Possible Antiviral Activity of 5-Aminolevulinic Acid in Feline Infectious Peritonitis Virus (Feline Coronavirus) Infection

Tomomi Takano, Kumi Satoh, and Tomoyoshi Doki
Original article: Possible Antiviral Activity of 5-Aminolevulinic Acid in Feline Infectious Peritonitis Virus (Feline Coronavirus) Infection; 10.2.2021; 18.4.2021

Abstract

Infectious feline peritonitis (FIP) is a life-threatening infectious disease of cats caused by virulent feline coronavirus (FIP: FIPV). Several effective antivirals have recently emerged for the treatment of FIP, but many are not available for practical use. 5-Aminolevulinic acid (5-ALA) is a low molecular weight amino acid synthesized in plant and animal cells. 5-ALA can be synthesized in large quantities and is widely used in the medical and agricultural industries. We hypothesized that 5-ALA inhibits FIPV infection. Therefore, we evaluated its antiviral activity against FIPV in whole felis catus fetal 4 cells and in primary feline macrophages. FIPV infection was significantly inhibited by 250 μM 5-ALA. Our study suggested that 5-ALA is useful for the treatment and prevention of FIPV infection.

Keywords: FIP, coronavirus, 5-aminolevulinic acid, antiviral drug, cat

Introduction

Feline Infectious Peritonitis (FIP) is a life-threatening infectious disease caused by feline coronavirus (FCoV) in domestic and wild Felidae species. FCoV is highly prevalent in cats throughout the world. FCoV is an enveloped single-stranded positive RNA virus. This virus belongs to the genus Alphacoronavirus of the subfamily Orthocoronavirinae of the family Coronaviridae (1). FCoV is divided into two serotypes according to the amino acid sequence of the spike protein (S), FCoV serotype I and FCoV serotype II (2). Serological and genetic studies have revealed that type I FCoV is dominant worldwide (3-5). FCoV is spread mainly by faecal-oral transmission (6). Most FCoV-infected cats are subclinical. However, several mutations have occurred in the VS protein that have led to the development of a virulent type called feline infectious peritonitis virus (FIPV) (7, 8). Serum in the peritoneal and pleural cavities and pyogranulomatous lesions in several organs are characteristic pathological findings of FIP in cats (9).

FIP is an immune-mediated and difficult-to-treat viral infection. Several effective antivirals for the treatment of FIP have recently been published (10, 11), but many are not available for practical use. Some anti-FCoV drugs, such as itraconazole, are available in animal kinetics, but their therapeutic effects are limited (12). Because FIP is a chronic and systemic disease, clinical remission is difficult to achieve. Therefore, it is desirable that therapeutic drugs for FIP have the following properties: (1) Few side effects for cats, (2) low cost, and (3) low pathogen mutagenesis.

5-Aminolevulinic acid (5-ALA) is a low molecular weight amino acid synthesized in plant and animal cells (13, 14). It is an intermediate in tetrapyrrole biosynthesis. Because 5-ALA is highly soluble in water and has low cytotoxicity, it is widely used in the medical and agricultural industries (15). Several studies have been published on the effects of 5-ALA on infectious diseases. Suzuki et al. stated that when 5-ALA and iron ion were administered orally to mice infected with a malaria parasite (Plasmodium yoelii), the mice survived (16). On the other hand, its effects on viral infection are unclear.

In veterinary medicine, photodynamic therapy (PDT) using 5-ALA has been studied in the treatment of tumors in dogs (17), but to our knowledge the effect of 5-ALA on infectious animal diseases has not been studied. We investigated whether 5-ALA could be administered as an anti-FCoV drug in vitro.

