The Inhibitory Effect of cLF-chimera, a Recombinant Antimicrobial Peptide, on Avian Influenza Virus Subtype H9N2

doi:10.21203/rs.3.rs-2480481/v1

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The Inhibitory Effect of cLF-chimera, a Recombinant Antimicrobial Peptide, on Avian Influenza Virus Subtype H₉N₂

https://doi.org/10.21203/rs.3.rs-2480481/v1

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Avian influenza subtype H9N2 is the most prevalent influenza virus in poultry worldwide. It imposes economic losses on the poultry industry and has zoonotic potential. Currently, there are two main groups of anti-influenza drugs in use; Adamantanes and Neuraminidase inhibitors. In recent years, there has been increasing resistance to existing anti-influenza drugs. Antimicrobial peptides are a group of potential drug candidates with broad-spectrum activity. cLF-chimera is an antimicrobial peptide synthesized from camel milk lactoferrin. This study's objective is to evaluate the inhibitory effects of cLF-chimera on avian influenza, subtype H9N2. For this purpose, one hundred and seventy 11-day-old embryonated eggs were randomly distributed in 17 groups. Different virus and peptide concentrations were injected into the eggs. The eggs were incubated for four days with daily candling for viability assessment. On the 4th day of incubation, each group's alive or dead embryos were sorted and evaluated for gross anomalies. Next, the chick embryos were fixed with 10% neutral buffered formalin for one week for histopathological studies. The MTT assay was also performed for the peptide and virus concentrations. Overall, the embryo viability results and macroscopic and histopathologic findings showed that the peptide has inhibitory effects against the virus. These findings agree with the MTT assay. Moreover, the peptide has proven effects against pathogenic bacteria that can be advantageous compared to common anti-influenza medications.

Avian Influenza

Subtype H9N2

Antimicrobial Peptide

cLF-chimera

Drug Candidate

Histopathology

Avian influenza viruses (AIVs) have a negative-sense RNA genome with eight segments in the Orthomyxoviridae family and Influenza A genus (Perez et al. 2019). They are categorized as high and low pathogenic based on the pathogenicity and molecular markers. They are also classified into 16 HA and 9 NA subtypes based on surface glycoproteins haemagglutinin (HA) and neuraminidase (NA) (Swayne et al. 2020). The H₉N₂ subtype is a low pathogenic AIV (LPAIV), which is the most prevalent avian influenza virus in poultry worldwide (Nagy et al. 2017). Its first reports was from turkeys in the USA in 1966 (Nagy et al. 2017). Later, it spread to Asia, especially in 1990s (Mehrabadi et al. 2019; Nagy et al. 2017). The first report on H₉N₂ in Iran was by Vasfi et al. in 1998. Since then, the virus has been endemic in Iran and reported frequently (Ahmadi et al. 2018).

The H₉N₂ AIV is important for following reasons: I: because of direct and indirect economic losses such as mortality, vaccination, and treatment costs (Swayne et al. 2020); II: the possible transmission to humans. The first zoonotic was reported in China in 1998 (Peacock et al. 2019). Fortunately, in humans it is sporadic with low severity, and there has been no report on transmission among the human population, but the virus can be considered a pandemic candidate (Pusch and Suarez 2018). III: the H₉N₂ can be a gene donor. To date, the role of H₉N₂ AIV in the emergence of at least three influenza subtypes, H₅N₁ (Peacock et al. 2019), H₇N₉ (Lam et al. 2013), and H₁₀N₈ (Chen et al. 2014), has been recognized.

Nowadays, the prevention strategy in poultry is based on vaccination and biosecurity (Shen H et al. 2014). However, vaccination is the primary strategy, its effectiveness is mainly influenced by the similarity of vaccine and virus HA glycoprotein (Song and Qin 2020). In addition, vaccination in birds with immunosuppressive diseases has no optimal effect (Sharif and Ahmad 2018).

In humans, anti-influenza drugs are used as treatment and prevention (Amarelle et al. 2017). The two main types of medicines, Adamantanes and Neuraminidase inhibitors, have been proven in many countries (Koszalka et al. 2017). The mechanisms of action for these drugs are blocking M₂ ion channel and neuraminidase glycoprotein, respectively (Watanabe and Kawaoka 2015).

