefore, ongoing efforts have focused on developing novel vaccines and discovering antiviral agents. The envelope (E) proteins of CoVs accumulate in the endoplasmic reticulum-Golgi intermediate compartment prior to virus budding. These E proteins assemble into viroporins, exhibiting ion channel activity that leads to cell membrane disruption, making them attractive targets for antiviral therapy.

In this study, we investigated the E proteins of IBV H-120, as well as IBV serotypes TW-I and TW-II. E protein expression resulted in inhibited bacteria growth, increased permeability of bacteria to β-galactosidase substrates, and blocked protein synthesis of bacteria by hygromycin B (HygB). Furthermore, in the presence of E proteins, HygB also impeded protein translation in DF-1 cells and damaged their membrane integrity. Collectively, these findings confirm the viroporin activity of the E proteins from IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. Next, the viroporin inhibitors, 5-(N,N-hexamethylene) amiloride (HMA) and 4,4’-diisothiocyano stilbene-2,2’-disulphonic acid (DIDS) were used to inhibit the viroporin activities of the E proteins of IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. In chicken embryos and chickens infected with IBV serotypes TW-I and IBV TW-II, no survivors were observed at 6 and 11 days post-infection (dpi), respectively. However, treatments with both DIDS and HMA increased the survival rates in infected chicken embryos and chickens and mitigated histopathological lesions in the trachea and kidney. Additionally, a 3D pentameric structure of the IBV E protein was constructed via homology modeling. As expected, both inhibitors were found to bind to the lipid-facing surface within the transmembrane domain of the E protein, inhibiting ion conduction. Taken together, our findings provide comprehensive evidence supporting the use of viroporin inhibitors as promising antiviral agents against IBV Taiwan isolates.

1. Introduction

Coronaviruses (CoVs) are large, positive-sense single-stranded RNA viruses enclosed in envelopes, which pose a significant threat to the health of both animals and humans. Among them, alphacoronaviruses such as human coronavirus 229E (HCoV-22E) and related CoVs cause common colds in humans, whereas betacoronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are responsible for severe respiratory diseases with high mortality rate in humans (Larson et al., 1980; Webb et al., 2022). Among animal coronaviruses, infectious bronchitis virus (IBV) is classified as a gammacoronavirus that causes substantial losses in the global poultry industry (Webb et al., 2022). IBV infects epithelial cells in the trachea, kidney, and oviduct, leading to respiratory symptoms, kidney diseases, and reproductive issues. This ultimately affects broiler growth efficiency and egg production/quality, while also increasing susceptibility to opportunistic infections (Cavanagh, 2007). The spike protein, a major structural protein of IBV, contains numerous epitopes that induce neutralizing antibodies. Crucially, even a slight 2 % change in the amino acid sequence of this protein due to recombination or genetic mutation can lead to the emergence of new serotypes, perpetuating continuous outbreaks of infectious bronchitis worldwide, particularly in Asian countries (Chen and Wang, 2010). The most prevalent IBV strains circulating in Taiwan include two IBV serotypes, IBV Taiwan-I (TW-I) and IBV Taiwan-II (TW-II), along with vaccine strains. Additionally, transmission through wild birds or poultry trade has extended the reach of these strains into China (Ma et al., 2012). These challenges highlight the pressing need for the continuous development of effective vaccines and antiviral treatments to combat this disease in the poultry industry.

Previous studies have demonstrated that the envelope (E) proteins of CoVs accumulate at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) before budding progeny viruses via the secretory pathway and subsequently releasing them from host cells (Krijnse et al., 1994; Westerbeck and Machamer, 2019). These CoV E proteins exhibit the ion channel activity, potentially increasing membrane permeability in bacteria, cultured cells, and artificial membranes (Xia et al., 2022). Such viral E proteins, possessing pore-forming activity, are known as viroporins. Viroporins are not exclusive to CoVs but are also encoded by other viruses such as poliovirus, hepatitis C virus (HCV), influenza virus (IAV), togavirus, picornavirus, and human immunodeficiency virus (HIV) (Guinea and Carrasco, 1994; Liao et al., 2004).

Cell rounding, syncytial cell formation, and lysis of CoV-infected cells are attributed to viroporins rather than the viral fusogenic protein (Gonzalez and Carrasco, 2003). Viroporins, characterized by an amphipathic α-helix and clustered basic residues, oligomerize to form channels. This results in the passage of small hydrophilic molecules across the cell membrane, disturbance of membrane potential and ionic gradient, and leakage of vital cell compounds (Hyser and Estes, 2015; Liao et al., 2004). These conditions favor intracellular ionic conditions conducive to virus replication, gradual damage to infected cell membranes, virus assembly, and the release and spread of the progeny viruses.

Viroporins have recently emerged as the antiviral targets, with numerous small molecules known to effectively disrupt or interfere with the ion channel activity of these viral proteins (Scott and Griffin, 2015). Several inhibitors, including amantadine, rimantadine, 5-(N,N-hexamethylene) amiloride (HMA), 4,4’-diisothiocyano stilbene-2,2’-disulphonic acid (DIDS), and iminosugar derivatives, have shown antiviral potency in various assay systems (Breitinger et al., 2021). Among these inhibitors, HMA has been reported to inhibit the replication of CoVs, including feline infectious peritonitis virus (FIPV) serotype I, mouse hepatitis virus (MHV), and HCoV-229E, in cultured cells (Takano et al., 2015). Patch clamp electrophysiology and cell viability assays have demonstrated the ability of HMA to inhibit the viroporin activity of the SARS-CoV E protein (Breitinger et al., 2021). DIDS also significantly reduced the titer of FIPV serotype II from cells. However, the inhibitory effects of HMA and DIDS varied among different serotypes of the virus (Takano et al., 2015).

In this study, we investigated and compared the viroporin activity of the E proteins from different IBV serotypes using bacterial and avian cell models. Additionally, we evaluated the therapeutic potential of HMA and DIDS against the circulating serotypes IBV TW-I and IBV TW-II using both chicken embryos and adult chickens. Our results provide comprehensive evidence supporting the efficacy of both viroporin inhibitors, HMA and DIDS, as promising antivirals against IBV Taiwan isolates, based on their ability to inhibit the viroporin activity of the E proteins.

2. Materials and methods

2.1. Bioinformatic analysis, structure modeling, and molecular docking

To classify the putative viroporin motifs of the IBV E protein, we employed the hydropathy analysis program from the transporter classification database to generate the Kyte and Doolittle hydropathy plots (Wilkins et al., 1999). Additionally, we utilized the PSIPred prediction analysis suite (http://bioinf.cs.ucl.ac.uk/introduction) to predict the secondary structure and membrane topology of the E protein (McGuffin et al., 2000). Helical wheel plots were generated using the Heliquest PepWheel analysis program (http://heliquest.ipmc.cnrs.fr) to identify the clustered basic residues within the putative viroporin motifs of the E protein (Gautier et al., 2008). The sequences of the IBV E protein (GenBank accession number BDN79603.1) were used as query sequences for three-dimensional (3D) structure modeling. The pentameric structures of the E protein were modeled using SWISS-MODEL with PDB 5 × 29 as the template (Surya et al., 2018; Waterhouse et al., 2018). Two-dimensional (2D) structures of HMA and DIDS were initially drawn and converted into 3D structures using the Discovery Studio software (v19.1.0.18287). Molecular docking was conducted using the Libdock tool within the Discovery Studio package. The binding pose with the highest LigScore was then selected, followed by visualization and analysis of ligand-protein interactions using PyMOL v2.2.3 and LigPlot+, respectively (Laskowski and Swindells, 2011).

2.2. Virus propagation, RNA preparation, and complementary DNA (cDNA) synthesis

The present study focused on IBV strains H-120, IBV TW-I (TW-I EU822336), and IBV TW-II (TW-II DQ646404). The names IBV TW-I (TW-I EU822336) and IBV TW-II (TW-II DQ646404) correspond to the IBV strains 3468/07 and TW2296/95, respectively (Chen et al., 2009; Huang and Wang, 2007). Virus propagation was conducted in 10-day-old specific-pathogen-free (SPF) embryonated eggs via the allantoic route. Allantoic fluid was harvested at 72 h post-inoculation and stored at −80 °C. Total RNA was extracted from the allantoic fluid using Trizol reagent following the manufacturer's instructions (Thermo Fisher Scientific, USA). RNA was reverse transcribed into cDNA using MMLV reverse transcriptase (ProTech, Taiwan). The cDNAs were synthesized from 50 ng RNA via reverse transcriptase (RT) polymerase chain reaction (PCR). The obtained cDNA fragments were then amplified via PCR. The protocol consisted of an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, and elongation at 72 °C for 30 s, with a final extension step at 72 °C for 10 min. The gene-specific primer sequences for RT-PCR are outlined in Table 1. For bacterial expression, the amplified products of the E protein genes of IBV H-120, IBV TW-I, and IBV TW-II were digested with SacI and EcoRI, purified, and cloned into the pET24a(+) plasmid previously linearized using the SacI and EcoRI restriction enzymes. For eukaryotic cell expression, the amplified products of the IBV H-120 E protein gene, the IBV TW-I E protein gene, and the IBV TW-II E protein gene were digested with EcoRI and EcoRV, purified, and cloned into the pcDNA 3.1 plasmid previously linearized with the EcoRI and EcoRV enzymes. After ligation with T4 DNA ligase, the products were transformed into Top10 E. coli competent cells. Plasmids were extracted and verified using the bidirectional sequencing.

Table 1.

Primers used in this study.

Primer Name	Sequence (5’-3’)	Nucleotide positionabc
IBV H120 E-Pet F primer	5’-TATTGGATCCATGATGAATTTATTGAATA-3’	24150-24170
IBV H120 E-Pet R primer	5′-AATAGAATTCTCAATGGTGGTGGTGATGATGAGAGTACAATTTGTTTCG-3′	24458-24479
IBV TW-I E-Pet F primer	5’-TATTGGATCCATGGCGAATTTATTGAAT-3’	3846-3864
IBV TW-I E-Pet R primer	5’-AATAGATATCTCAATGGTGGTGGTGATGATGAGTGTGCAATTTGTCGCG-3’	4155-4175
IBV TW-II E-Pet F primer	5’-TATTGGATCCATGATGAATTTATTGAATAA-3’	3840-3861
IBV TW-II E-Pet R primer	5’-AATAGATATCTCAATGGTGGTGGTGATGATGAGTGTGCAATTTGTCACA-3’	4148-4169
IBV H120 E-Pc F primer	5’-TATTGAATCATGATGAATTTATTGAATAA-3’	24150-24170
IBV H120 E-Pc R primer	5′-AATAGATATCTCAATGGTGGTGGTGATGATGAGAGTACAATTTGTTTCG-3′	24458-24479
IBV TW-I E-Pc F primer	5’-TATTGAATTCATGGCGAATTTATTGAAT-3’	3846-3864
IBV TW-I E-Pc R primer	5′-AATAGATATCTCAATGGTGGTGGTGATGATGAGTGTGCAATTTGTCGCG-3′	4155-4175
IBV TW-II E-Pc F primer	5’-TATTGAATTCATGATGAATTTATTGAATAA-3’	3840-3861
IBV TW-II E-Pc R primer	5’-AATAGATATCTCAATGGTGGTGGTGATGATGAGAGTACAATTTGT-3’	4148-4169
IBV H120 E-C1 deletion F primer	5’-AAGCATTTGTACAGGCTGGTGATGCTTGTTTATTTTGGTATAC-3’	24256-24282/24286-24302
IBV H120 E-C1 deletion R primer	5’-GTATACCAAAATAAACAAGCATCCACCAGCCTGTACAAATGCTT-3’	24256-24282/24286-24302
IBV H120 E-C2 deletion F primer	5’-ATTTGTACAGGCTGGTGATGCTTTATTTGGTATACATGGTTAG-3’	24261-243283/24290-24312
IBV H120 E-C2 deletion R primer	5’-CTAACCATGTATACCAAAATAAAGCATCACCAGCCTGTACAAAT-3’	24261-24283/24290-24312
IBV TW-I E-C1 deletion F primer	5’-TTGTACAAGTTGCTGACGCCTGTTTATTTTGGTATACTTGG-3’	3959-3979/3983-4003
IBV TW-I E-C1 deletion R primer	5’-CAAGTATACCAAAATAAACAGGCGTCAGCAACTTGTACAA-3’	3959-3979/3983-4003
IBV TW-I E-C2 deletion F primer	5’-TGTACAAGTTGCTGACGCCTTATTTTGGTATACTTGGG-3’	3960-3979/3986-4005
IBV TW-I E-C2 deletion R primer	5’-CCCAAGTATACCAAAATAAGGCGTCAGCAACTTGTACA-3’	3960-3979/3986-4005
IBV TW-II E-C1 deletion F primer	5’-AAGCATTTGTACAGGGTGGTGATGCTTGTTTATTTTGGTATA-3’	3947-3973/3977-3993
IBV TW-II E-C1 deletion R primer	5’-GTATACCAAAATAAACAAGCATCACCACCCTGTACAAATGCTT-3’	3947-3973/3977-3993
IBV TW-II E-C2 deletion F primer	5’-ATTTGTACAGGGTGGTGATGCTTTATTTTGGTATACATGGTTAG-3’	3951-3973/3980-4002
IBV TW-II E-C2 deletion R primer	5’-CTAACCATGTATACCAAAATAAAGCATCACCACCCTGTACAAAT-3’	3951-3973/3980-4002