Materials and methods

Cell cultures, animals and viruses

Felis catus (fcwf) -4 cat fetal 4 cells (kindly supplied by Dr. MC Horzink of Utrecht University) were grown in Eagles' MEM containing 50% L-15 Leibovitz media, 5% fetal calf serum (FCS), 100 U / ml penicillin and 100 μg / ml streptomycin. The maintenance medium had the same composition as the growth medium except for the FCS concentration (2%). Feline primary macrophages were selected for primary macrophages. Feline alveolar macrophages were obtained from four specific pathogen-free (SPF) cats aged 3-5 years by bronchoalveolar lavage with Hank's balanced salt solution. Feline primary macrophages were maintained in D-MEM supplemented with 10% FCS, 100 U / ml penicillin and 100 μg / ml streptomycin. SPF cats were maintained in an isolated temperature controlled facility. The animal experiment was approved by the President of Kitasato University by decision of the Institutional Committee on the Care and Use of Animals of Kitasato University (18-050) and conducted in accordance with the Guidelines for Animal Experiments of Kitasato University. Sample sizes were determined from our previous study and a minimum number of cats were used. The FCoV KU-2 type I strain (FIPV-I KU-2) was isolated in our laboratory. FCoV-II 79-1146 was kindly provided by Dr. MC Horzinek from Utrecht University. These viruses were cultured in fcwf-4 cells at 37 ° C.

Compounds

5-ALA and sodium ferrous citrate (SFC) were provided by Neopharma Japan (Tokyo, Japan). 5-ALA and SFC were dissolved in culture medium at 200 mM and 50 mM. The SFC solution was used as the 5-ALA solvent. On the day of the experiments, these compounds were diluted to the desired concentrations in the culture medium.

Cytotoxic effects of compounds

Fcwf-4 cells were seeded in 96-well plates. Compounds were loaded into wells in triplicate. After incubation for 96 hours, the culture supernatants were removed, WST-8 solution (Kishida Chemical, Osaka, Japan) was added and the cells were returned to the incubator for 1 hour. The absorbance of the formazan produced was measured at 450 nm using a 96-well spectrophotometric plate reader, according to the manufacturer's recommendations. Percent cell viability was calculated using the following formula: Cell viability (%) = [(OD of untreated compounds - cells treated with compound) / (OD of cells not treated with compound)] x100. 50% cytotoxicity concentration (CC50 ) was defined as the cytotoxic concentration of each compound that reduced the absorbance of treated cells to 50% compared to the absorption of untreated cells.

Antiviral effects of 5-ALA

Confluent monolayers of fcwf-4 cells were cultured in or without compound media at the indicated concentrations in 24-well multi-plate plates at 37 ° C for 24 or 48 hours. The cells were washed and the virus (MOI 0.01) was adsorbed into the cells at 37 ° C for 1 hour. After washing, cells were cultured in 1.5% carboxymethylcellulose (CMC) -MEM or MEM with or without compounds. For cells cultured in CMC-MEM, cell monolayers were incubated at 37 ° C for 48 hours, fixed and stained with 1% crystal violet solution containing 10% buffered formalin, and then the resulting plaques were counted. The percentage of decrease or increase in plaques was calculated using the following formula: Percent reduction of plaques (%) = [(number of plaques of compounds treated with compound) / (number of plaques of cells not treated with compound)] x100. EC50 was defined as the effective concentration of compounds that reduced the virus titer in the culture supernatant of infected cells to 50% compared to the virus control titer. For cells cultured in MEM, culture supernatants were collected 48 hours after infection and virus titers were measured by TCID assay.50.

Primary feline macrophages were cultured in medium with or without compounds at the indicated concentrations in 24-well multiple plates at 37 ° C for 48 hours. After washing with PBS, FIPV 79-1146 (1 x 104 TCID50) adsorbed into cells at 37 ° C with 5% CO2 for 1 hour. After washing with PBS, the cells were cultured in medium and the supernatants were collected. Virus titers were measured using the TCID assay50.

Statistical analysis

Data from only two groups were analyzed using Student's t-test (Welch t-test) and data from multiple groups were analyzed by one-way ANOVA followed by Tukey's test. A P value <0.05 was considered significant.

The results

Cytotoxic and antiviral effects of 5-ALA

A cytotoxicity assay was performed to determine the non-toxic concentration of 5-ALA against fcwf-4 cells (Figure 1). More than 75% fcwf-4 cells survived in the presence of 1,000 μM 5-ALA (maximum concentration in this experiment). The vehicle does not show any cytotoxic effects on fcwf-4 cells.

Figure 1.

