The study of anti‐rabies virus effect of Shougong powder

Rabies is a lethal infectious disease caused by rabies virus (RABV). The mortality rate is very high after the appearance of clinical symptoms, with a survival rate of almost 0%. There is presently no cure for rabies. In the present study, we investigated whether the extract of Shougong powder—a calcium powder prepared from gecko that has demonstrated immunomodulatory properties in mice—is an effective treatment for rabies. The antiviral effects of the extract were evaluated both in vitro and in vivo with the cytotoxicity and antiviral assays and by immunofluorescence analysis, quantitative real‐time (qRT)‐PCR, and western blotting. The results showed that Shougong powder and its extract increased survival rate in RABV‐infected mice is up to 60% and 50%, respectively, even in 20 times of LD50, whereas the control groups treated with isoprinosine (IPS) or saline are only 20% and 0% survival (p = .011). qRT‐PCR and western blotting analyses showed that the extract strongly inhibited viral mRNA expression and protein synthesis in vitro: expression of the N, P, M, G, and L genes of RABV was decreased by 28.8%–45.0% in the IPS group (p < .05) and by 50.1%–59.0% in the extract group (p < .05) relative to the control group. These results demonstrate that Shougong powder has certain antiviral effects against RABV and can potentially be used for the treatment for rabies.


INTRODUCTION
Viral diseases such as COVID-19, 1 rabies, 2,3 and Ebola 4 have become major threats to human health and there are at present no effective drugs for their treatment.Rabies is a highly lethal zoonosis caused by the rabies virus (RABV) that mainly occurs in developing countries, ranking third or fourth among infectious diseases in terms of global prevalence.RABV is usually transmitted from free-range animals and stray dogs and targets the central nervous system, causing encephalitis in humans and other mammals.It is estimated that about 59,000 people die each year from rabies globally 5 and over 15 million receive postexposure prophylaxis, with children accounting for 40% of cases. 6Rabies infection from transplanted organs has also been reported. 7Once neurologic symptoms appear, the mortality rate is almost 100%. 8ABV is a single-stranded negative-chain RNA virus.The 12-kb genome encodes five structural proteins including nucleoprotein (N), matrix protein (M), phosphoprotein (P), glycoprotein (G), and RNA-dependent RNA polymerase (L).Of these, G protein is the main antigen inducing the innate immune response in the host upon viral infection.There are several reports of cases that have survived RABV infection following treatment with the Milwaukee protocol; however, this was accompanied by severe neurologic sequelae. 9As such, there is a need for alternative treatment strategies for rabies.It was reported that arabinoside combined with interferon (IFN) has been effective in the treatment of patients with advanced rabies. 10Recently, studies involving photothermal therapy and photothermally triggered immunological effects provide a new potential solution for RABV treatment. 11ecko, also known as Shougong in China, is a small lizard found in warm regions 12 that is used in traditional Chinese medicine.The clinical efficacy of Shougong powder for the treatment of a variety of malignant tumors has been reported 13 ; moreover, it is less toxic and has fewer side effects than conventional chemotherapeutic drugs. 14The proprietary Chinese medicine Jinlong Gum capsules prepared from gecko is used for the treatment of colorectal, esophageal, gastric, and lung cancers 15 ; the combination of Xiaojie powder and Yiqi Sanjie soup containing Pihu is used for the treatment of esophageal cancer 16 ; and the combination of gecko and scorpion is used for the treatment of neck tumors. 17Thus, Shougong powder has both antitumor and antiviral effects.
In the present study, we investigated whether the extract of Shougong and Shougong powder-a calcium powder prepared from Japanese gecko (Gekko japonicas) that has demonstrated immunomodulatory properties in mice-is effective for rabies treatment using in vitro and in vivo models.

Animals
Female Kunming mice (specific pathogen-free) weighing 18-22 g were obtained from Changchun Institute of Biological Products (Changchun, China).The mice were handled in strict accordance with the standards for experimental animals set by the National Deployment Laboratory Animal Management Committee, and were allowed a 3-day acclimatization period in the laboratory before they were used for experiments.