Nucleotide position numbers of a) IBV H120 (GenBank accession No. MN548287), b) IBV TW-I IBV 3468/7 isolate (GenBank accession No. EU82236), and c) IBV TW-II TW2296/95 strain (GenBank accession No. DQ646404)

2.3. Site-directed mutagenesis

The involvement of two cysteine residues in the oligomerization of the IBV E protein was analyzed through site-directed mutagenesis, generating two mutants designated CI and CII. The CI mutant carries a point mutation at the C1 position, whereas the CII mutant carries mutations at both the C1 and C2 positions. The QuikChange II Site-Directed Mutagenesis Kit (Agilent, USA) was utilized to create the mutants. The PCR program consisted of an initial denaturation step at 95 °C for 2 min, followed by 18 cycles of denaturation at 95 °C for 20 s, annealing at 60 °C for 10 s, and elongation at 68 °C for 3 min, and a final extension step at 68 °C for 5 min according to the manufacturer's instructions. The primer sequences for mutant construction are outlined in Table 1. The PCR products were then digested with DpnI and transformed into XL1-Blue competent cells. Bidirectional DNA sequencing was employed to validate each clone. The extracted plasmids from the validated clones were subsequently transformed into the E. coli strain BL21 (DE3) cells for subsequent expression.

2.4. Growth kinetics of E. coli transformed with the IBV E gene plasmids

E. coli strain BL21 (DE3) cells transformed with pET-H120 E, pET-TW-I E, and pET-TW-II E, pET-H120 CI, pET-H120 CII, pET-TW-I CI, pET-TW-I CII, pET-TW-II CI, and pET-TW-II CII were cultured in LB medium supplemented with 30 mg/ml kanamycin overnight. Upon reaching an absorbance value (OD₆₀₀) of 0.6, induction of IBV E protein expression was initiated by adding 1 mM isopropyl-thio-β-D-thiogalactopyranoside (IPTG). For the inhibitor tests, HMA and DIDS were purchased from Sigma Aldrich (USA). A single colony was cultured in LB medium containing kanamycin until reaching an absorbance value of 0.6. Different concentrations of HMA (25 mM, 50 mM, and 75 mM) and DIDS (150 mM, 300 mM, and 450 mM) were separately added into the medium at 1 hour post-induction (hpi). The OD₆₀₀ values, representing the cell densities of cultured bacteria, were measured at the indicated time points post-induction.

2.5. Entry of o-nitrophenyl-β galactopyranoside (ONPG) into bacterial clones

The entry of ONPG (Sigma-Aldrich, USA) into different bacterial clones was measured to assess the effect of the expressed E proteins on cell permeability. E. coli strains harboring the pET-H120 E, pET-TW-I E, and pET-TW-II E, pET-H120 CI, pET-H120 CII, pET-TW-I CI, pET-TW-I CII, pET-TW-II CI, and pET-TW-II CII plasmids were cultured in LB medium supplemented with kanamycin. Once the OD₆₀₀ of the bacterial culture reached 0.6, E protein expression was induced by adding 1 mM IPTG. For the inhibitor tests, various concentrations of HMA (25 mM, 50 mM, and 75 mM) and DIDS (150 mM, 300 mM, and 450 mM) were separately added to the medium at 1 hpi. Next, 1 ml of bacterial broth was collected at the indicated time points post-induction. The pelleted bacteria were then suspended in 1 ml of M9 medium containing 2 mM ONPG. After incubation at 30 °C for 10 min, the reaction was terminated by adding 0.4 ml of 1 M sodium carbonate. Following centrifugation, the supernatant was collected, and its absorbance value at OD₄₀₅ nm was measured. This measurement was used to estimate the cleavage of ONPG inside the bacteria.

2.6. Western blotting for hygromycin B (HygB) assay

Hygromycin B (HygB) (Sigma-Aldrich, USA) is an antibiotic that inhibits protein synthesis once it enters bacterial cells. To detect the entry of HygB into the bacteria, E. coli strains carrying the pET-H120 E, pET-TW-I E, and pET-TW-II E, pET-H120 CI, pET-H120 CII, pET-TW-I CI, pET-TW-I CII, pET-TW-II CI, and pET-TW-II CII plasmids were cultured in LB medium with kanamycin. Upon reaching an OD₆₀₀ of 0.6, expression of the E protein was induced by adding 1 mM IPTG. After 50 min of induction, 2 mM HygB was added to the medium. For inhibitor tests, 75 μM HMA or 450 μM DIDS was also added at the same time. Following a 45-min treatment with 2 mM HygB, the bacteria were co-incubated with 10 mM puromycin (Clontech, USA) at 37 °C for 20 min. The bacteria were then pelleted via centrifugation and suspended in protein lysis buffer (250 mM NaCl, 20 mM Tris-HCl, 1 M phenylmethanesulfonyl fluoride, and 10 mM imidazole, pH 7.5) and 0.5 mg/ml lysozyme before the sonication, after which the supernatant was collected. To detect the entry of HygB into cells, DF-1 cells cultured in 24-well plates were transfected with pc-H120 E, pc-TW-I E, and pc-TW-II E plasmids mixed with Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, USA) for 24 h. For inhibitor tests, cells were pre-treated with 75 μM HMA or 450 μM DIDS prior to the transfection. The cells were either left untreated or treated with 25 μg/ml HygB for 2 h followed by 5 μg/ml puromycin labeling for 25 min. Next, the cells were lysed with the radio-immunoprecipitation assay buffer (150 mM NaCl, 1 % NP-40, 1 mM PMSF, and 50 mM Tris, pH 7.5), sonicated for 30 s, and centrifuged at 15,000 × g for 20 min. The supernatant was saved for later use. Protein samples extracted from bacteria or cells were resolved on 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane (Amersham Biosciences, USA), blocked at room temperature, and then probed with anti-puromycin antibody (Kerafast Inc, USA) or anti-His antibody (ProTec, Taiwan) overnight. After washing, the membrane was incubated with goat anti-mouse antibody conjugated with horseradish peroxidase (HRP) (ProTec, Taiwan). The membrane was then washed one more time, after which a chemiluminescence detection kit (ECL) (Thermo Fisher Scientific, USA) was used for development.

2.7. Oligomerization assay

E. coli strains carrying the pET-H120 E, pET-TW-I E, and pET-TW-II E plasmids were cultured in LB medium supplemented with kanamycin. Upon reaching an OD₆₀₀ of 0.6, protein expression was induced by adding IPTG. After 8 h of induction, the bacteria were pelleted via centrifugation and suspended in protein lysis buffer containing 0.5 mg/ml lysozyme before sonication. After centrifugation, the supernatant was incubated with the nickel-charged sepharose beads (GE, USA) at 4 °C overnight. After washing with washing buffer (500 mM NaCl, 50 mM Tris-HCl, and 40 mM imidazole, pH 7.5), the bound protein was eluted with elution buffer (250 mM NaCl, 20 mM Tris-HCl, and 500 mM imidazole, pH 7.5), dialyzed, and separated via 10 % SDS-PAGE in the presence of 10 mM iodoacetamide with or without 200 mM dithiothreitol (DTT), after which it was transferred to a PVDF membrane and probed with anti-His antibody. After washing, the membrane was hybridized with goat anti-mouse antibody conjugated with HRP. Finally, the membrane was developed using the ECL kit.

2.8. Immunofluorescence staining for HygB assay

DF-1 cells were transfected with pc-H120 E, pc-TW-I E, and pc-TW-II E plasmids mixed with Lipofectamine 2000 transfection reagent for 24 h. For inhibitor tests, cells were pre-treated with 75 μM HMA or 450 μM DIDS before transfection. One portion of the cells was left untreated to serve as a control, whereas the remaining cells were treated with 5 μM HygB for 2 h, followed by puromycin labeling at 5 μg/ml for 25 min. The cells were then fixed with 4 % paraformaldehyde for 5 min, followed by permeabilization with 0.5 % Triton X-100 for 10 min. To minimize background staining, the cells were blocked using 1 % bovine serum albumin for 1 h at 25 °C. After washing with PBS, the cells were co-stained with anti-puromycin antibody and anti-His antibody at 4 °C overnight. After washing, Alexa Flour 546-conjugated goat anti-mouse antibodies and FITC-conjugated goat anti-rabbit antibodies were used to co-stain the cells for 1 h, followed by DAPI counterstaining for 10 min. After a final wash, the cells were mounted and observed under an immunofluorescent microscope.

2.9. Flow cytometry

The integrity of the cells affected by the IBV E proteins and the viroporin inhibitors was measured as previously described (Ao et al., 2015). DF-1 cells were transfected with pc-H120 E, pc-TW-I E, and pc-TW-II E plasmids mixed with Lipofectamine 2000 transfection reagent for 48 h. For inhibitor tests, cells were treated with 75 μM HMA or 450 μM DIDS at the same time as transfection. Untreated DF-1 cells were used as the control group. The transfected cells were trypsinized, washed with PBS, and then re-suspended in binding buffer containing the propidium iodide (PI) according to the manufacturer's instructions. After washing with PBS, the cells were analyzed using flow cytometry. The BD FACSVerse software was used to calculate the percentage of PI-labeled cells.

2.10. Antiviral effect of viroporin inhibitors on chicken embryos

A total of 84 SPF chicken embryos (8-day-old) were randomly divided into 14 groups (n = 6 per group). These groups included a mock group, an IBV TW-I-infected group, an IBV TW-I-infected group treated with 150 mM DIDS, an IBV TW-I-infected group treated with 300 mM DIDS, an IBV TW-I-infected group treated with 450 mM DIDS, an IBV TW-I-infected group treated with 25 mM HMA, an IBV TW-I-infected group treated with 50 mM HMA, an IBV TW-I-infected group treated with 75 mM HMA, an IBV TW-II-infected group, an IBV TW-II-infected group treated with 150 mM DIDS, an IBV TW-II-infected group treated with 300 mM DIDS, an IBV TW-II-infected group treated with 450 mM DIDS, an IBV TW-II-infected group treated with 25 mM HMA, an IBV TW-II-infected group treated with 50 mM HMA, and an IBV TW-II-infected group treated with 75 mM HMA. Each embryo was inoculated through the allantoic cavity with 100 μl of allantoic fluid containing a viral load equal to 10⁶ 50 % egg infectious dose (EID₅₀). Additionally, each embryo received the respective inhibitor injection after 1 h of incubation. Embryo growth was monitored, and daily embryo deaths were documented until 6 days post-infection (dpi). Survival curves for each group were plotted using the GraphPad Prism 8.0 software. At the end of the experiments, the severity of lesions in each embryo from different groups, including toe curling, body hemorrhage, and dwarfing, were graded. The allantoic fluid from each embryo was then collected and subjected to virus titration.

2.11. Hemagglutination titration of IBV

This method was adapted from a previous study (Sun et al., 2010). The allantoic fluid collected from each embryo was centrifuged at 30,000 × g for 45 min. The pellet was then suspended in 0.01 M Tris-HCl (pH 6.4) and then incubated with an equal volume of phospholipase C type 1 (1 unit/ml) (Sigma-Aldrich, USA) at 37 °C for 30 min. The lysate containing the virus was serially diluted in a two-fold range and subsequently mixed with an equal volume of the chicken red blood cells (RBCs) at 4 °C for 1 h. The hemagglutination (HAU) titer was calculated as the reciprocal of the highest dilution of the virus resulting in complete agglutination of chicken RBCs. The HAU of each embryo from different groups was recorded.