Cytotoxic effects of 5-ALA on fcwf-4 cells. The viability of fcwf-4 cells was measured using the WST-8 assay. Black ring: 5-ALA. White circle: Vehicle (SFC). The vehicle (control solvent) was the same as the vehicle in the 5-ALA solution at each serial dilution. Results are presented as mean ± SE. The data represent three independent experiments (n = 3).

Effects of 5-ALA on FIPV infection in a feline cell line

The antiviral effects of 5-ALA against FIPV were evaluated in a plaque inhibition assay in fcwf-4 cells. Cells were treated with 5-ALA as follows: 24-hour pre-treatment (pre-24h), 24-hour pre-treatment followed by 49-hour FIPV treatment (pre-24h and co-49h) and 48-hour pre-treatment, after which followed by a 49-hour FIPV treatment (pre-48h and co-49h). At pre-24h, the percent plaque inhibition increased significantly at 500 μM or higher (Figures 2A, B). At pre-24h and co-49h, the percentage of plaque inhibition increased significantly at 125μM or higher (Figures 2C, D). At pre-48h and co-49h, the percentage of plaque inhibition in FIPV type I reached 125 μM 5-ALA 75% (Figures 2E, F). The control vehicle, SFC, showed no plaque inhibitory effects on FCoV under any conditions. According to the titration assay, FIPV type I and type II production was significantly reduced at 250 and 500 μM 5-ALA (Figure 3).

Figure 2.
FIPV plaque inhibition assay in 5-ALA-treated fcwf-4 cells. (A, B) Effects of 24-hour pretreatment on 5-ALA antiviral activity. Plaque inhibition rate of FIPV-infected fcwf-4 cells pretreated with 5-ALA for 24 hours. (C, D) Effects of 24-hour pre-treatment and 48-hour post-treatment on 5-ALA antiviral activity. Plaque inhibition rate of FIPV-infected fcwf-4 cells pre-treated for 24 hours and post-treated for 48 hours with 5-ALA. (E, F) Effects of 48-hour pre-treatment and 48-hour post-treatment on 5-ALA antiviral activity. Plaque inhibition rate of FIPV-infected fcwf-4 cells pretreated for 48 hours followed by 5-ALA for 48 hours. (A, C, E) type I FIPV. (B, D, F) type II FIPV. Black column: 5-ALA. White column: Vehicle (control solvent). Results are presented as mean ± SE. The data represent four independent experiments (n = 4). ** p <0.01 (* p <0.05) vs. vehicle.
Figure 3.
Inhibition of FIPV 5-ALA infection in fcwf-4 cells. (A, C) Effects of 48-hour pre-treatment and 48-hour post-treatment on antiviral activity of 250 μM 5-ALA. (B, D) Effects of 48-hour pre-treatment and 48-hour post-treatment on the antiviral activity of 500 μM 5-ALA. (A, B) 250 μM 5-ALA. (A, B) type I FIPV. (C, D) type II FIPV. Black ring: 5-ALA. White circle: vehicle (control solvent). Results are presented as mean ± SE. The data represent four independent experiments (n = 4). ** p <0.01 (* p <0.05) vs. vehicle.

Effects of 5-ALA on FIPV infection in a feline cell line

FIPV-infected macrophages are involved in the progression of FIP symptoms to severe. We investigated whether 5-ALA inhibits FIPV proliferation in macrophages. In this experiment, FIPV 79-1146 type II with a high ability to reproduce in primary cat macrophages was used. Virus production in FIPV-infected macrophages was reduced at 250 μM 5-ALA in three of the four cats (Figure 4).

Figure 4.
Inhibition of FIPV infection in macrophages. Effects on 5-ALA antiviral activity (250μM) in primary cat macrophages. Black column: 5-ALA. White column: Vehicle (control solvent). The data represent three independent experiments (n = 3).

Discussion

5-ALA is an intermediate in the synthesis of tetrapyrrole in animals, plants and microorganisms (13-15). The potential for 5-ALA efficacy in plants was reported in the 1980s (18), but it was difficult to produce 5-ALA in sufficient quantities for practical use because only a small amount is produced in microorganisms. After Nishikawa et al. introduced the method of mass production of 5-ALA using bacteria (19), the effectiveness of 5-ALA has been confirmed not only in agriculture but also in the medical and biological fields. 5-ALA is an inexpensive drug and is practically used as a supplement to improve animal performance and immune response in veterinary medicine (14, 20).

