Antiviral and virucidal activities of Lucilia cuprina maggots' excretion/secretion (Diptera: Calliphoridae): first work.

Maggots of Lucilia sericata and L. cuprina are a backbone of the maggot debridement therapy. Further, the excretion/secretion (E/S) of these maggots has antibacterial and antifungal activities, nevertheless the antiviral activity of E/S for these maggots still out the focus. This study aimed to evaluate the E/S of L. cuprina maggots against the Rift Valley Fever (RVF) and Coxsackie B4 (CB4) viruses for first time. After collection of the E/S, its cytotoxicity on Vero cells was evaluated and the safe concentration was determined which used to investigate the antiviral and virucidal effect of E/S on the selected viruses. The E/S decreased the titers of the tested viruses compared with that of untreated viruses. The outcome data refer to that the E/S of L. cuprina consider as a promising antiviral and virucidal agent.

Introduction

Maggot debridement therapy (MDT) is an expression referring to the treatment of wounds by using of maggots (Larvae of flies). The surgeon Ambroise Paré was the first person noted the beneficial effects of maggots on the soldiers’ wounds on the battlefield of Saint Quentin (1557) (Goldstein, 1931). The maggots of Lucilia sericata and L. cuprina are considered as a core of the MDT. The process of MDT done through three stages: debridement (Cleaning the necrotic tissues), disinfection (Antimicrobial effects) and stimulation of wound healing (Erdmann and Khalil, 1986; Chan et al., 2007; Sherman, 2014).

Excretion/secretion (E/S) produced by maggots possesses proteolytic enzymes and contains collagenase, allantoïn, sulfhydryl radicals, serine proteases, calcium, cysteine, glutathione, growth stimulating factors, leucine aminopeptidase, carboxypeptidases A & B and embryonic growth stimulating substance (Vistnes et al., 1981; Casu et al., 1994; Chambers et al., 2003; Nigam et al., 2006; Gupta, 2008). These proteolytic enzymes can dissolve the necrotic and infected tissues on the wound.

Despite the significance of medical uses of L. sericata and L. cuprina maggots or their E/S, the antiviral activity of E/S for these maggots still out the focus despite viruses such as Rift Valley fever virus, Dengue virus, Coxsackie virus…etc. represent dangerous pathogens worldwide.

According to World Health Organization (World Health Organization, 2018), severe form of RVF in humans was occurred in many countries such as; Republic of Niger (2016), Republic of Mauritania (2012), Republic of South Africa (2010), Madagascar (2008–2009), Sudan (2007), Kenya, Somalia & Tanzania (2006), Egypt (2003) and Saudi Arabia and Yemen (2000). The availability and abundance of the potential vectors, suitability of environmental conditions, importation of livestock's from infected countries and the close association of susceptible domestic animals with humans, the RVF virus could possibly occur and circulate (Kenawy et al., 2018).

In this study, depending on the significant effects of maggots' E/S against bacteria, fungi and Leishmania, the effect of Lucilia cuprina maggots’ E/S on Rift Valley Fever (RVF) and Coxsackie B4 (CB4) viruses was investigated for first time.

Materials and methods

Collection of E/S

The 3rd larval instar of Lucilia cuprina maggots was collected from maintained culture at the Laboratory of Medical Entomology, Animal House, Faculty of Science, Al-Azhar University. The collected maggots were washed with ethanol 70 % followed by sterile distilled water and then incubated with phosphate buffered saline (PBS) for 6 h with ratio 100 maggots/200 μL PBS at 25 °C in darkness. After that the E/S was collected and centrifuged at 10000 rpm for 10 min at 4 °C. The supernatant was sterilized by filtration through a 0.2 μm Millipore bacterial filter and stored at -20 °C.

Cells and viruses

Vero cells of adult African green monkey kidney (American tissue culture collection, USA) were supplied by Tissue Culture Department, VACSERA. The Vero cells were maintained in MEME media supplemented with fetal calf serum (10 %), penicillin (100 μg/ml) and streptomycin 10 (10 μg/ml) in CO2 incubator at 37 °C. Pantropic Rift Valley fever (RVF) virus Menya strain [Menya/Sheep/258] and Coxsackie B4 virus (CB4) were used to investigate the antiviral and virucidal activities of E/S on Vero cells using cytopathic effect inhibition assay. The viruses kindly provided by laboratory of Virology at VACSERA Company, Dokki, Giza.