Extract of Shougong
Shougong powder was purified by boiling in water for 30 min, treating with ethanol, and dialyzing against water.
The dialysate was subjected to ultrafiltration and then freeze-dried.The purified powder was dissolved in sterile water.isoprinosine (IPS) was purchased from Toronto Research Chemicals.Both the extract and IPS were dissolved in dimethyl sulfoxide.

Cytotoxicity assay
BHK-21 cells were seeded at a density of 2 × 10 5 cells/mL in 96-well plates and incubated at 37

Antiviral assay
BHK-21 cells were seeded at a density of 2 × 10 5 cells/mL in 96-well plates and incubated as described above, and then infected with CVS-11 (multiplicity of infection [MOI] = 0.1) at 37

Immunofluorescence analysis
BHK-21 cells were seeded at a density of 2 × 10 5 cells/mL in 96-well plates.The following day, the cells were infected with CVS-11 at MOI = 0.1 and incubated at 37 • C for 1 h.After washing with PBS, different concentrations of the extract (0, 2.74, and 5.47 mg) or IPS (0, 0.5, and 1 mM) were added.The cells were cultured at 37 • C and 5% CO 2 for 48 h and the medium was discarded.The cells were fixed with 50 μL/well of 80% cold acetone at −20 • C for 1 h, washed three times with PBS, and incubated at 37 • C for 1 h with 50 μL fluorescein isothiocyanate (FITC)-conjugated anti-RABV immunoglobulin diluted with PBS.After three washes with PBS containing 0.1% Tween-20, 90% glycerin buffer was added to the cells at 50 μL/well.The fluorescent signal was observed and photographed under a fluorescence microscope (IX73; Olympus).

Quantitative real-time-PCR
BHK-21 cells were seeded at a density of 2 × 10 5 cells/mL in 12-well plates and infected with CVS-11.The culture medium was replaced with fresh medium containing var- ious concentrations of the extract and incubated for 48 h.Total RNA was extracted from cells using TRIzol reagent (Takara Bio).Quantitative real-time (qRT)-PCR was performed with the Mx3000P quantitative (Q)-PCR system (Agilent-Stratagene) according to a previously described protocol. 18The primers used are listed in Table 1.The thermal cycling conditions were as follows: denaturation at 95 • C for 30 s, followed by 40 cycles of 95 • C for 5 s and 60 • C for 1 min.Melting curve analysis was performed to verify the specificity of amplification.Relative quantification of target genes was performed with the 2 −∆∆Ct method. 19Each reaction was prepared in triplicate, and the experiment was repeated at least three times.

Western blotting
BHK-21 cells were seeded at a density of 2 × 10 5 cells/mL in six-well plates and infected with CVS-11 for 1 h.The culture medium was replaced with fresh medium containing various concentrations of the extract, followed by incubation for 48 h.Total protein was extracted from the cells using radioimmunoprecipitation assay lysis buffer containing phenylmethylsulfonyl fluoride, and protein concentration was determined with the BCA Protein Quantification Assay Kit (Beyotime).Western blotting was performed as previously described. 19Protein band intensity was determined using an imaging system with Quantity One software (Bio-Rad) and was normalized to that of αtubulin.Polyclonal antibodies against CVS-11 G, N, P, and M proteins were produced in our laboratory.Rabbit polyclonal α-tubulin antibody (AF0001), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) (A0216), and HRP-conjugated goat anti-rabbit IgG (H+L) (A0208) were from Beyotime.
F I G U R E 1 Experimental scheme for evaluating the antirabies effect of Shougong powder and extract of Shougong in rabies virus (RABV)-infected mice.

