Antiviral activity of Piscidin 1 against pseudorabies virus both in vitro and in vivo

The peptides (Table 1) used in this study were synthesized on an automated solid-phase peptide synthesizer by Neweast Biosiences Inc. (Wuhan, China) [23]. The crude peptides were purified via a reverse-phase high-pressure liquid chromatography (RP-HPLC) using a C₁₈ column (Waters Xbridge). The elution was conducted using a water-acetonitrile linear gradient (0–80% of acetonitrile) containing 0.1% (V/V) trifluoroacetic aicd (TFA). Finally, the purity and accurate masses of the product peptides were determined using HPLC and mass spectrometry, respectively.

The PRV strains of Ea, HNXX, 152, and TGEV WH-1 were propagated in PK-15 cells based on the method reported in previous studies [24]. RV TM-a was propagated in Rhesus monkey kidney cell line (MA104) [25]. PRRSV YA was cultured in Marc-145 cells [26]. PEDV strain CH/YNKM-8/2013 was cultured in vero cells as described before [27]. The virus suspension was stored at − 80 °C until used.

In our initial topical screening assays, the viruses were pre-incubated with peptides (50 μg/ml) for 1 h at 37 °C before they were added to the target cells. Peptide-free controls (virus plus cell culture medium only) were incubated in parallel. After incubation, the TCID₅₀ of peptide-treated viruses and peptide-free viruses was measured. Residual virus was calculated according to the following formula, residual infectivity = b/a × 100% (“a” represent TCID50 of residual virus non-treated by the AMPs (control), “b” represent TCID50 of residual virus after treated by AMPs).

A 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cell proliferation assay was used to assess cell viability as described previously [28]. Briefly, PK-15 cells were added to 96-well tissue-culture plates (Coster, USA) and incubated overnight at 37 °C. Serial two fold dilutions of AMPs ranging from 200 to 3.12 μg/ml were added into the plate. The cells were incubated with AMPs at 37 °C for 48 h, and the morphology of the cells was evaluated under light microscope. Then, the cells were further incubated with MTT solution for 4 h and the cell pellets were collected for measurement of absorbance at 490 nm by an ELISA reader.

The antiviral activity was evaluated by plaque reduction assay [29] at different infection stages: pre-infection and post-infection, as well as direct inactivation of the viruses. Briefly, the direct effect of peptides on virus per se was analyzed by the following procedures. Monolayer of PK-15 was prepared in 12-well tissue culture plates. Prior to infection, 300 μl of different virus stocks with the titer of 10³ pfu/ml were incubated with 33 μl of peptides diluted in DMEM at concentrations of 25, 5, 1, 0.2 μg/ml at 37 °C for 1 h. After incubation, the peptide-virus mixture was further diluted in DMEM and was inoculated to the monolayer of PK-15. The absorption proceeded at 37 °C for 1 h. The virus-peptide mixture was then removed and replaced with the overlaid medium (4% FBS and 1.5% carboxymethyl cellulose in DMEM). The plaque formation was measured on the 3-4th days later by crystal violet solution staining.

To analyze the post-infection effect of the peptides on virus growth, cells were infected with the same amount of virus mentioned above at 37 °C for 1 h. Subsequently, the cells were washed 3 times with DMEM and treated by the peptides at different concentrations for 1 h at 37 °C. The peptides were removed from the cell culture and replaced with the overlaid medium. The resulting PFU titer was determined as described above.

To understand the pre-infection effect of the peptide on virus, the cells in 12-well tissue culture plates were treated with peptides at serial concentrations at 37 °C for 1 h. Then, cells were washed with DMEM for 3 times, followed by infection with viruses. The cells were covered with the overlaid medium for plaque assays.

Cell apoptosis was analyzed with Annexin V-FITC kit (Nanjing Keygen Biotech. Co., Ltd). Briefly, FITC-conjugated Annexin V (50 μl/well) and propidium iodide (PI, 50 μl/well) were added to the cells infected by AMP-treated viruses. Then, the obtained mixture was incubated at room temperature for 15 min in the dark prior to fluorescence observation. Cell apoptosis was determined on basis of the observation soon after initiating apoptosis. Cells translocated the membrane phosphatidylserine (PS) from the inner surface of the plasma membrane to the cell surface. Thereafter, PS can be easily detected by staining with a fluorescent conjugate of Annexin V (green), a protein that has a high affinity to PS. Cellular late apoptosis was determined by cell staining with PI (red). The results were analyzed by fluorescence microscope (Nikon, Japan) at 36 hpi.