2.12. Antiviral effect of viroporin inhibitors on chickens

A total of 66 one-day-old SPF chicks were obtained and randomly assigned to 11 experimental groups (n = 6 per group). These groups included a mock group, an IBV TW-I-infected group, an IBV TW-I-infected group treated with 150 mM DIDS, an IBV TW-I-infected group treated with 450 mM DIDS, an IBV TW-I-infected group treated with 25 mM HMA, an IBV TW-I-infected group treated with 75 mM HMA, an IBV TW-II-infected group, an IBV TW-II-infected group treated with 150 mM DIDS, an IBV TW-II-infected group treated with 450 mM DIDS, an IBV TW-II-infected group treated with 25 mM HMA, and an IBV TW-II-infected group treated with 75 mM HMA. Each chick was intranasally inoculated with 10⁶ EID₅₀ virus. After one day, each chick was gavaged with the respective concentration of each inhibitor dissolved in 0.5 ml PBS. Clinical signs and survival of the chicks were monitored and recorded daily until 11 dpi. The clinical scores of IBV-infected chickens were interpreted based on the previous study (Avellaneda et al., 1994). The clinical signs were evaluated as follows: 0 = no clinical signs; 1 = lacrimation, slight shaking, watery feces or tracheal rales; 2 = lacrimation, presence of nasal exudate, depression, watery feces, apparent sneezing or cough; 3 = same criteria as with 2 but with severe watery feces; 4 = death. Throughout the 11-day experiment period, the mean score of each group was calculated and compared with each other. The lesions in IBV-infected chickens were assessed based on a previous study (Li et al., 2020). The lesions in the trachea were evaluated as follows: 0 = no lesions; 1 = slight increase in mucin; 2 = large increase in mucin; 3 = large increase in mucin and mucosal congestion. The lesions in the kidney were evaluated as follows: 0 = no lesions; 1 = swelling with visible urate; 2 = swelling with urate; 3 = same as 2 with a large amount of urate deposition in the kidney. The average lesion scores for each group were calculated and compared with each other. Additionally, survival curves for each group were plotted using the GraphPad Prism 8.0 software.

2.13. Histopathology and immunohistochemical staining (IHC)

Trachea and kidney tissues were washed, fixed in 10 % formaldehyde, and embedded in paraffin wax followed by sectioning. The sections were then stained with hematoxylin and eosin (HE), after which histopathological changes were evaluated under the microscope. For IHC, paraffin was removed using xylene followed by rehydration with ethanol. Viral antigens were retrieved by treating the sections with 10 mM citric acid buffer at 95 °C followed by 3 % hydrogen peroxide. The sections were then probed with anti-IBV N protein monoclonal antibody (Novus Biologicals, USA) at 37 °C for 30 min after blocking with 1 % BSA. After washing, the sections were hybridized with HRP-conjugated goat anti-mouse antibodies and subsequently allowed to react with the peroxidase substrate (DAB). The slides were then subjected to HE counterstaining, fixed, and mounted. Lesion scores were interpreted based on the previous study (Alvarado et al., 2003). For the trachea, lesion scores were interpreted as follows: 1 = no lesion; 2 = mild lymphocyte infiltration with occasional loss of cilia and mucosal epithelial cell exfoliation; 3 = moderate epithelial cell exfoliation, lymphocyte infiltration, submucosal edema, and congestion; 4 = an extensive loss of cilia and mucosal epithelial cell exfoliation, severe lymphocytes infiltration with severe submucosal edema and congestion. For the kidney, lesion scores were interpreted as follows: 1 = no lesion; 2 = mild lymphocyte infiltration, vacuolar degeneration and necrosis; 3 = moderate lymphocyte infiltration, granular degeneration, necrosis and vacuolar degeneration; 4 = extensive lymphocyte infiltration, tubular epithelial cell exfoliation, severe vacuolar degeneration, and granular degeneration.

3. Results

3.1. Bioinformatic characterization of the viroporin activity of IBV E proteins

To assess whether the E proteins of IBV exhibit features of viroporin activity, we predicted the amphipathic domains and transmembrane domains (TMD) within the IBV E protein. The TMD contained pore-lining regions within their helices, as indicated by the membrane topology analysis. An amphipathic moment was identified in the E protein, with its hydrophobic region extending from aa 15 to 62. PSIPred prediction analyses suggested the presence of an α-helical region from aa 15 to 54 of the E protein. Accordingly, the α-helix proposed by the PSIPred program was located within the hydrophobic region encompassing the TMD. The formation of an ion channel by the oligomerization of IBV E proteins was facilitated by this α-helix region. The helical wheel plot indicated that TMD exhibited a hydrophobic moment (0.421) with distinct polar and nonpolar faces (Fig. 1).

Fig. 1.

Bioinformatic analysis of the viroporin activity of IBVs. Kyte-Doolittle hydropathy plot of IBV E protein predicted from the amino acid sequence. A 9-amino acid window size was used as the protscale (A). Helical wheel plot of the TMD of IBV E protein based on Heliquest (B). Prediction of secondary structure algorithm of IBV E protein based on PSIPred (C).

3.2. Growth inhibition, modification of the membrane permeability, and inhibition of the protein synthesis of E. coli by the expressed E proteins of different IBV serotypes

The impact of the IBV E protein expressed in E. coli on the bacterial cell growth was investigated using a well-known viroporin, AIV M2 protein, as a positive control. OD₆₀₀ values were measured at different time points post-IPTG induction to assess bacterial growth. Various degrees of growth retardations (all p<0.05 vs. the negative control) were observed in bacteria expressing the AIV M2 protein, IBV H120 E protein, IBV TW-I E protein, and IBV TW-II E protein, compared to the negative control group. The growth kinetics of the bacteria expressing IBV TW-II E protein closely resembled that of bacteria expressing AIV M2 protein, exhibiting the lowest absorbance value at 6 hpi (Fig. 2A). These results demonstrated that the growth of the bacteria was inhibited by the IBV E protein in a time-dependent manner, with varying inhibitory effects across different IBV serotypes. This further suggested that the membrane permeability of E. coli BL21 (DE3) cells was enhanced by the E protein of different IBV serotypes. Additionally, the expression of the AIV M2 and IBV E proteins was confirmed via western blot analyses. As expected, the sizes of the AIV M2, H120 E, TW-I E, and TW-II E proteins were 12, 13, 13, and 13 kDa, respectively (Fig. 2B). To assess whether the expressed E proteins of different IBV serotypes affect bacterial membrane permeability, the entry of ONPG into cells was analyzed. ONPG entry into the cell, indicated by its conversion to a colored product through β-galactosidase activity, was measured via OD₄₀₅ values. Bacteria expressing the AIV M2 protein, IBV H120 E protein, IBV TW-I E protein, and IBV TW-II E protein exhibited significantly higher ONPG uptake rates compared to the negative control group at every time point (all p<0.05 vs. the negative control) (Fig. 2C). ONPG entry for the cells expressing the IBV TW-II protein was similar to that of the cells expressing the AIV M2 protein, followed by those expressing IBV TW-I and IBV H120. These results further validate the enhanced membrane permeability induced by the IBV E proteins of different serotypes. HygB, an antibiotic that inhibits protein synthesis known to be impermeable to bacteria, was used to assess membrane permeability. After 45 min of IPTG induction, puromycin was used to label the bacteria. Our findings indicated that the expression of IBV H-120, IBV TW-I, IBV TW-II, and AIV M2 enhanced membrane permeability, allowing HygB entry and blocking protein synthesis in the host cell. In contrast, the control group (pET24a vector only) did not exhibit inhibition of protein synthesis because HygB was unable to enter the cells (Fig. 2D).

Fig. 2.

Growth retardation effect of the AIV M2 protein, IBV TW-I E protein, and IBV TW-II E protein on E. coli. In this study, E. coli transformed with pET24a only, pET24a IBV TW-I E, pET24a IBV TW-II E, and pET24a AIV M2 were incubated at 37 °C and then induced with IPTG. Bacteria carrying the pET24a AIV M2 and the pET24a only were respectively used as the positive and negative controls. Bacterial densities were calculated based on the absorbance value (OD 600 nm) measured in triplicate. The X-axis represents the hours post-induction (hpi) and the Y-axis represents the OD₆₀₀ nm values at the indicated hpi (A). The expression of the AIV M2 protein, IBV TW-I E protein, and IBV TW-II E protein was examined from the cell lysates with (+) or without (−) IPTG induction via western blot analysis (B). The entry of ONPG into bacterial cells was assessed using E. coli transformed with pET24a only, pET24a TW-I E, pET24a TW-II E, and pET24a AIV M2. The transformed bacteria were induced with 1 mM IPTG for 45 min. The bacteria were further incubated in the presence of 2 mM ONPG for 10 min at the indicated times. Finally, the β-galactosidase activity was determined by measuring the OD₄₀₅ nm value. AIV M2 protein and cells carrying the pET24a were used as the positive and negative controls, respectively. All data are presented as mean values ± standard error (SE) from three independent experiments. Different letters indicate significant differences (p-value<0.05) based on one-way ANOVA followed by Tukey's multiple comparison test (C). The membrane permeability of cells expressing the IBV TW-I protein and IBV TW-II protein was also assessed. E.coli transformed with the IBV TW-I E plasmid and IBV TW-II E plasmid were induced to express these proteins and then treated with HygB and puromycin. The bacteria were then lysed and western blot analyses were conducted using anti-puromycin antibodies to assess protein synthesis (D). Oligomerization of the IBV H120 E protein, IBV TW-I E protein, and IBV TW-II protein. The purified IBV H120 E protein, IBV TW-I protein, and IBV TW-II protein were separated without DTT (under non-reducing conditions) or with DTT (under reducing conditions) via SDS-PAGE followed by western blotting. Oligomers with different molecular weights (kDa) for reference are indicated on the left side of each panel (E).

3.3. Oligomerization of the expressed E proteins of different IBV serotypes

The expressed E proteins of different IBV serotypes were analyzed via reducing and non-reducing SDS-PAGE to investigate their potential to form oligomers. Following 3 h of IPTG induction, bacteria were lysed in lysis buffer with or without DTT. Western blot analysis revealed molecular weights of approximately 13 kDa, 26 kDa, 52 kDa, and 65 kDa for the E proteins of different IBV serotypes under non-reducing conditions. These bands corresponded to putative monomers, dimers, tetramers, and pentamers of the E proteins, respectively. Under reducing conditions, only the 13 kDa monomer was primarily detected. These findings suggest that the E proteins of IBV H-120, IBV TW-I, and IBV TW-II serotypes are able to form the homo-oligomers (Fig. 2E).

3.4. Effect of viroporin inhibitors on the bacterial growth, membrane permeability, and protein synthesis affected by the expressed E proteins of different IBV serotypes

Next, our study sought to assess the effect of viroporin inhibitors (HMA and DIDS) on the IBV E protein-induced inhibition of E. coli growth. Growth kinetics analysis was performed on bacteria expressing IBV H120 E protein, IBV TW-I E protein, and IBV TW-II E protein in the presence of varying concentrations of DIDS (0, 150, 300, and 450 μM) or HMA (0, 25, 50, and 75 μM). Significant growth retardation was observed in bacteria expressing all the IBV E proteins compared to the negative control. However, significant increases in growth were noted in bacteria expressing IBV H120 E protein with at least 150 μM DIDS. Similarly, significant growth enhancement was observed in bacteria expressing IBV TW-I protein with 300 μM DIDS at 6 hpi. The cell density of bacteria expressing the IBV TW-II E protein treated with 150 μM DIDS was not significantly different from that of bacteria expressing the IBV TW-II E protein only. However, significant growth enhancement was observed in bacteria expressing IBV TW-II protein when treated with at least 300 μM DIDS. Similarly, significant increases in cell growth were observed in bacteria expressing IBV H120 E protein when treated with at least 25 μM HMA (p<0.05). Additionally, treatment with at least 25 μM HMA resulted in significant increases in cell density of bacteria expressing IBV TW-I E protein or IBV TW-II E protein at 6 hpi. These results indicate that the inhibitory effect of IBV E protein on E. coli growth can be mitigated to various degrees by the presence of DIDS or HMA in a dose-dependent manner. DIDS exerted similar levels of abolishment of bacterial growth inhibition in the IBV H-120, TW-I, and TW-II serotypes. However, although there was a significant difference, the mitigation of growth inhibition induced by IBV E protein was only slightly weaker using HMA. Our findings demonstrated that the nullification of bacterial growth inhibition by HMA was largely similar among the IBV H-120, IBV TW-I, and IBV TW-II serotypes (Fig. 3A). Next, we examined the effect of viroporin inhibitors on the membrane permeability of E. coli induced by the IBV E protein. Significant increases in ONPG uptake were observed for bacteria expressing IBV H120 E protein compared to the negative control group. Bacteria expressing the IBV H120 E and IBV TW-I E proteins exhibited significant reductions in ONPG uptake upon treatment with 300 μM and 450 μM DIDS. Similarly, DIDS significantly decreased ONPG entry in cells expressing the IBV TW-II E protein in a dose-dependent manner. For HMA, significant decreases in ONPG entry were observed in cells expressing the IBV H120 E protein, IBV TW-I E protein, and IBV TW-II E protein at HMA concentrations as low as 25 μM. Furthermore, the entry of ONPG into bacteria expressing these IBV E proteins was dose-dependently reduced at HMA concentrations of 50 μM and 75 μM. Although both DIDS and HMA significantly nullified the increased uptake of ONPG by cells expressing the IBV E proteins, DIDS showed higher potency compared to HMA. Nevertheless, the performance of both inhibitors was generally very similar (Fig. 3B). Collectively, these findings suggested that the increased membrane permeability in bacteria induced by the E proteins of different IBV serotypes can be reversed by both viroporin inhibitors. Additionally, the inhibition of host protein synthesis by the HygB was observed in bacteria expressing IBV H120, TW-I, and TW-II proteins. However, the reductions in host protein synthesis induced by the IBV E proteins were reversed in the presence of 450 μM DIDS. Similarly, when the viroporin activities of IBV E proteins of the IBV H120, TW-I, and TW-II strains were inhibited by 75 μM HMA, the protein synthesis of bacteria returned to normal levels (Fig. 3C).