Vaccination and biosecurity in poultry cannot completely prevent the disease outbreakes. Currently, there is no specific treatment for poultry, and the use of current human anti-influenza drugs are prohibited in many countries (Ahmadi et al. 2018; Tsuruoka et al. 2017). On the other hand, drug resistance, especially for Adamantanes, has increased in recent years (Hussain et al. 2017). In addition, prescribing these drugs 48 hours after the onset of symptoms, is not considered effective (Lehnert et al. 2016). It is therefore required to search for new anti-influenza agents as a drug that can also be used in poultry that antimicrobial peptides are one of many candidates (Ahmadi et al. 2018; De Angelis et al. 2021).

Antimicrobial peptides (AMPs) or host defense peptides (HDPs) are small amino acid molecules (usually less than 100 amino acids) that are part of the innate immune system (Kang et al. 2017; Lei et al. 2019). These peptides exist in many organisms (Lei et al. 2019) and have a broad-spectrum effect on bacteria, fungi, and cancer cells (Ong et al. 2014; Yeaman and Yount 2003). The peptides are also an acceptable candidate in preventing and treating viral diseases, with proven antiviral activity against some DNA and RNA viruses (Bahar and Ren 2013).

A group of AMPs is derived from larger proteins. The N-terminal of lactoferrin has hydrophobic and cationic AMPs with antibacterial, antiviral, and anti-fungal activities (Tanhaiean et al. 2018a; Tanhaieian et al. 2018b). Chimeric peptide cLF-chimera is derived from camel milk lactoferrin. The peptide is synthesized from the linkage of lactoferricin (cLFcin) C-terminus and lactoferampin (cLFampin) N-terminus by a lysine amino acid (Pirkhezranian et al. 2020; Tanhaieian et al. 2018a) and expressed recombinantly in E. coli and L. lactis (Tanhaieian et al. 2018a; Tanhaieian et al. 2018b). The peptide has an antibacterial effect against E. coli, S. Enteritidis, S. Typhi, P. aeruginosa, and S. aureus in vitro and E. coli and C. perfringens, in vivo (Daneshmand et al. 2019; Daneshmand et al. 2020; Roshanak et al. 2020; Tanhaieian et al. 2020).

There are some researches on natural and synthetic AMPs which focus on antiviral drugs in humans and birds. However, most researches concentrate on human pathogens like MERS-CoV, SARS-CoV, HIV, hepatitis C virus, and measles (Zhao et al. 2020); others have focused on avian pathogens. One of these important potential zoonotic pathogenes is AIV. Different peptides have been tested on different subtypes of AIV, Like H₁, H₃, H₅, H₇ (Zhao et al. 2016; Li et al. 2011) and H₉N₂ (Arbi et al. 2022; Rajik et al. 2009).

In this study, we also decided to predict the interaction of the so-called peptide with its poteantially related receptros (HA, NA and M2 ion channel). In order to accomplish this task, we used computational modeling technique. Computational modeling has the benfits of reducing wet lab practices costs and minimizing blind experiments (Meng et al. 2011). On the other hand, molecular docking has been accepted as a modeling technique that could elucidate the interaction between molecules (Pagadala et al. 2017). This method explains ligand and target protein's most favorable binding mode (Tripathi and Bankaitis 2017). Therefore, it can be used in molecular biology and drug design (Meng et al. 2011; Pagadala et al. 2017).

The objective of this study was to evaluate the probable inhibitory effects of cLFchimera on H₉N₂ subtype of influenza virus to prevent and treat influenza both in humans and poultry. Moreover, computational modeling predicts the interaction between the peptide and the virus surface projections (HA, NA, M₂).

Virus, cells, and embryonated eggs

Low Pathogenic AIV (LPAIV) strain A/chicken/Iran/UT-Barin/2017 (H9N2) was used in this study. The virus was provided by the Department of Avian Diseases, Faculty of Veterinary Medicine, University of Tehran. Madin-Darby canine kidney (MDCK) cells were purchased from Razi Vaccine & Serum Research Institute, Iran, and maintained in Dulbecco's minimal essential medium (DMEM; Invitrogen), with 10% fetal bovine serum (FBS; Invitrogen) at 37 centigrade degrees and 5% CO₂ atmosphere. Eleven-day-old embryonated eggs (Ross 308) were also used to evaluate the possible virus-peptide interactions.

cLF-chimera peptide

The chimeric peptide is derived from camel-milk lactoferrin. It is synthesized from the linkage of cLFcin C-terminus (amino acids 17–30) and cLFampin N-terminus (amino acids 265–284) by a lysine amino acid. There is also a six-aminoacids-histidine tag linked to lactoferampin (Tanhaiean et al. 2018a; Tanhaiean et al. 2018b). Figure 1 shows the cartoon shape and amino acid sequence of the peptide.