Treatment of RABV-infected mice with Shougong powder and the extract
The protocol for animal experiments was reviewed and approved by the Ethics Committee of Animal Experiments at Shanghai Center for Clinical Laboratory.The experimental scheme is shown in Figure 1.Mice were randomly divided into eight groups (n = 10); four groups were intracerebral injection challenged with a 20 times of lethal dose of CVS-11 (20× LD 50 ) and were treated 1 h later with saline (control), Shougong powder, the extract (8.75 mg/day), or IPS (1000 mg/kg) by intramuscular injection into the hind legs once a day.The feeding group was given processed rat food (equivalent to 1 g of purified powder) daily for 11 consecutive days.The other four groups were used as healthy controls and treated in an identical manner but without virus infection.All animals were observed daily for 21 days.In the experiments, the brains of dead mice were taken for immunohistochemical analysis of monoclonal anti RABV nuclear protein antibodies labeled with FITC.Fluorescence signals were observed under a fluorescence microscope and photographed.The tail vein blood of surviving mice was collected after the experiments.FAVN (fluorescent antibody virus neutralization test) was utilized to detect neutralizing antibody levels in the serum.

Statistical analysis
Data are expressed as mean ± SEM.Significant differences between groups were evaluated by one-way analysis of variance followed by the Bonferroni F test, or with the Student's t-test or chi-squared test.p < .05 was considered statistically significant.

Cytotoxicity of the extract of Shougong powder
The toxicity of IPS and the extract in BHK-21 cells was evaluated with the MTS assay.The optical density of each sample was measured and used to calculate survival rate relative to untreated control cells.Cell proliferation was inhibited in a time-and dose-dependent manner by treatment with 10.94 mg the extract and 2 mM IPS (Figure 2A).We selected 5.47 mg/mL the extract and 1 mM IPS as working concentrations for subsequent experiments as they had no significant effects on cell proliferation.In the virus titer detection experiment, the drug concentration was used as the horizontal coordinate and the virus titer (Log1 0TCID 50/mL) was plotted as the longitudinal coordinate.The results are shown in Figure 2B.Different concentrations of Shougong powder extract and IPS treatment on BHK-21 cells infected with CVS-11 showed inhibition of virus titer to a certain extent.

Antiviral effects of the extract in vitro
The antiviral effect of the extract in BHK-21 cells infected with CVS-11 was evaluated by immunofluorescence analysis, qRT-PCR, and western blotting.Treatment of infected cells with the extract or IPS resulted in a concentrationdependent reduction of the green fluorescent signal (Figure 3A), indicating that viral replication was suppressed.Additionally, the extract and IPS reduced the expression of CVS-11 N, P, M, G, and L genes in a concentration-dependent manner by 28.8%-45.0%(p < .05)and 50.1%-59.0%(p < .05),respectively, relative to the control group, with 12.2%-34.0%(p < .05)lower transcript levels in the extract group compared to the IPS group (Figure 3B).Similar trends were observed for M, N, G, and P protein levels (Figure 3C).These results indicate that like IPS, the extract inhibits both RABV gene expression and protein synthesis.

Antiviral effects of Shougong in vivo
Mice in the four experimental groups infected with CVS-11 showed significant weight loss from days 5 to 9, which was accompanied by loss of limb coordination, trembling, paralysis, and death (Figure 4).These symptoms were alle-viated in mice treated with the extract or IPS and in the feeding groups as compared to untreated control mice.Thus, the extract and IPS alleviate weight loss and clinical symptoms associated with RABV infection.The four groups of mice that were not infected with CVS-11 remained in good condition and gained weight normally, with all but four mice in the IPS group surviving until the end of the experiment; this suggested that Shougong powder and the extract were less toxic to mice than IPS.In contrast, among the four groups of mice that were infected with CVS-11, all mice from the saline group died on day 8 of the experiment, while the number of surviving mice in the feeding, the extract, and IPS groups was 6, 5, and 2, respectively, at the end of the experiment.The survival rate in mice treated with the extract was 50% as compared to the rates of 20% in the IPS group and 60% in the feeding group.The chi-squared test showed that the survival rates of the feeding and the extract groups were significantly different from that of the control group (p = .011and .033,respectively), with a higher rate in the former.The results of nucleic acid gel electrophoresis and immunohistochemical analyses showed that mice from the four experimental groups infected with CVS-11 all died from rabies, whereas no CVS-11 was detected in uninfected mice from the IPS group (20% mortality).Immunofluorescence staining was performed on the brain tissue of dead mice in the experiment, and the results were observed under fluorescence microscope.Some experimental results are presented in Figure 5, and specific fluorescent antibody staining reac-tions were observed in the hippocampus, cortex, and other parts.FAVN was used for detection and the results showed that the neutralizing antibody in the serum of surviving mice turned positive and reached a protective level (>0.5 International Unit (IU)).