Fig. 3.

Effect of viroporin inhibitors on the growth of E. coli expressing the IBV H120 E protein, IBV TW-I E protein, and IBV TW-II E protein. Bacteria were incubated at 37 °C and then induced with IPTG in the presence or absence of 150 μM DIDS, 300 μM DIDS, and 450 μM DIDS or 25 μM HMA, 50 μM HMA, and 75 μM HMA. Bacteria carrying the pET24a plasmid were used as the negative control. The X-axis represents the hpi and the Y-axis represents the OD₆₀₀ nm values at the indicated hpi (A). Effect of the viroporin inhibitors on the entry of HygB into the bacterial cells expressing the IBV H120 E protein, IBV TW-I E protein, and IBV TW-II E protein. When the OD₆₀₀ of the bacteria expressing the IBV H120 E protein, IBV TW-I E protein, and IBV TW-II E protein reached 0.6, E protein expression was induced by IPTG in the absence or the presence of 150 μM DIDS, 300 μM DIDS, and 450 μM DIDS or 25 μM HMA, 50 μM HMA, and 75 μM HMA. The bacteria were further incubated in the presence of 2 mM ONPG for 10 min at the indicated time. β-galactosidase activity was determined by measuring the OD₄₀₅ value. The bacteria carrying the pET24a plasmid served as the negative control. All data are reported as the mean ± SE from three independent experiments. Significant differences between the negative controls vs. the H120, TW-I, and TW-II groups without treatments are indicated by *p<0.05. Significant differences between each treatment group vs. the H120, TW-I, and TW-II groups without treatments are indicated by **p<0.05 (B). Cell membrane permeability of E.coli modified with the IBV H120 E protein, IBV TW-I E protein, and IBV TW-II E protein. E.coli cells transformed with the IBV H120 E, IBV TW-I E, and IBV TW-II E plasmids were induced to express the respective proteins and then treated with HygB and puromycin in the absence or the presence of 450 μM DIDS or 75 μM HMA. The bacterial cells were then lysed and western blot analyses with anti-puromycin antibodies were conducted to assess protein synthesis (C).

3.5. Functional role of cysteine residues of the E proteins of different IBV serotypes

The effect of two cysteine residues at the amino acid positions 45 (C1) and 46 (C2) of the IBV E protein was investigated. Mutant CI, which contains a mutation at the C1 position, and mutant CII, which contains mutations at both the C1 and C2 positions, were analyzed. The bacteria expressing the IBV H120 E protein exhibited growth retardation compared to the negative control (p<0.05). The growth kinetics of bacteria expressing the IBV H120 CI protein were similar to those expressing the IBV H120 E protein. However, the cell density of bacteria expressing IBV H120 CI remained significantly different compared to that of IBV H120 E protein at 6 hpi. Interestingly, the growth kinetics of bacteria expressing the IBV H120 CII protein closely resembled that of the negative control. Furthermore, significant differences were observed between bacteria expressing IBV H120 CII and those expressing IBV H120 E protein at all time points. The growth kinetics of bacteria expressing the IBV TW-I CI protein and the IBV TW-II CI protein were similar to those of bacteria expressing the IBV H120 E protein, and no statistical difference was identified compared to the cells expressing the IBV H120 E protein at 6 hpi. Additionally, the growth kinetics of bacteria expressing the IBV TW-I CII protein and IBV TW-II CII protein resembled that of the negative control, with significant differences observed between them and bacteria expressing IBV H120 E protein at all time points (Fig. 4A). Regarding the uptakes of ONPG, bacteria expressing the IBV H120 E protein exhibited higher uptake compared to the negative control group. The amount of ONPG uptake for bacteria expressing IBV H120 CII remained similar to that of the negative control but significantly differed from the uptake of bacteria expressing IBV H120 E protein. Furthermore, the uptake of ONPG by bacteria expressing IBV H120 CI protein were similar to that of the bacteria expressing IBV H120 CI protein was similar to that of bacteria expressing IBV H120 E protein, despite significant differences observed at all time points. The expression of the IBV TW-I CII protein or the IBV TW-II CII protein led to significant decreases in the ONPG uptake levels, which were similar to that of the negative control but significantly different from bacteria expressing the IBV TW-I E protein or IBV TW-II E protein. Conversely, the uptake of ONPG by bacteria expressing the IBV TW-I CI protein resembled that by bacteria expressing the IBV TW-I protein, despite significant differences observed at each time point between them. Similar results were also found for bacteria expressing the IBV TW-II CI protein. These findings further confirmed that the membrane permeability of the bacteria is enhanced by the E proteins of different IBV serotypes (Fig. 4B). As expected, the expression of the IBV E proteins of the H120, TW-I, and TW-II serotypes in the presence of the HygB caused an almost complete blockage of the protein synthesis. Similar to the control group, no effect on bacterial protein translation was observed for the expressed IBV H120 CII protein, IBV TW-I CII protein, and IBV TW-II CII protein. However, significant reductions in protein synthesis were observed for the expression of IBV CI proteins of the H120, TW-I, and TW-II serotypes in the presence of HygB. Collectively, these results suggest that the cysteine residue at the C1 position may not to be involved in the viroporin activity of the E protein, but the presence of two cysteine residues at the C1 and C2 positions is necessary for maintaining the viroporin activity of the IBV E protein (Fig. 4C).

Fig. 4.

Effect of the E mutants on the growth of E. coli. The growth rates of E.coli expressing the IBV H120 E protein, IBV H120 CI protein, IBV H120 CII protein, IBV TW-I E protein, TW-I CI protein, TW-I CII protein, IBV TW-II E protein, IBV TW-II CI protein, and IBV TW-II CII protein were measured. The bacteria were incubated at 37 °C and then induced with IPTG. Bacteria carrying the pET24a were used as the negative control. The X-axis represents the hpi and the Y-axis represents the OD₆₀₀ nm values at the indicated hpi (A). The entry of HygB into the bacterial cells expressing the IBV H120 E protein, IBV H120 CI protein, IBV H120 CII protein, IBV TW-I E protein, IBV TW-I CI protein, IBV TW-I CII protein, IBV TW-II E protein, IBV TW-II CI protein, and IBV TW-II CII protein were measured. The transformed bacteria were induced with 1 mM IPTG for 45 min. Bacteria were further incubated in the presence of 2 mM ONPG for 10 min at the indicated times. Finally, β-galactosidase activity was determined by measuring the OD₄₀₅ value. The bacteria carrying the pET24a served as the negative control. The data are presented as the mean ± SE from three independent experiments. Significant differences between the negative controls vs. the H120, TW-I, and TW-II groups without treatments are indicated with *p<0.05. Significant differences between the CI and CII proteins of each group vs. the H120, TW-I, and TW-II serotypes are indicated with **p<0.05 (B). The expression of the IBV H120 E protein, IBV H120 CI protein, IBV H120 CII protein, IBV TW-I E protein, IBV TW-I CI protein, IBV TW-I CII protein, IBV TW-II E protein, IBV TW-II CI protein, and IBV TW-II CII protein in the transformed E.coli cells was induced with 1mM IPTG, after which the cells were treated with HygB and puromycin. The bacteria were lysed and the host protein synthesis was monitored via western blot analyses using anti-puromycin antibodies (C).

3.6. Effect of viroporin inhibitors on the membrane permeability and protein synthesis of DF-1 cells affected by the expression of E proteins from different IBV serotypes

The HygB permeability assay conducted in DF-1 cells revealed that cells transfected with the pc-H120 E, pc-TW-I E, and pc-TW-II E plasmids showed significant inhibition of cellular protein synthesis when treated with HygB and puromycin. The results of our immunofluorescence analyses demonstrated a notable reduction in red fluorescent signals in cells transfected with these plasmids in the presence of HygB, indicating blocked protein translation. In contrast, no such effects were observed in DF-1 cells as the negative control or in transfected cells in the absence of HygB. Furthermore, when cells transfected with the pc-H120 E, pc-TW-I E, and pc-TW-II E plasmids were treated with 75 μM HMA or 450 μM DIDS, the red fluorescent intensity reverted to levels similar to those of the negative control cells (Fig. 5A). Western blot analysis corroborated these findings, showing minimal global protein synthesis in cells transfected with the pc-H120 E, pc-TW-I E, and pc-TW-II E plasmids in the presence of HygB. However, protein synthesis remained normal in the negative control and transfected cells without HygB. Treatment with 75 μM HMA or 450 μM DIDS restored protein synthesis to normal levels (Fig. 5B). Taken together, these results suggested that the membrane permeability of the DF-1 cells was enhanced by the E protein of different IBV serotypes, thereby allowing the entry of HygB into the cells and blocking cellular protein translation. Conversely, this effect can be reversed by the viroporin inhibitors, HMA or DIDS.

Fig. 5.

Effect of viroporin inhibitors on the membrane permeability and protein synthesis of eukaryotic cells expressing the IBV H120 E protein, IBV TW-I E protein, and IBV TW-II E protein. DF-1 cells were treated with viroporin inhibitors (DIDS and HMA) prior to transfection of pc-H120 E, pc-TW-I E, and pc-TW II E plasmids. The cells were treated with or without 5 μM HygB and then labeled with 5 μg/ml puromycin. The cells were doubly labeled with anti-puromycin antibody and anti-His antibody, followed by Alexa Flour 546-conjugated goat anti-mouse (red) and FITC-conjugated goat anti-rabbit (green) antibodies in parallel. DAPI (blue) was used to counterstain the nucleus, after which the cells were observed using an immunofluorescent microscope (A). DF-1 cells were treated with the viroporin inhibitors (DIDS and HMA), transfected with pc-H120 E, pc-TW-I E, and pc-TW II E plasmids, treated with or without HygB, and finally labeled with puromycin. The host protein synthesis was examined via western blotting using anti-puromycin antibodies (upper panel). The expression of IBV H120 E protein, IBV TW-I E protein, and IBV TW-II E protein was verified via western blotting using anti-His antibodies (bottom panel) (B).

3.7. Effect of viroporin inhibitors on the membrane integrity of DF-1 cells affected by the E protein of different IBV serotypes

The effects of E proteins from different IBV serotypes on membrane integrity were assessed using PI staining. Membrane permeabilization was indirectly represented by the fluorescence intensity of PI, as shown in Q2 of the flow cytometry plot. The PI-positive percentages of the control cells, the cells transfected with the IBV E protein gene of H120, the cells transfected with the IBV E protein gene of H120 treated with 450 μM DIDS, the cells transfected with the IBV E protein gene of H120 treated with 75 μM HMA, the cells transfected with the IBV E protein gene of TW-I, the cells transfected with the IBV E protein gene of TW-I treated with 450 μM DIDS, the cells transfected with the IBV E protein gene of TW-I treated with 75 μM HMA, the cells transfected with the IBV E protein gene of TW-II, the cells transfected with the IBV E protein gene of TW-II treated with 450 μM DIDS, and the cells transfected with the IBV E protein gene of TW-II treated with 75 μM HMA were 0.93±0.03, 42.2±0.75, 5.1±0.78, 4.37±1.05, 60.97±1.67, 7.17±0.23, 6.83±0.54, 39.63±2.58, 4.43±0.19, and 3.27±0.53 (p<0.05 vs. control or both treatments vs. IBV E H120, IBV E TW-I, and IBV E TW-II only). These results suggest that IBV E proteins from H120, TW-I, and TW-II serotypes exhibit viroporin activity, as demonstrated by the induction of membrane permeability. Among them, the IBV E protein of the TW-I serotype exhibited the strongest viroporin activity in DF-1 cells. Furthermore, all viroporin activities of different IBV serotypes could be significantly reversed by treatment with DIDS or HMA (Fig. 6A and B). This finding supports the idea that increased membrane permeability of DF-1 cells triggered by E proteins from different IBV serotypes can be abolished by both viroporin inhibitors.

Fig. 6.