Experimental design

The study was conducted in 6 steps as below:

Titration of the virus by the EID₅₀ method

First, a ten-fold dilution of the virus was prepared. Serial dilution continued to 10^− 9. Then, each dilution was injected into five embryonated eggs. After incubation at 37°C and 75% humidity for 72 hours, the number of live and dead embryos was counted in each group. Then, the chorioallantoic fluid (CAF) of all eggs was harvested. The fluids were evaluated for the presence of the virus by HA test. To increase the accuracy, HA test was performed in duplicate. To reach 10², 10⁴, and 10⁶ EID₅₀ of the virus, Reed and Muench method was performed (Villegas 2008).

Injection of the peptide and titrated virus into embryonated eggs

Eleven-day-old embryonated eggs from a 42-week-old broiler breeder flock (Ross 308) were randomly distributed into 17 groups (each group with ten eggs). After peptide dilutions were prepared (40, 80, and 160 µg) (Tahmoorespur et al. 2020; Torres et al. 2013), 0.1 ml of each dilution was mixed with 0.1 ml of the virus dilutions (10², 10⁴, and 10⁶ EID₅₀). The mixture remained at room temperature for 30 minutes. Then, each inoculum was injected into the allantoic sac of embryonated eggs (Tare et al. 2015). There were three groups : positive, negative, and injection control. All treatment groups are summarized in Table 1.

Table 1

All groups of embryonated egg injection.
Groups	Treatment
C	No treatment for egg control
CS	Normal saline injection for injection shock control
CV1	10² EID₅₀ unit of the virus injection for virus control
CV2	10⁴ EID₅₀ unit of the virus injection for virus control
CV3	10⁶ EID₅₀ unit of the virus injection for virus control
CP1	Control for 40 µg of the peptide
CP2	Control for 80 µg of the peptide
CP3	Control for 160 µg of the peptide
V1P1	10² EID₅₀ unit of the virus + 40 µg of the peptide
V1P2	10² EID₅₀ unit of the virus + 80 µg of the peptide
V1P3	10² EID₅₀ unit of the virus + 160 µg of the peptide
V2P1	10⁴ EID₅₀ unit of the virus + 40 µg of the peptide
V2P2	10⁴ unit of the virus + 80 µg of the peptide
V2P3	10⁴ unit of the virus + 160 µg of the peptide
V3P1	10⁶ EID₅₀ unit of the virus + 40 µg of the peptide
V3P2	10⁶ EID₅₀ unit of the virus + 80 µg of the peptide
V3P3	10⁶ EID₅₀ unit of the virus + 160 µg of the peptide

Evaluation of macroscopic and histopathologic lesions of the embryos

The eggs were incubated for four days (Arbi et al. 2022) in a GALLENKAMP® incubator model at 37°C and 75% humidity. During this period, all eggs were daily candled for viability. The deaths in the first 24 hours were assumed as injection error or probable bacterial infection and were eliminated from the results (Perez et al. 2019). On the 4th day of incubation, each group's alive or dead embryos were sorted and evaluated for gross anomalies. Next, the chick embryos were fixed with 10% neutral buffered formalin for one week. Finally, 4 µm tissue sections were provided from the whole chick body, including head, thorax, and abdomen contents, then were embedded in paraffin and stained with hematoxylin and eosin (H&E) for microscopic studies.

Cytotoxicity assay

Ninety-six-well culture plates with MDCK cells were incubated at 37 centigrade degrees with a 5% CO₂ atmosphere for a day. Cells were then treated with various concentrations of the peptide, virus, and peptide-treated virus for 24 hours with the same condition of the previous step (Table 2 shows all 14 groups in detail). Next, a cell viability assay was performed by MTT method to determine the cytotoxicity of all the treatment groups. In brief, 10 µL of MTT solution was added to each well and incubated for four hours at 37°C. Then, 50 µL of DMSO solution was added to the wells. Finally, the solution's optical density (OD) was measured at 570 nm by ELISA reader (anthos 2020^®) (Bahuguna et al. 2017; Mehrbod et al. 2018).