DISCUSSION
The earliest record of rabies in China is in the book Zuo Zhuan, which dates back at least 2500 years.Despite the longstanding awareness of this disease, there is no effective cure.Previous studies have identified a variety of compounds that show inhibitory effects on RABV including ribavirin, IFN-α, ketamine, amantadine 20 and new antiviral agents in development like immunotherapeutics in the form of monoclonal antibodies targeting specific RABV proteins [21][22][23] but their therapeutic benefits are limited and require further research. 24rmaseptins secreted by amphibian skin have been shown to inhibit RABV in vitro and enhance survival in infected mice. 25IPS inhibits RABV in vitro and is used as a positive control when screening anti-RABV drugs. 26PS at a dose of 500-1000 mg/kg increased survival in mice by 20%-30% 18 ; we therefore used an IPS dose of 1000 mg/kg in our study.However, the mortality rate of 20% in mice treated with IPS indicates a certain degree of toxicity.Shougong is a type of terrestrial reptile belonging to the Gekkonidae family that has the capacity for tissue regeneration and adhesion to surfaces.27,28 In the ancient Chinese books Compendium of Materia Medica and Peaceful Holy Benevolent Prescription, Shougong powder is recommended for the treatment of apoplexy.29 Our results demonstrate that Shougong powder and its extract have inhibitory effects on RABV that are superior to those of IPS in mice, and that it can alleviate clinical symptoms while reducing mortality from rabies. Th safety of the powder and the extract was highlighted by our observation that neither was toxic in control mice.
In summary, the results of our study indicate that Shougong powder can improve survival in mice infected with RABV by inhibiting viral replication, while having minimal toxicity.These findings provide evidence for the therapeutic value of Shougong powder for the treatment of rabies, although additional studies are needed to elucidate the mechanistic basis for its effects.

A C K N O W L E D G M E N T S
The authors thank the following individuals from Military Veterinary Institute, Academy of Military Medical Sciences.Professor Rongliang Hu and Professor Shoufeng Zhang provided the laboratory that we used to conduct our experiments.Teng Chen and Lijuan Mi provided technical support for western blotting.Jinjin Yang provided technical support in the animal experiments.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The datasets supporting the conclusions of this article are included within the article and its additional files and will be freely available to any scientist wishing to use them for non-commercial purposes upon request via e-mail with the corresponding author.

TA B L E 1
Sequences and the sizes of forward (F) and reverse (R) primers of target genes for quantitative real-time (qRT)-PCR.

F I G U R E 2
Cytotoxicity of isoprinosine (IPS) and Shougong extract at different concentrations in BHK-21 cells.**p < .01versus control; ##p < .01extract of Shougong versus IPS.(A) The cytotoxicity of IPS and extract of Shougong treated BHK-21 cells with different concentration.(B) Anti-rabies virus (anti-RABV) effect of IPS and extract of Shougong.

F I G U R E 4 F I G U R E 5
Anti-rabies virus (anti-RABV) effect of Shougong in mice.Survival was significantly longer in the feeding and extract of Shougong groups than in the control group (p = .011and .033,respectively).Brain smear of infected mice.