Effect of the IBV E protein on the membrane permeability. DF-1 cells were transfected with the plasmids expressing the E protein of IBV H120, IBV serotype TW-I, and IBV serotype TW-II for 48 h followed by the propidium iodide (PI) staining. Q2 indicates the percentage of cells with the positive PI fluorescence (A). The percentage of PI-positive cells in each group is shown in the bar graph (B). The data are presented as mean values ± SE derived from three independent experiments. Significant differences were indicated as *p<0.05.

3.8. Effect of viroporin inhibitors on the lesion and survival rate of embryos infected by different IBV serotypes and HAU titer of allantoic fluids of the IBV-infected embryos

Given that the IBV H120 strain has been used as a vaccine for IBV in many countries, including Taiwan, only pathogenic strains such as IBV TW-I and IBV TW-II serotypes isolated in Taiwan were used in the embryo and chicken tests. Different degrees of therapeutic effects were observed in the infected embryos treated with 150 μM DIDS, 300 μM DIDS, and 450 μM DIDS or 25 μM HMA, 50 μM HMA, and 75 μM HMA. When chicken embryos were infected with IBV TW-I and IBV TW-II, all embryos in the positive groups infected with viruses at 6 dpi exhibited similar lesions, including toe curling, body hemorrhages, and dwarfing, all of which are recognized as representative symptoms of IBV infection. When the IBV TW-I-infected embryos were treated with 450 μM DIDS, two embryos exhibited body hemorrhage. However, when 75 μM HMA was administered to the IBV TW-I-infected embryos, a half of the embryos appeared similar to the negative control. When TW-II-infected embryos were treated with 450 μM DIDS, no lesions were detected in any of the embryos. Additionally, when IBV TW-II-infected embryos were treated with 75 μM HMA, three embryos exhibited dwarfing (Table 2). The IBV TW-I and IBV TW-II-infected embryos treated with DIDS were much closer to normal compared to those treated with HMA (Fig. 7A). For the HAU titer of IBV, the titer of the IBV TW-I-infected embryos ranged from 2⁷ to 2⁸, with an average of 2^7.6. This indicated the propagation of IBV TW-I, resulting in the death of all embryos, all of which exhibited disease-specific lesions. The average titers for the IBV TW-I-infected embryos treated with 150 μM DIDS, 300 μM DIDS, and 450 μM DIDS were 2^3.8, 2^3.3, and 2^1.6, respectively. The average titers for the IBV TW-I-infected embryos treated with HMA also decreased in a dose-dependent manner. Thus, our findings confirmed that the propagation of IBV TW-I in embryos was dose-dependently inhibited by both drugs. No virus was detected in the embryos of the negative control group, with a titer of 2⁰ equivalent to 0. Next, the HAU titer of the IBV TW-II-infected embryos, ranging from 2⁷ to 2⁸, with an average of 2^7.5, was close to that of the IBV TW-I-infected embryos. The average titers of all IBV TW-II-infected embryos treated with 150 μM, 300 μM, and 450 μM DIDS were 2^3.1, 2^1.5, and 2⁰, respectively. No viruses were detected in the allantoic fluids of any IBV TW-II-infected embryos treated with 450 μM DIDS. The average titers for the IBV TW-II-infected embryos treated with 25 μM HMA, 50 μM HMA, and 75 μM HMA were 2^4.6, 2^4.1, and 2^1.8, respectively (Table 3). The antiviral effect of DIDS was better than that of HMA, as reflected not only in the severity of lesions in the infected embryos but also in the viral titer of the allantoic fluids collected from the infected embryos. Embryos infected with IBV TW-I also began to die at 2 dpi and no survivors were found at 6 dpi. The survival rates of embryos infected with IBV TW-I and treated with 150 μM DIDS, 300 μM DIDS, and 450 μM DIDS were 50 %, 50 % and 83.3 %, respectively. The highest mortality of the embryos infected with IBV TW-II was also observed at 6 dpi. Compared with the IBV TW-II, the survival rates significantly increased to 33.3 %, 66.7 %, and 100 %, respectively. Similarly, embryos infected with IBV TW-I also began to die at 2 dpi, and no survivors were found at 6 dpi. The survival rates of embryos infected with IBV TW-I with treatments of 25 μM, 50 μM, and 75 μM HMA were 16.7 %, 33.3 %, and 50 %, respectively. The peak mortality of the embryos infected with IBV TW-II was also observed at 6 dpi. Compared with IBV TW-II, the survival rates significantly increased to 16.7 %, 33.3 %, and 66.7 %, respectively (Fig. 7B). Collectively, these findings demonstrate that DIDS and HMA could increase the survival rates of chicken embryos infected with different IBV serotypes. DIDS was much more effective than HMA in increasing the survival rates of chicken embryos infected with IBV. Regarding the serotype of IBV, the IBV TW-I serotype seemed more resistant to the antiviral effects of DIDS and HMA, as the survival rates increased less compared to the IBV TW-II serotype.

Table 2.

Severity of lesions of chicken embryos infected with both IBV serotypes with inhibitor treatments.

Lesion	Severity	NC	TW-I	TW-I + 150 μM DIDS	TW-I + 300 μM DIDS	TW-I + 450 μM DIDS	TW-I + 25 μM HMA	TW-I + 50 μM HMA	TW-I + 75 μM HMA	TW-II	TW-II + 150 μM DIDS	TW-II + 300 μM DIDS	TW-II + 450 μM DIDS	TW-II + 25 μM HMA	TW-II + 50 μM HMA	TW-II + 75 μM HMA
Toe curling	No change	6/6	0/6	3/6	4/6	5/6	0/6	1/6	3/6	0/6	2/6	4/6	6/6	0/6	0/6	3/6
	Mild	0/6	0/6	1/6	1/6	1/6	0/6	2/6	2/6	0/6	1/6	1/6	0/6	0/6	2/6	0/6
	Moderate	0/6	0/6	1/6	1/6	0/6	1/6	2/6	1/6	0/6	1/6	1/6	0/6	1/6	2/6	0/6
	Severe	0/6	6/6	1/6	0/6	0/6	5/6	1/6	0/6	6/6	1/6	0/6	0/6	5/6	2/6	3/6
Body hemorrhage	No change	6/6	0/6	3/6	4/6	4/6	0/6	1/6	3/6	0/6	2/6	5/6	6/6	0/6	0/6	3/6
	Mild	0/6	0/6	1/6	1/6	2/6	0/6	3/6	2/6	0/6	3/6	1/6	0/6	0/6	3/6	1/6
	Moderate	0/6	0/6	1/6	1/6	0/6	3/6	2/6	1/6	0/6	1/6	0/6	0/6	1/6	1/6	0/6
	Severe	0/6	6/6	1/6	0/6	0/6	3/6	0/6	0/6	6/6	0/6	0/6	0/6	5/6	2/6	2/6
Dwarfing	No change	6/6	0/6	3/6	4/6	5/6	0/6	1/6	3/6	0/6	2/6	4/6	6/6	0/6	0/6	3/6
	Mild	0/6	0/6	2/6	2/6	1/6	0/6	1/6	2/6	0/6	3/6	2/6	0/6	0/6	2/6	1/6
	Moderate	0/6	0/6	1/6	0/6	0/6	1/6	2/6	1/6	0/6	1/6	0/6	0/6	2/6	3/6	0/6
	Severe	0/6	6/6	0/6	0/6	0/6	5/6	1/6	0/6	6/6	0/6	0/6	0/6	4/6	1/6	2/6

*Positive samples/total samples.

Fig. 7.

Anti-IBV effects of viroporin inhibitors (HMA and DIDS) on chicken embryos infected with the IBV TW-I and IBV TW-II serotypes. IBV-specific pathological states such as hemorrhages, toe curling, and embryo dwarfing were observed. Morphological abnormalities similar to the normal phenotype were observed in each embryo of the negative control and in the IBV-infected groups, both with and without 150 μM DIDS, 300 μM DIDS, 450 μM DIDS, 25 μM HMA, 50 μM HMA, and 75 μM HMA treatment. The time of death was recorded at the bottom of each dead embryo before or at the end of the experiment (A). One-day-old chicken embryos were inoculated with allantoic fluid containing a viral load equal to 10⁶ 50 % egg infectious dose (EID₅₀) with or without 150 μM DIDS, 300 μM DIDS, and 450 μM DIDS or 25 μM HMA, 50 μM HMA, and 75 μM HMA treatment. The survival rates were recorded daily until 6 days post-infection (dpi) (B).

Table 3.

Hemagglutination titer of IBV in chicken embryos infected with both IBV serotypes with inhibitor treatments.

Group	Hemagglutination titer						Average
NC	20	20	20	20	20	20	20
TW-I	28	27	28	27	28	28	27.6
TW-I+150 μM DIDS	26	26	25	25	21	20	23.8
TW-I+300 μM DIDS	26	26	24	24	21	20	23.3
TW-I+450 μM DIDS	23	23	21	23	20	20	21.6
TW-I+25 μM HMA	27	27	25	25	21	21	24.3
TW-I+50 μM HMA	27	26	26	24	21	21	24.1
TW-I+75 μM HMA	26	27	25	25	20	21	24
TW-II	28	27	27	27	28	28	27.5
TW-II+150 μM DIDS	24	25	25	21	21	23	23.1
TW-II+300 μM DIDS	23	24	21	20	20	21	21.5
TW-II+450 μM DIDS	20	20	20	20	20	20	20
TW-II+25 μM HMA	26	26	25	24	24	23	24.6
TW-II+50 μM HMA	27	25	25	23	22	23	24.1
TW-II+75 μM HMA	24	23	22	20	21	21	21.8

3.9. Effect of viroporin inhibitors on the clinical symptoms, survival rates and lesion scores of chickens infected with different IBV serotypes