The 6 to 8 week-old specific-pathogen-free BALB/c mice were purchased from the Experimental Animal Center of Zhongnan Hospital of Wuhan University (China) and randomly divided into six groups consisting of 10 mice each. Individuals in each group of mice were anesthetized with 1–3% isoflurane gas and challenged by the intra-footpad injection with 50 μl of DMEM containing 5 × 10³ TCID₅₀ of PRV-Ea in the presence or absence of 10, 5, 1, 0.2 μg/ml piscidin. After 14 days, the surviving mice were challenged with 5 × 10³ TCID₅₀ of PRV again. Mice viability and behaviors were monitored on a daily basis. In the first 2 days after the challenge, the mice commonly did not exhibit severe symptoms. However, mice gradually showed neurological symptoms in the subsequent days during the monitoring period and the post-challenged mice were monitored since day 3 post PRV infection once every 6–8 h for 8 days to obtain the survival information of the mice. The level of PRV infection symptoms was scored for every mouse by the following system: 0 = posture normal, absence of neurological symptoms, 1 = mild neurological symptoms: excitation, unrest, occasional itching, and foot swollen; 2 = severe neurological symptoms: ataxia, severe pruritus, and self-mutilation, biting and bleeding of the footpad. Mice were euthanized in the chamber with CO2 gradually filled when the score reached 2.

On day 6, three mice from control group and non-control groups were euthanized and brain tissues were collected and transferred to 4% paraformaldehyde. The tissues were dehydrated in ascending grades of ethyl alcohol i.e. 70, 90 and 100% ethanol. Afterwards, the tissues were cleared with xylene. Slides were stained with Haematoxylin and Eosin (H&E) stain and analyzed through a light microscope.

All of the animal experimental protocols were approved by the Ethics Committee of Huazhong Agricultural University according to Hubei province Laboratory Animal Management Regulations (HZAUMO2015–0015). During the experiments, mice were offered ad libitum access to water and food in a controlled environment of a 12 h light/dark cycle. All efforts have been made to reduce suffering of animals.

The antiviral effects of the peptides (maculatin, caerin, piscidin, lactoferricin B, indolicidin) were investigated in vitro against several viral pathogens that severely threaten the porcine industry. Four enveloped viruses were used in this assay including the PRV Ea strain, PEDV YNKM strain, TGEV WH-1 strain, and PRRSV YA isolate, as well as one non-enveloped RV TM-a strain. The peptides exhibited different inhibitory activities against these viruses (Fig. 1). The relative potency of the peptides against PRV was: piscidin≈caerin>macualtin>indolicidin> lactoferricin B. Piscidin and caerin showed strong virucidal activity with the residual infectivity being around 2.3% while indolicidin and lactoferricin B exhibited mild antiviral activity. In descending order of potency, the peptides against PEDV ranked as follows: caerin>piscidin> maculatin>lactoferricin B > indolicidin. Caerin had the most potent activity with the residual infectivity being 0.2%. The assay results indicated, that the order of potency versus PRRSV was: caerin>lactoferricin B > piscidin>maculatin>indolicidin. Caerin exhibited the best inhibitory activity with the residual infectivity being 41.1%, separately. The assay results of antivirus activity against TGEV indicated the activity order as follows: piscidin>lactoferricin B > indolicidin>maculatin>caerin. Piscidin was the most potent peptide versus TGEV with the residual infectivity being 41.5%. The assay result also indicated that PRRSV and TGEV were less sensitive to the tested peptides, compared with other viruses (Fig. 1). When it comes to RV, the relative potency order was: piscidin>caerin>maculatin>lactoferricin B > indolicidin. Piscidin displayed the most potent activity against RV with residual infectivity being 7.4%. Piscidin and caerin exhibited activity against almost all the viruses with the residual infectivity being lower than 50% with exception of piscidin versus PRRSV and caerin versus TGEV. Caerin showed the greatest inhibitory activity against PEDV with the residual infectivity being 0.2%, which was the lowest value among all the viruses tested. Maculatin exhibited strong activity against PRV (10.1%) and mild activity against RV (43.4%). However, it showed limited activity against PEDV, PRRSV, and TGEV. Lactoferricin B and indolicidin were not as potent as other peptides to fight against viruses. Lactoferricin B showed stronger inhibitory activity against TGEV than against other tested viruses with residual infectivity being 54.1%, and residual infectivity of indolicidin versus PRV was 34.6%.

We examined the effects of AMPs on cell viability after incubating PK-15 cells with different concentrations of each peptide. Cytotoxicity was evaluated using MTT method (Fig. 2). The non-linear regression analysis result indicated that the 50% toxic concentration (TC₅₀) of caerin, maculatin, piscidin, and indolicidin were 67.8, 76.6, 65.2, and 110.2 μg/ml, respectively. Indolicidin was less cytotoxic than piscidin, caerin and maculatin. Lactoferricin B exhibited the weakest cytotoxic activity and it did not exhibit obvious cytotoxic activity even at maximum concentration of 200 μg/ml.