Table 2

All groups of cell culture system.
Groups	Treatment
C	No treatment for cell control
CV	Control of the virus (10⁶ EID₅₀)
CP1	Control for 40 µg of the peptide
CP2	Control for 80 µg of the peptide
CP3	Control for 160 µg of the peptide
V1P1	10² EID₅₀ unit of the virus + 40 µg of the peptide
V1P2	10² EID₅₀ unit of the virus + 80 µg of the peptide
V1P3	10² EID₅₀ unit of the virus + 160 µg of the peptide
V2P1	10⁴ EID₅₀ unit of the virus + 40 µg of the peptide
V2P2	10⁴ EID₅₀ unit of the virus + 80 µg of the peptide
V2P3	10⁴ EID₅₀ unit of the virus + 160 µg of the peptide
V3P1	10⁶ EID₅₀ unit of the virus + 40 µg of the peptide
V3P2	10⁶ EID₅₀ unit of the virus + 80 µg of the peptide
V3P1	10⁶ EID₅₀ unit of the virus + 160 µg of the peptide

Computational modeling

SWISS-MODEL web server modeled the HA, NA, and M₂ projections (Waterhouse et al. 2018). PEP-FOLD web server also modeled the peptide (Shen Y et al. 2014). Then, docking complexes, prepared with ClusPro web server (Porter et al. 2017), were opened with Pymol software (Schrödinger and DeLano 2020). Afterwards, photos were taken in the ligand-receptor interaction site.

Statistical analysis

Statistical analysis and diagrams were performed by Graph Pad Prism software, version 8.0. The data represent the mean ± standard deviation for each sample. The two-way analysis of variance (ANOVA) was used to compare the means.

Injection of peptide and titrated virus to embryonated eggs

After four days of incubation, eggs were candled to identify the livability of the embryos. The results showed 100% vitality for all peptide concentrations (CP1, CP2, and CP3). There was also nearly 100% viability in all V1 (V1P1, V1P2, and V1P3) and V2 (V2P1, V2P2, and V2P3) groups (except a death related to injection error in V2P2 group). All embryos in V3P1 and V3P2 were dead, but V3P3 had very high livability (except two deaths related to injection error in V3P3). All embryos were dead in virus control groups (CV1, CV2, and CV3), as expected. Table 3 shows the results in detail.

Table 3

Number of live and dead embryos in each group.
Groups	Number of live embryos	Number of dead embryos	Livability (percent)
C	10	0	100
CS	10	0	100
CV1	0	10	0
CV2	0	10	0
CV3	0	10	0
CP1	10	0	100
CP2	10	0	100
CP3	10	0	100
V1P1	10	0	100
V1P2	10	0	100
V1P3	10	0	100
V2P1	10	0	100
V2P2	9	1	90
V2P3	10	0	100
V3P1	0	10	0
V3P2	0	10	0
V3P3	8	2	80

Macroscopic and histopathologic lesions observed in embryos

After four days of incubation and determining the number of live and dead embryos, they were all checked for visible gross lesions. Based on the findings, the embryos in virus controls (CV1, CV2, CV3), V3P1, and V3P2 were dwarf, featherless, and with visible hemorrhagic lesions in some cases. In contrast, embryos were larger, feathered, and had no visible lesions in the remaining groups (all V1, all V2, and V3P3). Figure 2 shows prominent macroscopic findings in some groups.

After checking for gross lesions, all the embryos were processed and stained with hematoxylin and eosin for microscopic studies. The results showed lesions in different organs in the virus control groups (CV1, CV2, CV3), V3P1, and V3P2. The lesions were more severe in the virus control groups,while no significant histopathologic lesions were observed in other groups. Table 4 and Fig. 3 indicate the histopathological findings in detail.