Chickens infected with the IBV TW-I serotype exhibited tracheal rales and lacrimation at 1 dpi, followed by sneezing and coughing at 3 dpi, and severe diarrhea, leading to chicken deaths starting at 5 dpi. Chickens infected with IBV TW-I serotype and treated with 150 μM DIDS displayed clinical signs at 1 dpi, with some chickens beginning to die at 5 dpi. However, the clinical symptoms in this group were milder compared to the positive control group. Chickens infected with IBV TW-I serotype and treated with 450 μM DIDS exhibited mild symptoms at 1 dpi, with only one chicken dead by 8 dpi. Additionally, chickens infected with IBV serotype TW-I and subsequently treated with 25 μM and 75 μM HMA exhibited minor symptoms at 2 dpi, with only one dead chicken was found at 5 dpi and 4 dpi, respectively. Only slight symptoms were observed in the remaining chickens until the end of the experiment. Chickens infected with IBV serotype TW-II displayed the described clinical signs at 2 dpi, with chicken deaths occurring at 5 dpi. Chickens infected with IBV serotype TW-II and treated with 150 μM and 450 μM DIDS began showing clinical symptoms at 5 dpi and 4 dpi, respectively. Death occurred in chickens treated with 150 μM DIDS at 7 dpi, but no deaths were observed in chickens treated with 450 μM DIDS. Moreover, only mild symptoms were observed at 3 dpi and 5 dpi, respectively, in chickens infected with IBV serotype TW-II and treated with 25 μM and 75 μM HMA. All chickens survived when infected with IBV serotype TW-II and treated with 25 μM or 75 μM HMA. Regarding the clinical score, the mean clinical score for the IBV TW-I was 1.9. Administration of 150 μM DIDS and 450 μM DIDS to IBV TW-I infected chickens significantly decreased the scores to 1.2 and 0.8, respectively (both at least p<0.005 vs. the TW-I). The mean clinical scores for the IBV TW-II, the IBV TW-II and treated with 150 μM DIDS, and the IBV TW-II and treated with 450 μM DIDS were 1.6, 0.8, and 0.5, respectively (both p<0.0001 vs. the TW-II). When 25 μM HMA and 75 μM HMA were administered to chickens infected with IBV TW-I, the scores significantly decreased to 1.3 and 0.9, respectively (both at least p<0.005 vs. the TW-I). The mean clinical scores for the IBV TW-II, the IBV TW-II and treated with 25 μM HMA, and the IBV TW-II and treated with 75 μM HMA were 1.5, 0.7, and 0.1, respectively (both p<0.0001 vs. the TW-II). Notably, a significant difference in clinical score was observed between the 25 μM HMA and 75 μM HMA treatments (Fig. 8A). In terms of survival rate, deaths in chickens infected with IBV TW-I began at 5 dpi, with no survivors by 11 dpi. Compared to IBV TW-I, the survival rates increased to 33.3 % and 83.3 % with the 150 μM DIDS and 450 μM DIDS treatments, respectively. Similarly, chickens infected with IBV TW-II began dying at 7 dpi, with no survivors by 11 dpi. The survival rates of chickens infected with IBV TW-II and treated with 150 μM DIDS and 450 μM DIDS were 33.3 % and 83.3 %, respectively. Compared to IBV TW-I, the survival rates increased to 66.7 % and 83.3 % with the 25 μM HMA and 75 μM HMA treatments, respectively. The survival rates of chickens infected with IBV TW-II and treated with 25 μM HMA and 75 μM HMA were 83.3 % and 100 %, respectively (Fig. 8B). Overall, these findings demonstrate that both DIDS and HMA could increase the survival rates of chickens infected with both IBV serotypes. HMA was more effective than DIDS in increasing the survival rates of chickens. Similarly, the IBV TW-I serotype appeared to be more resistant to the antiviral effects of HMA, as evidenced by a lower increase in survival rate compared to the IBV TW-II serotype. The lesions in IBV TW-I infected chickens included serous exudates and mucosal congestion in the trachea, swollen and pale kidneys with large amounts of urate deposition, and distended ureters with urates. Similar lesions were also observed in the IBV TW-II infected chickens. When 450 μM DIDS was given to IBV TW-I infected chickens, much less mucin was observed in the trachea. Large and slight increases in mucin were observed when IBV TW-I infected chickens were given 25 μM and 50 μM HMA, respectively. When IBV TW-II infected chickens were given 450 μM DIDS, a slight increase in mucin was observed in the trachea. A large increase and a very small amount of mucin were respectively found in the trachea of IBV TW-II infected chickens given 25 μM and 50 μM HMA, respectively (Fig. 9A). When IBV TW-I infected chickens were treated with 150 μM DIDS, urate deposits remained in the swollen kidneys and ureters. The morphology of the kidneys was close to that of the negative control when IBV TW-I infected chickens were treated with 450 μM DIDS. Swelling with urate was observed in the kidneys of IBV TW-I infected chickens given 25 μM HMA, but milder lesions were visible when the HMA dose was increased to 75 μM. The kidneys of IBV TW-II infected chickens treated with 150 μM DIDS were swollen and had urate deposits, which could also be observed in the distended ureters. However, the outline of the kidneys was similar to that of the negative control when 450 μM DIDS was given to the IBV TW-II infected chickens. Swollen kidneys with urea deposits were found in the IBV TW-II infected chickens treated with 25 μM HMA. When IBV TW-II infected chickens were treated with 75 μM HMA, the kidneys were only slightly swollen (Fig. 9B). Regarding the lesion scores of the trachea, the average scores of different groups are listed in Supplementary Table 1. The average score significantly decreased only in the group treated with 450 μM DIDS compared to the IBV TW-I group (p<0.0001). However, both 25 μM HMA and 75 μM HMA treatments significantly decreased the average scores compared to the IBV TW-I group (both at least p<0.001 vs. the TW-I). Similarly, only the treatment with 450 μM DIDS significantly decreased the average score compared to the IBV TW-II group (p<0.0001). The average scores for the IBV TW-II groups treated with 25 μM HMA group and 75 μM HMA decreased significantly compared to the IBV TW-II group (both HMA-treated groups p<0.0001 vs. the TW-II) (Fig. 9C). For the lesion scores of the kidneys, the average scores of different groups are listed in Supplementary Table 2. Only the treatment with 450 μM DIDS significantly decreased the average score compared to the IBV TW-I group (p<0.0001 vs. the TW-I). The average scores for the IBV TW-I group treated with 25 μM HMA and 75 μM HMA decreased significantly compared to the IBV TW-I group (both at least p<0.005 vs. the TW-I). The treatment with 450 μM DIDS significantly decreased the average score compared to the IBV TW-II group (p<0.0001 vs. the TW-II). Treating the IBV TW-II infected chickens with 25 μM HMA and 75 μM HMA significantly decreased their average scores compared to the IBV TW-II group (both at least p<0.005 vs. the TW-II) (Fig. 9D).

Fig. 8.

Anti-IBV effects of the viroporin inhibitors (HMA and DIDS) on the chickens infected with IBV serotypes TW-I and TW-II. The clinical score was recorded daily for each chick based on the evaluation criteria of the clinical signs and presented as mean scores. Significant differences were represented as **p<0.005; ***p<0.001; ****p<0.0001 (A). One-day-old chicks were inoculated with 10⁶ EID₅₀ viruses with or without 150 μM DIDS, 450 μM DIDS, 25 μM HMA, and 75 μM HMA treatment. The survival rates were recorded daily until 11 dpi (B).

Fig. 9.

Anti-IBV effects of the viroporin inhibitors (HMA and DIDS) on chickens infected with IBV serotypes TW-I and TW-II. The figure illustrates representative trachea (A) and kidney (B) lesions for each group. Lesion scores were recorded for each chick based on the evaluation criteria and presented as mean scores. The mean lesion scores of the trachea (C) and kidney (D) for each group are shown. Significant differences are indicated as **p<0.005; ***p<0.001; ****p<0.0001.

3.10. Histopathology and IHC

At 11 dpi, histopathological examinations were conducted on both tracheal and kidney lesions in chickens infected with different IBV serotypes. The structure of the tracheal epithelium appeared intact with normal cilia. However, chickens infected with IBV TW-I exhibited significant pathological abnormalities in the trachea, including cilia detachment, pronounced infiltration of inflammatory cells, submucosal edema, and epithelial hyperplasia. Some areas of the mucosal epithelial cells in the trachea showed exfoliation and disappearance. Similar but less severe lesions were observed in the trachea of chickens infected with IBV TW-II, with milder sloughing of tracheal cilia and epithelial cells. As the dosage of DIDS and HMA increased, the severity of tracheal lesions markedly decreased. Tracheal structures appeared increasingly normal, albeit with more evident submucosal edema and epithelial hyperplasia, and reduced infiltration of inflammatory cells in the DIDS treatment groups. Notably, the HMA treatment groups exhibited much less submucosal edema and intact tracheal cilia, with the tracheal morphology even resembling that of the negative control. Thus, compared to DIDS, HMA was more effective in alleviating the damage to the trachea caused by IBV TW-I and IBV TW-II serotypes (Fig. 10A). In the non-infected group, normal renal tubular epithelial cells with well-defined renal tubular compartments were observed. However, in chickens infected with IBV TW-I and IBV TW-II serotypes, exfoliated, vacuolar degenerated, and necrotic renal tubular epithelial cells were noted, along with dilated collecting tubules, and urate deposits in the tubular lumen. Diffuse edema and sporadic infiltration of lymphocytes in the interstitium were also observed. The severity of renal lesions in chickens infected with the IBV TW-I serotype was comparable to that of the IBV TW-II serotype. The morphological changes in the kidney decreased gradually as the dosages of DIDS and HMA increased. The number of necrotic, disorganized, and disintegrated renal tubular cells gradually decreased, although focal infiltrations of inflammatory cells persisted. In chickens infected with IBV serotype TW-I, the structure of renal tubules became more complete, with fewer lymphocyte infiltrations in the interstitium observed in the DIDS treatment groups. Similarly, there were fewer red blood cells in the kidney stroma, and the renal tubule structure of chickens infected with IBV serotype TW-I also became more intact after HMA treatment. Both DIDS and HMA were more effective in improving kidney damage caused by IBV TW-II serotype compared to that caused by IBV TW-I serotype (Fig. 10B). Collectively, DIDS and HMA effectively ameliorated the histopathological lesions of the trachea and kidney in chickens infected with IBV TW-I and IBV TW-II serotypes. These results were consistent with the improved survival rates of IBV-infected chickens observed with both inhibitors. For the histopathological scores of the trachea, the scores for each parameter and the total scores of different groups were listed in Supplementary Table 1. Treatment with 150 μM DIDS and 450 μM DIDS significantly decreased the total scores compared with those of the IBV TW-I group (both at least p<0.0001 vs. the TW-I). Similarly, the total scores for the IBV TW-II groups treated with 150 μM DIDS and 450 μM DIDS also decreased significantly compared with those of the IBV TW-II group (both DIDS treated groups p<0.0001 vs. the TW-II). Treatment with 25 μM HMA and 75 μM HMA also significantly decreased the scores compared with those of the IBV TW-I group (both at least p<0.0001 vs. the TW-I). The total scores for the IBV TW-II groups treated with 25 μM HMA group and 75 μM HMA decreased significantly compared with those of the IBV TW-II group (both HMA treated groups p<0.0001 vs. the TW-II) (Fig. 10C and Supplementary Table 3). Supplementary Table 2 outlines the histopathological scores of the kidney samples for each parameter and total scores of different groups. Treatment with 150 μM DIDS and 450 μM DIDS significantly decreased the total kidney scores compared with those of the IBV TW-I group (both at least p<0.0001 vs. the TW-I). Similarly, the total scores for the IBV TW-II groups treated with 150 μM DIDS group and 450 μM DIDS decreased significantly compared with those of the IBV TW-II group (both p<0.0001 vs. the TW-II). Next, treating the IBV TW-I infected chickens with 25 μM HMA and 75 μM HMA significantly decreased their scores compared with the IBV TW-I group (both at least p<0.0001 vs. the TW-I). The total scores for the IBV TW-II groups treated with 25 μM HMA and 75 μM HMA also decreased significantly compared with those of the IBV TW-II group (both p<0.0001 vs. the TW-II) (Fig. 10D and Supplementary Table 4). Furthermore, the IBV antigen, which exhibited a tan color, was found in the trachea of chickens infected with IBV TW-I and IBV TW-II serotypes. IBV antigens were observed within several types of cells, including desquamated and hyperplastic epithelial cells, infiltrated inflammatory cells in the submucosa, and detached epithelial cells in the lumen. Most viral antigens were detected in the cytoplasm and no viral antigens were found in the trachea of chickens in the negative control group. When chickens infected with the IBV serotypes TW-I or IBV TW-II were administered DIDS, fewer antigen-positive cells with lower labeling intensity were observed. The number of antigen-positive cells decreased in a dose-dependent manner. Much fewer viral antigens with weaker intensity were observed in the trachea of IBV TW-I serotype- or IBV TW-II serotype-infected chickens treated with HMA. Notably, the expressed viral antigens became almost undetectable in IBV TW-I serotype- or IBV TW-II serotype-infected chickens treated with a higher concentration of HMA (75 μM) (Fig. 11A). Similarly, numerous antigen-positive renal tubular epithelial cells were observed in the kidney of chickens infected with IBV serotype TW-I or IBV serotype TW-II. Intense labeling was also seen in the sloughed and dissolved epithelial cells falling into the lumen. The number and labeling intensity of antigen-positive cells in the kidney tissue of chickens infected with the IBV TW-I serotype were comparable to those of the IBV TW-II serotype. Significant decreases in the antigen-positive renal tubular cells with milder labeling were seen in the kidneys of IBV TW-I serotype-infected chickens treated with DIDS. As with previous results, these improvements were dose-dependent. Only a few renal tubular cells were positive for the IBV antigen in the kidney of IBV TW-I serotype-infected chickens treated with HMA. The expression of IBV antigens was much lower in the kidney tissues of IBV TW-II serotype-infected chickens treated with DIDS. This effect was also dose-dependent. When the IBV TW-II serotype-infected chickens were administered 75 μM HMA, the viral antigens were almost undetectable (Fig. 11B). Regarding the IHC results, the integrated optical density (IOD) of IBV antigens in the trachea of chickens infected with IBV serotype TW-I serotype or IBV serotype TW-II was significantly higher compared with the negative control (p<0.0001). The IOD values of IBV-positive antigens in the trachea of chickens infected with IBV serotype TW-I or IBV serotype TW-II and treated with 150 μM DIDS or 25 μM HMA decreased significantly (p<0.005 vs. the positive control). A significant difference (p<0.001) was observed in the trachea of chickens infected with IBV serotype TW-I or IBV serotype TW-II and treated with 450 μM DIDS, and a significant difference (p<0.0001) was found in the trachea of chickens infected with IBV serotype TW-I or IBV serotype TW-II and treated with 75 μM HMA compared with the positive control (Fig. 11C and Supplementary Table 5). The IOD values of IBV antigens in the kidney of chickens infected with IBV serotype TW-I or IBV serotype TW-II increased significantly compared with the negative control (p<0.0001). The IOD values of IBV-positive antigens in the kidney of chickens infected with IBV serotype TW-I or IBV serotype TW-II and treated with 150 μM DIDS or 25 μM HMA decreased significantly (p<0.005 vs. the positive control). A significant difference was observed in the kidney of chickens infected with IBV serotype TW-I or IBV serotype TW-II and treated with 450 μM DIDS (p<0.001). Significant difference (p<0.0001) was also identified in the kidneys of chickens infected with IBV serotype TW-I or IBV serotype TW-II and treated with 75 μM HMA compared with the positive control (Fig. 11D and Supplementary Table 6).

Fig. 10.