Cell viability

Cytotoxicity of the E/S was assessed on Vero cells by the MTT assay (Bahuguna et al., 2017). Briefly; the Vero cells were seeded in a 96-well microtiter plate and incubated for 24 h at 37 °C in a 5 % CO2 incubator. The cells were divided in to sets, one treated with two-fold serial dilution from the E/S which diluted with culture media, while in other set, the culture media were added to cells and considered as control. The cells were re-incubated for 24 h in the same conditions. After that, MTT was added (10 μl/well) and incubated for 4 h at 37 °C in a CO2 incubator. Then DMSO (50 μl/well) was added for 5 min at 37 °C in a CO2 incubator. Finally, the optical density was measured with ELISA reader at 570 nm for treated cells at each dilution and cells without treatment as control. Cell viability % was calculated by the following equation:Cell viability % = (mean OD of tested cells/mean OD of control cells) ×100where OD is optical density.

Antiviral activity assay

Vero cells were seeded in tissue culture plate 96-well and divided into 2 sets. The 1st set was treated with the safe concentration of E/S (the safe concentration was adjusted by dilute E/S with culture media) as treaded group, while the other set was treated with the free media of E/S as control group. Both groups were incubated at 37 °C for 24 h. After incubation, the media were discarded, and D-PBS was used for washing. The virus (multiplicity of infection (MOI) = 0.1) was added into both sets to obtain the control and treated groups. After that, both groups were incubated at 37 °C for 24 h. Titers of control and treated RVF and CB4 viruses were determined according to the standard TCID50 method (Reed and Muench, 1938).

Virucidal activity assay

Virucidal activity of E/S against RVF and CB4 viruses (MOI = 0.1) was conducted on monolayer of Vero cells. The virus was mixed with equal volume from the safe concentration of E/S and incubated at 37 °C for 1, 3, 6 and 24 h to obtain treated group. The negative control group was obtained by mixing the virus with equal volume from the E/S’ free media and incubated at 37 °C for the same time intervals. Both groups were added into previously cultured Vero cells and incubated at 37 °C for 24 h. Titers of control and treated viruses were determined according to the standard TCID50 method (Reed and Muench, 1938).

Statistical analysis

Data were expressed as the mean ± standard error of the mean (S.E.M). The significance of differences between the values of control and treated groups was assessed using a two-tailed independent Student's t-tests and performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA. Statistically significant was set at P ≤ 0.05.

Results

Regarding the Cytotoxicity evaluation of the E/S, the viability of Vero cells is inversely proportional with the E/S concentration. The concentration of E/S (100 %) induced a decrease in cell viability and showed approximately 30 % cell viability. While concentrations of 50, 25, 12.5, 6.25 and 3.13 % showed 34, 48, 78, 99 and 100 % cell viability, respectively (Fig. 1). The safe concentration of the E/S, which used in the antiviral and virucidal investigations, was considered as 5 %.

Fig. 1

Cytotoxicity of the Lucilia cuprina maggots' excretion/secretion on the Vero cells.

Titers of control viruses

Infectivity titers of control RVF and CB4 viruses were 5.8 and 4.7 log (10)/0.1 ml, respectively.

Antiviral activity

Safe concentration of E/S (5 %) significantly reduced the infectivity titers of RVF and CB4 viruses. This concentration significantly reduced the infectivity titer of RVF virus to 5.5 log (10)/0.1 ml (P < 0.05) and CB4 virus to 3.5 log (10)/0.1 ml (P < 0.01) as shown in Fig. 2. The reduction percent was approximately 5 % and 26 % for RVF and CB4 viruses, respectively.

Fig. 2

Antiviral activity of Lucilia cuprina maggots' excretion/secretion against Rift Valley Fever and Coxsackie B4 viruses. The statistical significance is analyzed by independent t-test (*P < 0.05 and **P < 0.01).