Table 4

Histopathological lesions of the groups. V: Virus: 3 to 1 maximum to minimum concentration (3: High concentration, 2: Medium concentration, 1: Low concentration), C: Control, P: peptide: 3 to 1 maximum to minimum concentration (3: High concentration, 2: Medium concentration, 1: Low concentration), Pathologic observation: (3: Intensive, 2: Moderate, 1: Mild, 0: none)
Tissue/Group	C	CS	CV1	CV2	CV3	CP1	CP3	V1P1	V1P2	V1P3	V2P1	V2P2	V3P1	V3P2	V3P3
CNS	0	0	3	3	3	0	0	1	1	0	1	1	3	2	0
Eyeball	1	0	3	3	3	1	1	2	1	1	1	1	3	2	0
Lung	0	0	3	3	3	0	0	1	0	0	1	0	3	3	0
Liver	0	0	3	3	3	0	0	0	0	0	0	0	3	3	0
Kidney	0	0	3	3	3	0	0	0	0	0	0	0	3	3	0
Skin	0	0	3	3	3	0	0	1	0	0	0	1	3	2	1
Proventriculus	0	0	0	1	1	0	0	0	0	0	0	0	0	0	0
Intestine	0	1	0	1	1	0	0	0	0	0	0	0	3	2	0
Spleen	0	0	3	3	3	0	0	1	1	1	0	0	3	2	1

Histopathologic evaluation demonstrated target organs being affected significantly in CV1, CV2, CV3, V3P1, and V3P2, according to Table 4:

The major histopathological findings in brains of control group were included of large vacuolation of neuropil (status spongiosis), which established by edema. Moreover the blood vessels were obviously congested, and ischemic neuronal changes were scattered throughout the parenchyma.

Massive hemorrhage and congestion in large and small blood vessels appeared in renal and perirenal adipose tissues. The intensive tubular necrosis with deep eosinophilic staining was an outstanding finding. Glomeruli were relatively spared from influential damage, and the interstitium was invaded by a large population of inflammatory cells, chiefly mononuclear cells.

Intensive hemorrhages with focal to diffuse hepatocellular degeneration or necrosis and congestion of central veins, small-sized blood vessels and sinusoids were obvious in the liver, Large pulmonary vascular congestion with hemorrhage in the interstitium, which was accompanied by large amounts of inflammatory cells were determined in the pulmonary parenchyma. Furthermore, primitive parabronchi had highly-exfoliated epithelial cells in their lumen.

In the eyeball, the continuous hemorrhages along the choroid layers with extensive edema were striking. Moreover, severe disruption of retinal pigmented epithelium or even in some embryosit completely detached, which not identified in microscopic evaluation. Eventually, hemorrhage and congestion were revealed in dermal and hypodermal layers with degraded collagen fibers in the integument. Other organs with minimal changes were the spleen and intestine, in which the red pulps were filled with erythrocytes in the spleen, and necrotic villi was detected in only some intestinal tissues.

No noticeable changes were observed in other organs like the gizzard, proventriculus, and musculoskeletal system. In peptide or peptide-virus treatment groups, all organs had normal architecture similar to the negative control group. However, mild to moderate congestion was seen in V1P1-3, V2P1-3, and V3P3 groups, and scant infiltration of inflammatory cells were present around central veins with mild hyperplasia of bile ducts in the liver.

Cytotoxicity assay

We have tested different peptide and the virus concentrations and studied the mixtures at 570 nm. The results (Fig. 4) showed that the peptide concentration increased in V1 (V1P1, V1P2, and V1P3) and V2 (V2P1, V2P2, and V2P3) groups leading to a decrease in cell viability. All cell viability percentages in V1 groups were lower than V2. In V3 group (V3P1, V3P2, and V3P3), unlike V1 and V2, the increase in the peptide concentration caused an increase in cell viability.

In the peptide controls (CP1, CP2, and CP3), there was a decrease in cell viability with an increase in the peptide amount. The cell viability was the lowest in virus control group (Fig. 4).

Computational modeling

We have applied SWISS-MODEL and PEP-FOLD web servers to estimate the viral projections and peptide shapes, respectively. Then we used ClusPro web server to evaluate the docking complexes. Figure 5 shows the docking complexes and putative interactions between receptor and ligand amino acids. Molecular docking showed 5, 7, and 13 possible interactions between the peptide and viral M₂ ion channel, HA, and NA glycoproteins.