To determine whether the inhibitory activity against PRV by maculatin, caerin, piscidin was strain-specific or not, we tested the activity of these peptides against different PRV isolates including PRV-HNXX (a newly PRV isolate in China in 2012) and PRV-152 (modified Bartha strain). We found that all the three peptides displayed inhibitory activities against all the tested strains (PRV HNXX, PRV 152, and PRV-Ea) (Fig. 3a). This finding indicated that the activity of the peptides versus PRV was not strain-specific. When the concentration of the peptides increased from 50 to 100 μg/ml, the residual infectivity of PRV decreased (Fig. 3b). This trend was the most obvious for maculatin with the residual infectivity decreasing from 10.11 to 0.0017%, while the groups treated with piscidin and caerin exhibited a decrease by more than 30 folds in the residual infectivity. These data indicated that these peptides inhibited the virus in a dose-dependent manner.

In order to better understand the inhibitory effect of AMPs on the propagation of PRV-Ea, we examined whether AMPs directly damaged virus particles or indirectly interacted with the host cells pre- or post-infection. Plaque-reduction assay was also performed to confirm the peptides’ antiviral activity.

The viruses were incubated with the peptides prior to infection. The result showed that all the three peptides inhibited the virus infection in a dose-dependent manner (Fig. 4). At the concentration of 25 μg/ml, pisicidin, caerin and maculatin were found to inhibit most of the PRV particles. Pisicidin exhibited the strongest activity and obvious inhibitory ability at concentrations above 5 μg/ml. The 50% effective concentration (EC₅₀) values of the peptides were calculated using probit analysis by IBM SPSS (version 21, New York, USA). The analysis revealed that EC₅₀ values of caerin and maculatin were 2.63 μg/ml and 1.09 μg/ml, respectively. EC₅₀ of piscidin was the lowest (0.23 μg/ml) among the peptides tested, which indicated that piscidin was the most potent antiviral peptide against PRV in this study.

Subsequently, the experiments were conducted to further determine whether the peptides could function at post- (curative) or pre-infection (prophylactic) stage. The cell monolayers were treated with piscidin, caerin, and maculatin before (prophylactic) or after (curative) the virus binding stage, separately. No inhibitory effect of the three peptides on PRV could be detected (the obtained data not shown). This implied that these peptides probably inhibited PRV infection by directly interacting with the virus particles.

The cell apoptosis assay showed that PRV could induce both early and late apoptosis of PK-15 cells (Fig. 5). The late apoptosis rate of cells infected by the peptides-treated viruses decreased as the concentration of the peptide increased (Fig. 5b). The inhibition of late cellular apoptosis was obvious at the concentration of 25 μg/ml (Fig. 5a).

In the control group of the in vivo assay, mice (n = 10) were injected with 50 μl of piscidin (200 μg/ml) by the intra-footpad route. All mice survived and behaved normally. PRV was injected into mice with or without piscidin at various dosages (0.5, 2.5, 5, 10 μg/ml). And the surviving mice were re-challenged with PRV on day 14 (Fig. 6). The mice survival rate was recorded for 28 days. PRV infected mice [Piscidin (0 μg/ml) + PRV] began to show clinical signs around 72 h post infection, characterized by swelling of the inoculated foot and occasional itching. By 144 h post infection, most of the PRV inoculated mice showed constant tremors in inoculated leg and distinctive PRV symptoms of excitation, scratching and biting of the foot. By 168 h post infection, severe neurological symptoms of severe pruritus, ataxia, biting and bleeding of the footpad could be observed. The mice in the piscidin treated groups also showed similar symptoms, but symptoms appeared later and not so severe in piscidin treated mice than those in PRV infected mice. And the mice in the control group [Piscidin (10 μg/ml)] remained asymptomatic, posture normal without signs of neurological symptoms. In the group [Piscidin (0 μg/ml) + PRV], 90% of the mice (n = 10) died within 10 days showing typical symptoms of PRV infection. Mice (n = 10) that received piscidin at concentration of above 5 μg/ml were obviously protected against PRV infection (Fig. 6). However, one mouse from the 2.5 μg/ml peptide-treated group and nine mice from the 0.5 μg/ml group died within 10 days. To further characterize the prevention effect of piscidin against brain damage induced by PRV, the brain sections of mice from the control group, PRV-infected, and co-injection group were collected respectively and subjected to pathological examination. The blood stasis was observed in the brain of mice from PRV-infected group (Fig. 6a). The brain of mice from co-injection group showed no specific symptom. After 14 days, the surviving mice were re-challenged with PRV at 5 × 10³ TCID₅₀. The groups co-injected with piscidin and PRV failed to provide protection with the survival rates dropping to 30% at the end of the experiment and the mice in the 5 μg/ml peptide treated group all died.