Virucidal activity

The E/S (5 %) showed a significant reduction in infectivity titer of RVF virus. The virus titer became 4.3 log (10)/0.1 ml (P < 0.01) after incubation with the concentration of E/S for 1 h. While the titer of RVF virus was reduced to 3.8, 3.3 and 2.5 log (10)/0.1 ml (P < 0.001) after incubation with E/S concentration for 3, 6 and 24 h, respectively (Fig. 3). Actually, the reduction percent of infectivity titer was approximately 26, 35, 44 and 57% for 1, 3, 6 and 24 h, respectively.

Fig. 3

Virucidal activity of Lucilia cuprina maggots' excretion/secretion against Rift Valley Fever and Coxsackie B4 viruses, CPE = Cytopathic effect. The statistical significance is analyzed by independent t-test (*P < 0.05, **P < 0.01 and ***P < 0.001).

In the same time, the infectivity titer of CB4 virus was significantly reduced after intervals times’ incubation with the 5 % E/S (P < 0.001). After 1 h incubation, the infectivity titer was 2.25 log (10)/0.1 ml with reduction nearly 53 %, while, it became 1.75 and 1.25 log (10)/0.1 ml with reduction nearly 63% and 74% after incubation for 3 and 6 h, respectively. The infectivity titer disappeared after incubation for 24 h, where, no cytopathic effect (CPE) was detected (Fig. 3).

Discussion

Globally, the insects and their extracts play a great role in the traditional medicine, where, natural components from insect bodies, secretions and excretions showed an activity against bacteria, fungi, viruses and parasites (Wachinger et al., 1998; De Clercq, 2000; Muharsini et al., 2000; Meylaers et al., 2004; Huberman et al., 2007; Slocinska et al., 2008; Meenakshisundaram et al., 2009; Lu et al., 2014; Coelho et al., 2015; Faruck et al., 2016). Broad spectrum antimicrobial peptides produced from most insects during innate immune responses and have a great antimicrobial activity (Bulet and Stocklin, 2005; Poppel et al., 2015; Xu et al., 2006).

Maggot therapy, whether the uses of Lucilia sericata and/or L. cuprina maggots itself or its excretion/secretion, is considered as a modern technique in managements of the wound and infection. The effect of E/S of L. cuprina maggots gave antimicrobial effects, where, the E/S of L. sericata and/or L. cuprina maggots have antibacterial activity (Bexfield et al., 2004; Kerridge et al., 2005; van der Plas et al., 2007; Jiang et al., 2012; Valachova et al., 2014; Hassan et al., 2016), antifungal activity (Poppel et al., 2014; Hassan et al., 2016) and anti-leishmanial activity (Polat et al., 2012; Sanei-Dehkordi et al., 2016; Laverde-Paz et al., 2018).

The relationship between cell viability and concentration E/S of L. cuprina in this study is similar with the findings of Sanei-Dehkordi et al. (2016), where the cell viability decreased with increasing the concentration of E/S from L. sericata.

In this study, RVF virus was affected by the E/S of L.cuprina maggots which showed antiviral and virucidal activities, where, the virus titer was decreased. Also, the same finding was observed with CB4 virus. The antiviral and virucidal effects of E/S might be due to the presence of some constituent compounds such as serine proteases, glutathione and cysteine. As published in literatures, these compounds have remarkable effects on some viruses; serine proteases limit the Dengue virus type 2 infection (Brackney et al., 2008). While, glutathione inhibits the viral production of dengue virus infection (Tian et al., 2010) and may be has a value in human immunodeficiency virus (HIV) treatment (Kalebic et al., 1991). In addition, cysteine derivative inhibits the H5N1 replication (Geiler et al., 2010).

Conclusion

In conclusion, the effect of Lucilia cuprina maggots’ excretion/secretion (E/S) on viruses was investigated for first time. The E/S of L. cuprina maggots reduced the infectivity titer of Rift Valley fever (RVF) and Coxsackie B4 (CB4) viruses. Therefore, this study recommended that E/S of L. cuprina maggots may serve as additional therapeutic options affecting on viral diseases. Future studies should focus on the role of E/S of L. cuprina maggots in the treatment of viral diseases and mode of action in vitro and in vivo levels.

Declarations

Author contribution statement

Mohammad R. K. Abdel-Samad: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Competing interest statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.