The H₉N₂ subtype of AIV can cause direct and indirect losses in poultry industry and can be transferred to humans, which is a public health matter (Ali et al. 2019; Mostafa et al. 2018; Perez et al. 2019; Swayne et al. 2020). Since the virus has a highly negative nature, resistance to current chemical drugs is a common finding (Jones et al. 2006). Since chemical drugs have several side effects (Anand et al. 2019), there is an immediate need for a new group of anti-influenza drugs with less viral resistance and side effects (Sala et al. 2018). For this purpose, many studies have focused on different drug candidates; one group is antimicrobial peptides which have anti-influenza properties with broad-spectrum activity (Kang et al. 2017). This study evaluated the anti-influenza effects of the novel chimeric peptide, cLF-chimera, on H₉N₂ subtype in embryonated eggs and MDCK cells.

In ovo model is a standard method to evaluate the probable anti-influenza activity of different drug candidates. It is an ethical way in contrast to laboratory animal models and is on the edge of in vitro and in vivo models (Ghoke et al. 2018). In this study, the data from embryonated egg injection (Table 3) showed that the embryos were highly livable without distinct macroscopic lesions (Fig. 2) in peptide control groups. This result is comparable with Michálek et al.'s results that have used melittin as an antimicrobial peptide in 2 µM concentration which did not severely affect embryo vitality, and the embryos did not have macroscopic lesions (Michálek et al. 2015). In addition, the histopathological findings in peptide control groups were similar to negative control and saline control without any significant changes (Table 4 & Fig. 3). Based on the above data, it is inferable that the peptide was not toxic to embryos in given doses.

We have observed that in virus control groups, embryo mortality was 100% (Table 3), and the embryos were dwarf, featherless with visible hemorrhagic lesions in some cases (Fig. 2). Knowing that Avian Influenza viruses cause pathological changes in chicken embryos through apoptosis and necrosis (Ahmadi et al. 2018), the histopathological findings conribute to the viral damage. In our study, histopathological results in virus control groups revealed that (Table 4 & Fig. 3), except for the gastrointestinal tract with minimal lesions, the other main infected organs were prominently affected and severely damaged. This result is comparable with Shah et al.'s study who evaluated the potential effect of three herbal extracts on H₉N₂ subtype in embryos. In their study, positive controls were severely damaged in different organs (Liver, spleen) (Shah et al. 2021). All in all, it is clear that the virus is very harmful to embryos in given doses.

Our results also revealed that the embryos were very livable in all V1 and all V2 groups (Table 3), without distinct macroscopic lesions (Fig. 2). The histopathological findings in all of these groups showed mild lesions in some cases (Table 4 & Fig. 3). According to the results, the peptide in given doses in all V1 and V2 groups could prevent the adverse effect of the different virus concentrations in ovo. This data (especially embryo survival rate) is comparable with the study by Sauerbrei et al. on H₉N₂ subtype (Sauerbrei et al. 2006). In this research, four anti-influenza drugs were evaluated against 10² and 1 EID₅₀ units of the virus: Amantadine, Rimantadine, Oseltamivir, and Zanamivir, with the highest embryo survival rate of 21.9% for adamantanes and 50% for neuraminidase inhibitors (Sauerbrei et al. 2006).

In V3P1 and V3P2 groups, the embryo survival rate is zero (Table 3), and embryos are dwarf, featherless, with apparent macroscopic lesions (like hemorrhagic lesions) in some cases. At histopathology, severe lesions were detected in these groups (like virus controls, but the lesions were less severe, and V3P2 group had less severe lesions than V3P1) (Table 4 & Fig. 3). The data also indicated that the low and medium peptide concentrations could not entirely prevent destructive viral effects in the embryos. In V3P3 group, the survival rate is very high (except the deaths due to injection error) (Table 3), and the embryos have no noticeable macroscopic lesions (Fig. 2). From the histopathological point of view, the findings are similar to peptide control, negative control, saline control, and all V1 and V2 groups (Table 4) without any significant changes (Fig. 3). Based on the data obtained from V3 groups, it is deducible that the peptide could prevent the adverse effect of the virus in a dose-dependent manner.