Anti-IBV effects of the viroporin inhibitors (HMA and DIDS) on IBV-induced tissue lesions. Histopathological examination of the negative control group, the IBV TW-I-infected chickens, the IBV TW-I-infected chickens treated with 150 μM DIDS, the IBV TW-I-infected chickens treated with 450 μM DIDS, the IBV TW-I-infected chickens treated with 25 μM HMA, the IBV TW-I-infected chickens treated with 75 μM HMA, the IBV TW-II-infected chickens, the IBV TW-II-infected chickens treated with 150 μM DIDS, the IBV TW-II-infected chickens treated with 450 μM DIDS, the IBV TW-II-infected chickens treated with 25 μM HMA, and the IBV TW-II-infected chickens treated with 75 μM HMA. The figure illustrates representative trachea sections of each group stained with hematoxylin and eosin (HE). The pathological abnormalities of the trachea included the loss of cilia, exfoliation of mucosal epithelial cells, inflammatory cell infiltration, submucosa edema, and epithelial hyperplasia (A). Representative kidney sections of each group stained with HE are presented. The pathological abnormalities of the kidney included exfoliation, vacuolar degeneration, necrosis of renal tubular epithelial cells, renal tubule expansion, urate deposits, and inflammatory cell infiltration (B). The histopathologic scores were recorded for each chick based on the evaluation criteria and presented as mean scores. The mean histopathologic scores of the trachea (C) and kidney (D) of each group are shown. Significant differences were indicated as ****p<0.0001.

Fig. 11.

IHC detection of IBV antigens on the tissues of IBV-infected chickens treated with or without viroporin inhibitors (HMA and DIDS). IHC of the negative control group, the IBV TW-I-infected chickens, the IBV TW-I-infected chickens treated with 150 μM DIDS, the IBV TW-I-infected chickens treated with 450 μM DIDS, the IBV TW-I-infected chickens treated with 25 μM HMA, the IBV TW-I-infected chickens treated with 75 μM HMA, the IBV TW-II-infected chickens, the IBV TW-II-infected chickens treated with 150 μM DIDS, the IBV TW-II-infected chickens treated with 450 μM DIDS, the IBV TW-II-infected chickens treated with 25 μM HMA, and the IBV TW-II-infected chickens treated with 75 μM HMA. Representative IHC slides of the trachea section of each group with viral antigens are presented (A). The representative IHC slides of the kidney sections from each group with viral antigens are presented (B). Integrated optical density (IOD) values were recorded for each chick and presented as the mean score. The mean IOD values of the trachea (C) and kidney (D) of each group are presented. Significant differences are indicated as **p<0.005; ***p<0.001; ****p<0.0001.

3.11. Interaction between HMA or DIDS and the TMD of IBV E protein

The E protein of CoVs exhibits viroporin activity by forming ion channels in the host cell membrane through a conserved TMD. For IBV, our bioinformatic analyses demonstrated that the TMD is located within the residues from E11 to Q40 of the E protein (Fig. 12A). Next, homology modeling was employed to reconstruct the 3D pentameric structure of the E protein of different IBV serotypes. As illustrated in Fig. 12B, the TMD of the E protein self-oligomerizes to create a pentameric ion channel and our predictions indicated that residues F15, V21, F25, L28, G32, Q36, and V39 are responsible for lipid-facing surface formation. These modeled structures were then utilized for molecular docking to explore how HMA and DIDS interact with the TMD of the E protein. As depicted in Fig. 12C and Fig. 12D, both compounds were found to bind to the lipid-facing surface within the TMD of IBVs via the hydrophobic interactions. In the HMA binding site, the main residues contributing to the hydrophobic interactions were Y29, G32, R33, and F38 (Fig. 12C), whereas DIDS was hydrophobically stabilized by F25, L28, Y29, L31, G32, R33, and Q36 (Fig. 12D). The binding of drugs to the lipid-facing surface of the ion channel may block the ion conduction, thereby inhibiting viroporin activity.

Fig. 12.

Domain architecture of the IBV E protein and sequence alignment of the TMD of IBVs, SARS-CoV, and SARS-CoV-2. Highly conserved residues are indicated in red. NTD, N-terminal domain; TMD, transmembrane domain; CTD, C-terminal domain (A). Cartoon representation of the pentameric E protein of IBVs. The structures of IBV TW-I, TW-II, and H120 serotypes are indicated in blue, orange, and cyan, respectively. The organization of the E protein TMD is illustrated on the right. The lipid-facing residues in the SARS-CoV-2 E protein corresponding to the IBV E protein are labeled and represented as sticks (B). Representative docking poses of HMA (C) and DIDS (D) when interacting with the IBV E protein. The pink sticks represent the drugs interacting with the lipid-facing surface. The residues involved in hydrophobic interactions between the drugs and protein are labeled and represented as sticks.

4. Discussion

The roles of the CoV E protein in the progeny virus assembly, the release of mature virions, and pathogenicity have been linked to its function in virus budding into the ERGIC prior to exocytosis. Previous studies on the IBV E protein revealed two key findings. Firstly, mutant IB viruses with the impaired viroporin activity yielded fewer viruses compared to wild-type IBV, as they became trapped inside cells post-infection (Westerbeck and Machamer, 2019). Secondly, the IBV E protein induced a higher pH in the lumen of the Golgi apparatus and ERGIC, crucial for enhancing progeny virus yield, which was mediated by the monomeric form of IBV E independently of its ion channel activity. The cytoplasmic tail of the IBV E protein interacts with syntenin and a tight junction protein through both threonine 16 and alanine 26, located in its single hydrophobic domain (HD), thereby altering luminal pH and disrupting the secretory pathway (Westerbeck and Machamer, 2015). This latter finding suggests that signals trafficking through the vesicular system, initiated by intracellularly allocated E proteins, may indirectly open pores in cells (Gonzalez and Carrasco, 2003). However, the viroporin activity of the IBV E protein was first characterized in this study using standard techniques, including bacterial growth assays, membrane permeability assays in bacteria and cells, and puromycin labeling for protein synthesis in bacteria and cells. Significant inhibition of bacterial growth, similar to previous findings involving SARS-CoV and MHV proteins, was found with the expressed IBV E proteins of IBV H120 and TW-II serotypes. Additionally, the entry of HygB and ONPG into the bacteria was stimulated by the expressed IBV E protein, suggesting that these E proteins might form thydrophilic pores in the bacterial membrane through their oligomerization. Our study characterized the pentamers formed from the monomers of IBV E proteins of different serotypes, and our findings indicated that all the E proteins of IBV H-120, IBV serotype TW-I, and IBV serotype TW-II could form oligomers, leading to the leakage of intracellular substances necessary for bacterial survival through the membrane pores. In turn, the β-galactosidase inside the bacteria either reacted with ONPG entering the cells or exited the bacteria to react with the ONPG outside the cells. Moreover, increased membrane permeability of eukaryotic cells was evidenced by hindered cellular protein translation with HygB and cellular entry of PI due to defective cellular integrity caused by the E protein of different IBV serotypes. These results were similar to those triggered by the viroporin activity of the SARS-CoV E protein in artificial membranes (Madan et al., 2005). Apart from accumulating at the ERGIC, CoV E proteins were also located at various host cell membranes, including the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, and plasma membrane. Thus, the altered permeability of the cellular membrane by the IBV E proteins likely resulted from their interaction, which directly generated pores on the plasma membrane (Gonzalez and Carrasco, 2003). This mechanism has been proposed by MHV, and brefeldin A prevented the movement of the E protein from the ER to the plasma membrane, resulting in the loss of viroporin activity of the MHV E protein (Madan et al., 2005).

Although the α-helical TMD of the E protein is flanked by a juxtamembrane cluster of cysteine residues, the pentamerization of the SARS-CoV E protein was primarily attributed to the TMD. This is why even though there are no cysteine residues on the NS7a protein of PDCoV, it can still self-form oligomers (Xia et al., 2022). Previous studies indicated that the self-assembly of CoV E proteins into oligomers is promoted by multiple factors, including mutual interaction between the leucine-isoleucine zipper and GxxxG motif, repeated leucine-isoleucine residues within the TMD, and the presence of cysteine residues on the E protein (Cao et al., 2021). These cysteines of the E protein do not directly form disulfide bonds but are partially involved in inter-monomeric contacts through their hydrophobic side chains for the oligomerization of the SARS-CoV E protein (Parthasarathy et al., 2012). Perfluorooctanoic acid (PFO) electrophoresis revealed more pentameric forms of the SARS-CoV E protein when cysteine residues were replaced by alanine residues, which was attributed to the increased helicity of the secondary structure of the E protein. Notably, pentameric forms of the IBV E protein were observed when the IBV E protein or IBV E protein with cysteine substituted by alanine at both positions 45 and 46 were examined in PFO electrophoresis (Parthasarathy et al., 2012). Single and double deletions of cysteine residues of the IBV E protein were generated to clarify the role of these residues in the viroporin activity of the E protein. Interestingly, the results of viroporin activity assay, including bacterial growth, membrane permeability, and puromycin labeling, all suggested that the viroporin activity of the IBV E protein could not be blocked when one cysteine residue at position 45 was deleted, but it was lost when both cysteine residues (at positions 45 and 46) were omitted. This pattern was consistent across all IBV serotypes. Accordingly, similar to other CoVs, two cysteine residues at the C-terminus of the E protein may partially contribute to the normal structural topology of the TMD of the IBV E protein (Lopez et al., 2008). Double deletions of cysteine residues of the IBV E protein may result in improper folding of the E protein oligomers, leading to loss of viroporin activity.

To analyze the biochemical activity of proteins expressed in bacteria, it is crucial to ensure their solubility, especially considering that most viroporins exhibit varying levels of hydrophobicity. Therefore, preventing the aggregation of these proteins in the cytoplasm rather than the cell membrane is essential for characterizing their viroporin activity when expressed in bacteria. To address this challenge, a glutathione S-transferase tag was added at the N-terminus of the NS7a protein of the porcine deltacoronavirus (Xia et al., 2022). This approach was successful in overcoming solubility issues, similar to how the same tag was utilized for the E protein of FIPV, where viroporin activity was observed both in bacteria and feline cells (Takano et al., 2015). The tag effectively mitigated the higher hydrophobicity of the viroporin without interfering with its activity. Likewise, a His-tag was incorporated into the IBV E protein expressed in bacteria. Both the NS7a protein and IBV E protein exhibited inhibition of bacterial growth, as evidenced by low absorbance values in a time-dependent manner. Additionally, to enhance the solubility of the 2B protein of foot and mouth disease virus (FMDV), a SUMO tag was added to its N-terminus. Furthermore, due to the high toxicity of the 2B protein to bacteria, C43 (DE3) pLysS cells were chosen to express the FMDV 2B protein instead of BL21 (DE3) pLysS cells. Notably, the choice of bacterial strain is crucial, as it can significantly impact the outcomes of such assays (Ao et al., 2015).

Although mutant IAV lacking the M2 protein can still replicate in cultured cells, the presence of M2 is crucial for optimal progeny virus yield. Viroporins such as HCV p7 play vital roles in viral assembly and release (Steinmann et al., 2007). However, mutant IAV lacking the M2 protein cannot be recovered from inoculated mice, indicating the indispensable role of M2’s viroporin activity for productive replication in vivo. Viroporin inhibitors, like amantadine, have been shown to effectively inhibit IAV replication in cells, achieving substantial virus yield inhibition (<5 %) at a 50 μM concentration (Sugrue and Hay, 1991; Watanabe et al., 2001). Even with mutated M2 proteins, residual viroporin activity within the virus may sustain replication to some extent. This highlights the importance of viroporin inhibitors as antivirals, as they can target various viroporins across different viruses. For example, the viroporin activity of PDCoV, a type of CoV, is not only mediated by its E proteins but also by other accessory proteins such as NS7a (Xia et al., 2022). Additionally, the antiviral treatment not only inhibits virus growth but also facilitates virus clearance by the host immune system, leading to a more pronounced antiviral effect in vivo.