5-ALA inhibited FIPV growth in fcwf-4 cells. The metal complexes of the 5-ALA metabolite, protoporphyrin IX (PpIX), were found to have antiviral activity (21-23). The PpIX metal complex, heme, inhibits the multiplication of dengue virus (21). On the other hand, the multiplication of Zika virus is not inhibited by heme (23). It is not clear whether heme inhibits FCoV proliferation. Generally, an increase in intracellular heme stimulates the production of hemeoxygenase-1 (HO-1), a heme degrading enzyme. HO-1 has been reported to induce antiviral activity (24, 25). However, in a preliminary experiment, we confirmed that the expression level of HO-1 mRNA in cells treated with 250μM 5-ALA remained unchanged (data not shown). Based on this finding, 5-ALA-induced inhibition of FIPV infection occurs due to a factor other than heme and HO-1.

There are already many studies on therapeutic drugs in FIP. Many drugs effective in FIP have been identified in vitro, and some have been shown to have therapeutic effects when administered to cats with FIP (10-12). However, the effects of all drugs were weak in cases with neurological manifestations. Poor transmission of these drugs to the central nervous system was thought to be the cause; therefore, there is a need for a drug that has antiviral effects against FIPV capable of entering brain tissue. 5-ALA is an amino acid with low molecular weight and possible good penetration ability into brain tissue (26). It is further reported that the diffusion of 5-ALA from the blood into normal brain tissue is very low (27), suggesting that it has fewer adverse effects. FIP can only be unambiguously diagnosed by detecting the FCoV antigen in the lesion (28). However, when treatment is not started until a clear diagnosis has been made, the symptoms have meanwhile progressed to a state where, in many cases, there is no longer a response to treatment. Therefore, if there is a drug that can be given prophylactically before the diagnosis of FIP, it is possible to prevent the progression of symptoms, for which 5-ALA may be an ideal means. However, in our experiment with FIPV target cells, macrophages, the antiviral effects of 5-ALA were not observed in some cats. Therefore, when 5-ALA is used as a therapeutic drug for FIP, anti-FCoV drugs such as GS-441524 (29), GC-376 (30), U18666A (31) and itraconazole (32) or anti-inflammatory drugs such as anti-TNF-alpha antibody (33) should be used concomitantly.

FECV transmission mainly occurs in cats in the field, while horizontal FIPV infection between cats is considered rare (34). FIPV has been found to be generated by a genetic mutation of FECV. Thus, if there are means to prevent FECV infection on a daily basis, the development of FIP can be prevented. No vaccine has been developed to prevent FECV infection. Addie et al. reported that viral gene secretion in faeces ceased in FECV-infected cats treated with a synthetic adenosine analog (35). Therefore, elimination of FECV infecting the gut is expected by administering FECV antivirals to infected cats. However, synthetic adenosine analogs can induce coronavirus gene mutations (36). In addition, although the level of the FECV gene in feces has fallen below the limit of detection in cats treated with a synthetic adenosine analog, it is possible that FECV has latently infected the gut or other tissues. Long-term administration of a synthetic adenosine analog is required to prevent FECV gene mutation and reliably eliminate FECV infection, but this is not realistic due to its high cost. On the other hand, 5-ALA is practically used as a dietary supplement. 5-ALA has low toxicity to animals and plants, which strongly suggests the possibility of long-term administration of 5-ALA to cats. In the future, it is necessary to examine whether 5-ALA is useful as an adjunct to prevent the development of FIP in cats infected with FECV.

In this study, we confirmed the possibility that 5-ALA inhibits FIPV proliferation and TNF-alpha production. Because 5-ALA is an amino acid present in the body, immediate administration is possible. However, it is necessary to administer 5-ALA to cats with FIP and to monitor whether therapeutic effects can be achieved. It is further investigated whether long-term administration of 5-ALA eliminates the virus and inhibits FIP development in FECV-infected cats.

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