MTT is a color-based assay that evaluates the metabolic activity of cells. The assay is routine, easy, and advantageous for different animal cell lines (Tolosa et al. 2015). According to the results obtained from MTT assay in peptide control groups (Fig. 4), the cell viability decreases as the peptide dose increases. This result suggests that the peptide is toxic to the cell line in a dose-dependent manner. This data agrees with previous studies on different antiviral peptides for different cell lines (De Angelis et al. 2021; Sala et al. 2018). Overall, our results indicated that the peptide was toxic for the cell line but not for the embryo. This difference can result from lack of defense mechanisms and higher sensitivity of cells in the cell culture system compared to the body (Hartung 2007).

We have also discovered that in the virus control group, the pecenage of cell viability was the lowest of all groups (Table 5). The low percentage can be due to cytopathic effects (CPE) of the virus (Chen et al. 2021). Based on the data, it is obvious that both the peptide and the virus harmed cell livability, but the effect of the virus is much higher.

Our study showed that in V1 groups, the cell viability decreases as the peptide dose increases, and V1P1 had the best cell livability (Fig. 4). The cell viability percentage of these groups is lower than the asame dose of peptide controls, but is higher than virus control (except V1P3 which is higher than P3 alone, but with a minimum difference) (Fig. 4). It seems that the peptide in V1P3 group can block the viral effect, and the remaining peptide have lower toxicity than P3 alone. As the viral dose is constant in these groups, it is more probable that the decrease in cell viability is much related to peptide toxicity. Still, at the same time, the peptide can partially prevent the destructive effects of the virus.

The data from V2 groups are similar to that of V1, but all cell viability percentages are higher (Fig. 4). As in these groups, like V1, the virus amount is constant; it seems that the decrease in cell vitality is related to peptide toxicity. Compared to the virus control, in V2 groups, the peptide can partially inhibit the harmful effects of the virus.

In V3 groups, our data showed that unlike V1 and V2, the increase in the peptide dose causes an increase in cell viability (Fig. 4). All the percentages were higher than the virus control but lower than the peptide controls. Because the virus concentration is constant, and the decrease in V3P1 and V3P2 is much higher than in previous groups and it is close to the virus control, it might primarily result from viral CPE. However, even in this group, the peptide can partly block the virus's adverse effects.

Based on the data obtained in these three groups, it is inferable that the peptide can inhibit the destructive viral effects in all the groups. Our study also revealed that the optimum dose of the peptide in all V1, V2, and V3 groups were V1P1, V2P1, and V3P3, respectively.

Molecular docking is a drug design procedure that anticipates binding form and mimics the molecular interaction of ligand and receptor (Fan et al. 2019). In our study, we have also evaluated the interaction between the peptide, viral surface glycoproteins (HA and NA), and M₂ ion channel by molecular docking in order to determine the probable mechanism of action for the peptide. According to the docking results (Fig. 5, a & b), M₂ ion channel has five possible interactions with the peptide. According to the previous studies on Adamantane's action mechanism, amino acid residue ASP-44 is a site of action for these drugs (Özbil 2019; Rosenberg and Casarotto 2010). Thus, the peptide may mimic Adamantane's action mechanism by blocking M₂ ion channel (Fig. 5, a & b).

The docking complex between the peptide and viral HA showed seven potential peptide attachment residues. Based on previous researches on drugs that block HA glycoprotein, amino acids ASN-153 and ARG-131 are in the receptor-binding site of HA₁ (Yang et al. 2013). Therefore, the peptide may prevent the attachment of the virus to cellular receptors (Fig. 5, c & d).

Our docking results also showed 13 possible interaction sites between the peptide and viral NA glycoprotein (Fig. 5, e & f). Based on previous studies on NA blockers, none of these interaction sites have a role in blocking NA glycoprotein. However, as for the high number of potential attachment sites, further studies are required which can be a subject for later studies on cLF-chimera.

Finally, antimicrobial peptides like cLF-chimera can have different potential modes of action. Some action mechanisms include: inhibiting virus attachment and cell membrane fusion, disrupting viral envelope, inhibiting viral replication, and other probable mechanisms (Skalickova et al. 2015). Therefore, the anti-influenza effect of the peptide in this study can be a combination of the mentioned mechanisms.

cLF-chimera has also proven the effects against bacteria participating in respiratory complexes (Roshanak et al. 2020; Tanhaiean et al. 2018a; Tanhaiean et al. 2018b; Tanhaiean et al. 2020). This study also suggests that the peptide has potential anti-influenza properties. Since H9N2 subtype can mainly cause respiratory disease in poultry (Nili and asasi, 2003; Swayne et al. 2020) and flu-like, mild, primarily respiratory illness in human populations (Liu et al. 2018; Song and Qin 2020),the peptide can affect the virus and other secondary bacteria. Compared to common anti-influenza drugs, this broad-spectrum impact can be an advantage for the peptide.