A previous study demonstrated the inhibition of FIPV (a type of CoV) replication using the viroporin inhibitors, HMA and DIDS, in feline cells. While HMA significantly inhibited the replication of FIPV serotype I, its effects on FIPV serotype II were only slight. Conversely, DIDS markedly reduced the titer of FIPV serotype II but not that of FIPV serotype I, indicating that different serotypes of the same virus may not be equally sensitive to the same viroporin inhibitor (Takano et al., 2015). In this study, rather than testing FIPV inhibitors exclusively in cultured cells, both inhibitors were examined in chicken embryos and chickens. When chicken embryos infected with different IBV serotypes were treated with DIDS or HMA, the survival rates of embryos significantly increased. IBV serotype TW-II was found to be more sensitive to both HMA and DIDS compared to IBV serotype TW-I. Additionally, DIDS was more effective than HMA in increasing the survival rates of chicken embryos. Considering the developmental stage of the chicken embryos and the immature state of their immune responses during our IBV challenge experiments (8 to 14-day-old), the increased survival rates were likely due to the antiviral effects of HMA and DIDS. Particularly, these immature immune responses were limited to only a weak interferon response detected in 6-day-old embryos (Seto, 1981). Coincidently, results from bacterial growth and membrane permeability assays using IBV E protein from different serotypes also suggested that DIDS was more effective than HMA. Furthermore, both DIDS and HMA significantly increased the survival rates of chickens infected with different IBV serotypes, with higher survival rates observed in chickens compared to embryos. In contrast to the embryo experiments, the antiviral effect of HMA was higher than that of DIDS in chicken experiments, with the survival rates of chickens infected with IBV TW-II serotypes reaching 100 %. Additionally, IBV serotype TW-I remained more lethal than IBV serotype TW-II. Notably, DIDS and HMA act as chloride and potassium channel blockers, respectively. While the viroporin activities of FIPV E proteins from serotypes I and II are predominantly affected by different channel blockers, the effectiveness of both DIDS and HMA against IBV TW-I and IBV TW-II serotypes suggests that the viroporins of these IBV serotypes may be influenced by both chloride and potassium channel blockers (Lane et al., 1992; Nelson et al., 1997; Takano et al., 2015).

The inhibitory potential of the amiloride derivative HMA on viroporin activity has been tested across different groups of CoVs, including group 1 (HCoV-229E), group 2 (MHV), and group 3 (IBV), using ion channel conductance assays in planar lipid bilayers. These assays revealed that the ion selectivity of the IBV E protein exhibited the following order: Na⁺>K⁺>Cl⁻ (Wilson et al., 2006). This further supported the dominance by both chloride and potassium channels. HMA unexpectedly failed to significantly inhibit the ion channel conductance of IBV E protein. However, HMA effectively inhibited the ion channel conductance of HCoV-229E and MHV E proteins, which was consistent with its blocking effect on the replication of these viruses in cells. Therefore, these findings provide crucial insights into the inhibitory effect of HMA on IBV. Despite the lack of an evident electrophysiological effect on IBV E protein viroporin activity, HMA demonstrated potent anti-IBV activity in more physiologically relevant systems, including chicken embryos and chickens. This underscores the importance of evaluating antiviral efficacy in authentic models. Moreover, the diverse protein quaternary structures of E proteins across different CoVs suggest that ion channel properties are largely determined by individual topology, influencing sensitivity to viroporin inhibitors (Wilson et al, 2006). Viroporins are classified into two major groups, class I and class II, based on the number of transmembrane domains they possess. Computer-aided analysis has indicated that the E protein of IBVs belongs to class I viroporins, characterized by a single α-helical TMD. This classification aligns with the typical characteristics of CoV E proteins. Regarding the orientation of the E protein within the plasma membrane, studies on SARS-CoV have demonstrated that its E protein is inserted into the membrane with its N-terminus facing the cytoplasm and its C-terminus facing the extracellular domain (Pervushin et al., 2009). However, determining the precise topology of the IBV E protein may require further experiments, as the orientation of CoV E proteins in the membrane can be influenced by factors such as the expression levels of the protein and its oligomerization pattern (Kuo et al., 2007).

Although the comparison between the lipid-bilayer-based structural model of the SARS-CoV-2 E protein and the micelle-derived structural model revealed certain similarities, substantial differences remained due to the inherent sensitivity to the membrane microenvironment (Mandala et al., 2020). Therefore, in the absence of experimentally determined structures for IBV E protein, the 3D pentameric structures of different IBV serotypes based on homology modeling provided valuable preliminary information. Recent research has highlighted the presence of a (E/D/R)₈x(G/A/V)₁₀xxhh(N/Q)₁₅ motif with a hydrophobic residue at the N-terminus of the SARS-CoV-2 E’ transmembrane domain (ETM), which acts as a cation filter responsible for cation conduction (Mandala et al., 2020). Similarly, a E₈xGxxhT₁₄ motif was identified in the ETM of all IBV serotypes, albeit with only a hydrophobic residue in front of a polar residue, T, whereas this amino acid position is occupied by an N residue in SARS-CoV-2. Both the ETMs of SARS-CoV-2 and IBV share an E residue at the N-terminus, serving as a selective filter for channel entrance. The structural flexibility of the N-terminus depends on the third residue of the ETM, G (glycine), which is highly conserved among SARS-CoVs and IBVs. The last residues of these motifs for SARS-CoVs, N, and those for IBVs, T, can both contribute to hydrogen bonding through their polar side chains to secure the channel structure (Mandala et al., 2020). The open cavity, which enables ion interaction, is made up of residues A32 and T35 on the C-terminus of the ETM of SARS-CoVs, and residues A34 and Q36 by IBVs, with both T and Q being classified as polar residues. Similar to SARS-CoVs, mostly hydrophobic residues occupy the channel pore, resulting in a less hydrated status of the E protein. Additionally, a previous study suggested that the N-terminal lumen of the ETM of SARS-CoV-2 was superficially inserted by HMA, leading to dispersed orientations and steric obstruction of the pore, as demonstrated by molecular docking analyses. However, HMA appeared to intercalate deeply into the N-terminal lumen of the ETM of IBVs, leading to the inhibition of cation conduction. This conclusion was drawn from the observation that residues T11, V14, and N15 of the ETM of SARS-CoV-2 were bound by HMA, whereas the HMA-bound residues in the ETM of IBVs were Y29, G32, R33, and F38 (Mandala et al., 2020). In addition to the aforementioned polar and basic amino acid residues, one extra non-polar amino acid residue also participates in the binding of HMA with the ETM of IBVs compared to SARS-CoVs. In fact, a previous study identified a binding site of the E protein for HMA, which exhibited an N residue for SARS-CoV and HCoV-229E and Q for MHV (Pervushin et al., 2009). Crucially, these amino acids are characterized by a long polar side chain. The docking region for DIDS within the N-terminal lumen of the ETM of IBVs appeared to be the same as that for HMA, albeit with some changes in the amino acid sequences. To the best of our knowledge, our study is the first to conduct docking analyses to characterize the interaction between the DIDS molecule and the ETM of CoVs.

The characteristics of avian CoV, IBV, including its high mutation rates and recombination tendency, have led to the generation of various IBV serotypes (Laconi et al., 2019). Different serotypes may coexist in the same geographic area, such as Taiwan within the East Asian region, and the co-circulation of multiple serotypes poses difficulties in eliminating this disease due to the lack of satisfactory cross-protection among vaccines made from different IBV serotypes. Therefore, the aim of this study was to identify efficient antivirals targeting the viral protein against all IBV serotypes. Unlike a previous study using the IBV M41 strain, which is well-adapted in cultured cells, the effect of antivirals on viral replication in cells could be measured by tissue culture infective doses (Chen et al., 2019). The evaluation criteria of antivirals in this study focused on the ability of these compounds to alleviate disease-related lesions and maximize the survival rate of embryos and chickens. Similar to many avian CoV wild isolates, the IBV serotypes TW-I and TW-II isolated in Taiwan cannot easily adapt to cell culture conditions with a stable titer. In contrast, assessing antiviral effects within the context of a living organism with an immune system provides a much more realistic estimation compared to an in vitro approach. Previous studies evaluating antivirals have relied on quantitative RT-PCR, which only detects genetic materials but not live viral particles (Chen et al., 2019). Due to these limitations, this approach was not applied in the present study. Similar to indirect immunofluorescence, which was previously used to detect the levels of viral antigens in cells after viral infection in the presence or absence of antivirals, IHC using an anti-IBV nucleocapsid antibody was employed to detect the presence of IBV antigen in IBV-infected chickens with or without viroporin inhibitors. Previous studies have identified a divergence of up to 25 % among the amino acid sequences of spike proteins of different IBV strains, resulting in an evolutionary distance of up to 30 % (Khanh et al., 2018). To avoid false negatives due to insufficient cross-reaction between IBV antigens and antibodies, it is more reliable to use the antibody against the nucleocapsid of IBV, which is the most conserved viral protein among different serotypes. Compared with the detection of viral antigens in cultured cells, tissue-level IHC analyses provide a more reliable method to evaluate the antiviral potential of a drug candidate. Not only does IHC analysis yield tissue-specific data derived from an in vivo model (e.g., chickens), but it also offers both qualitative and quantitative information about how inhibitors affect viral replication and histological distribution within the target organ.

The mortality rate of chickens infected with different IBV serotypes was positively correlated with the severity of clinical signs. Interestingly, our findings demonstrated that new IBV Taiwan isolates not only caused more serious clinical signs but also higher death rates in chickens compared to the classical Taiwan IBV lineages (Li et al., 2020). Interestingly, only differences in clinical scores greater than a certain threshold affected their survival rates. For instance, when the difference between the mean clinical score of the IBV TW-I serotype with 25 µM HMA and that of the IBV TW-II serotype with 25 µM HMA was 0.6, the survival rates increased from 66.7 % to 83.3 %. A larger decrease in the mean clinical score, 0.8, resulted in the survival rates of IBV serotype TW-I with 25 µM HMA and IBV serotype TW-II with 25 µM HMA increasing from 83.3 % to 100 %. Moreover, no survival of chickens infected by IBV was observed when the mean clinical score was 1.6, whereas no mortalities were observed when the mean clinical score was 0.1. Additionally, the relationship between the clinical score and the histopathology results was noted in this study. The clinical scores of chickens infected with IBV serotype TW-I or IBV serotype TW-II treated with HMA were smaller than those of chickens infected with serotypes TW-I or TW-II with DIDS. These findings were supported by the histopathological lesions of tracheas in chickens infected by both IBV serotypes treated with HMA, which were milder than those in chickens infected by both IBV serotypes treated with DIDS. The total histopathological scores of the trachea in chickens infected with the IBV TW-I serotype or IBV TW-II serotype treated with HMA were indeed lower than those in chickens infected with the IBV TW-I serotype or IBV TW-II serotype treated with DIDS. Moreover, the severity of histopathological lesions in the kidneys of chickens infected with the IBV TW-II serotype treated with DIDS or HMA was less than that in chickens infected with the IBV TW-I serotype treated with DIDS or HMA, which also reflected in the clinical score. The total histopathological scores of the kidneys in chickens infected with the IBV TW-I serotype or IBV TW-II serotype treated with DIDS were larger than those in chickens infected with the IBV TW-I serotype or IBV TW-II serotype treated with HMA. All observations were mutually supported by the statistical analysis of survival rates, clinical scores, and histopathological scoring. Furthermore, the IHC signals of IBV antigens in the renal lesions were stronger than those in the upper respiratory tract, such as the trachea. This suggests that IBV TW-I and IBV TW-II serotypes may replicate more efficiently in the kidney than in the trachea. Although the upper respiratory tract remains a prime site for the infection and replication of IBV, both IBV TW-I and TW-II serotypes, classified as nephropathogenic strains similar to the IBV QX-like SD strain, IBV T strain, IBV B1648 strain, and IBV AZ2374 strain, can induce severe nephritis, resulting in higher mortality (Ignjatovic et al., 2002). This is supported by the intense labeling signals found in the kidneys of chickens infected by both serotypes. Antigen-positive cells were still detected at 11 dpi, suggesting a longer persistence in target organs for both serotypes. This differs from the scenario where only histopathological lesions without the presence of viral antigens are induced by IBV M41 strain or the peak at 7 dpi and subsequent disappearance of antigen-positive cells in the kidneys of chickens infected by IBV QX-like strains (Benyeda et al., 2010). The significant reduction of positive cells in both the trachea and kidney of chickens infected by IBV TW-I and TW-II serotypes after treatment with DIDS and HMA further verifies the anti-IBV activity of both viroporin inhibitors.

Research involving human and animal rights

This study was carried out in accordance with the recommendations in the guide for the care and use of laboratory animals of the animal ethics committee and approved by National Chung Hsing University, Taiwan. The animal ethics committee approval number is IACUC No.112-048.

Informed consent

No human subjects were used in this study.

Funding

This work was funded by Grants from the Ministry of Science and Technology, Taiwan (MOST 111-2313-B-005-039-MY3) and the iEGG and Animal Biotechnology Center from the Feature Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education in Taiwan.

CRediT authorship contribution statement

Mikael Cristofer Sitinjak: Writing – original draft, Project administration, Investigation, Data curation. Jui-Kai Chen: Methodology, Investigation, Data curation. Fang-Lin Liu: Resources, Data curation. Ming-Hon Hou: Validation, Methodology. Shan-Meng Lin: Methodology, Formal analysis, Data curation, Conceptualization. Hung-Jen Liu: Software, Resources, Methodology. Chi-Young Wang: Writing – review & editing, Writing – original draft, Validation, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• Data will be made available on request.