In most viral respiratory diseases, a primary viral agent is accompanied by secondary infections (Swayne et al. 2020; Seto et al. 2013). However, further studies are needed to explain the potential pros and cons of the peptide as a drug candidate. This study elucidates some peptide characteristics with some undefined questions regaring its novelty: how to use the peptide in vivo? how to find its precise dose? and what is the exact mechanism of action? These topics are beyond the scope of this paper, but can be evaluated in future studies.

AIV = avian influenza virus; HA = haemagglutinin; NA = neuraminidase; LPAIV = low pathogenic avian influenza virus; AMP = antimicrobial peptide; HDP = host defense peptide; cLFcin = camel lactoferricin; cLFampin = camel lactoferampin; MDCK = Madin-Darby canine kidney; DMEM = Dulbeco’s minimal essential medium; FBS = fetal bovine serum; EID₅₀ = embryo infective dose50; CAF = chorioallantoic fluid; HA test = haemagglutination test; H&E = hematoxylin and eosin; MTT = mitochondrial toxicity test; DMSO = Dimethyl sulfoxide; OD = optical density; ELISA = enzyme-linked immunosorbent assay; ANOVA = analysis of variance; CPE = cytopathogenic effect

Acknowledgment

This work was supported by the University of Tehran under Grant number IR.UT.VETMED.REC.1401.004. The authors, also would like to thank the Rastegar Central Laboratory, University of Tehran, for technical support.

Statement of animal ethics

This study was approved by the animal ethics committee of the University of Tehran under approval number IR.UT.VETMED.REC.1401.004.

Conflict of interest statement

The authors state no conflict of interest.

Funding

This work was supported by the University of Tehran under Grant number IR.UT.VETMED.REC.1401.004.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

JR has proposed the idea of the study and also designed it. M KH has done the study with the contribution of MHS in designinig the peptide, AB in embryonic and cell culture parts, and OD in histopathology. MKH drafted the initial manuscript. All authors reviewed the drafted manuscript for critical content. All authors approved the final version of the manuscript.

Data Availability

The data sets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics Approval

In this study, ethics approval was proved by the Ethics Committee of University of Tehran (Date: 2022.05.10; No: IR.UT.VETMED.REC.1401.004).

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The Inhibitory Effect of cLF-chimera, a Recombinant Antimicrobial Peptide, on Avian Influenza Virus Subtype H₉N₂

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Virus, cells, and embryonated eggs

cLF-chimera peptide

Experimental design

Titration of the virus by the EID₅₀ method

Injection of the peptide and titrated virus into embryonated eggs

Evaluation of macroscopic and histopathologic lesions of the embryos

Cytotoxicity assay

Computational modeling

Statistical analysis

Results

Injection of peptide and titrated virus to embryonated eggs

Macroscopic and histopathologic lesions observed in embryos

Cytotoxicity assay

Computational modeling

Discussion

Abbreviations

Declarations

References

table 5

Additional Declarations

Status:

Version 1

The Inhibitory Effect of cLF-chimera, a Recombinant Antimicrobial Peptide, on Avian Influenza Virus Subtype H9N2

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Virus, cells, and embryonated eggs

cLF-chimera peptide

Experimental design

Titration of the virus by the EID50 method

Injection of the peptide and titrated virus into embryonated eggs

Evaluation of macroscopic and histopathologic lesions of the embryos

Cytotoxicity assay

Computational modeling

Statistical analysis

Results

Injection of peptide and titrated virus to embryonated eggs

Macroscopic and histopathologic lesions observed in embryos

Cytotoxicity assay

Computational modeling

Discussion

Abbreviations

Declarations

References

table 5

Additional Declarations

Status:

Version 1

The Inhibitory Effect of cLF-chimera, a Recombinant Antimicrobial Peptide, on Avian Influenza Virus Subtype H₉N₂

Titration of the virus by the EID₅₀ method