| Chinese soft-shelled turtle (Trionyx sinensis) is a very important species in aquaculture due to its rich nutritional and medicinal value. However, intensive farming has led to frequent outbreaks of bacterial and viral diseases, causing significant economic losses to the turtle farming industry. Viral parotitis is one of the more harmful viral diseases for T. sinensis in recent years. Its pathogen is the T. sinensis hemorrhagic syndrome virus (TSHSV). TSHSV is an enveloped single-stranded RNA virus with a diameter of 60-80 nm and shares homology with arteriviruses. The main clinical symptoms of TSHSV infection include multi-organ hemorrhage, with a high mortality rate. However, there are currently no effective treatments for TSHSV. Since the discovery of TSHSV virus, some researches have conducted preliminary explorations of the virus' invasion mechanism and host resistance mechanism using lung tissue transcriptomics as well as liver proteomics. Notably, after viral infection, the antiviral genes rsad2 and mx2 mRNA are significantly upregulated, indicating that rsad2 and mx2 are involved in the antiviral immune response of T. sinensis. Studies have shown that vaccines have achieved outstanding results in preventing viral diseases in aquatic animals. Unfortunately, TSHSV exhibits antibody-dependent enhancement (ADE), where the stimulation by polyclonal antibodies significantly upregulates the antiviral genes rsad2 and mx2, as well as the virus copy number. Therefore, there is an urgent need to find other effective technical approaches to prevent TSHSV.
The Chinese herbal medicines, due to their safety, efficacy, minimal toxic side effects, low risk of resistance development, and reduced drug residues, meet the requirements of harmless aquaculture. Additionally, they are beneficial to consumer health, thus gaining widespread application in the prevention and treatment of diseases in aquatic animals. Echinacea purpurea is one of the popular herbs in the international herbal market, known for its antibacterial, antiviral, anti-inflammatory, and immunomodulatory effects. Among its components, polysaccharides are considered to be one of the main active ingredients responsible for Echinacea purpurea's medicinal properties. Luetlig et al. discovered that different concentrations of Echinacea purpurea polysaccharide (EPP) can stimulate macrophages to release tumor necrosis factor α (tnf-α) and interferon β (ifn-β), thereby enhancing immune activity. It is noteworthy that existing studies have also found that EPP can alleviate viral infections both in vitro and in vivo. For example, in vivo experiments showed that EPP significantly inhibited lesions caused by influenza A virus infection in mice. In vitro experiments demonstrated that L926 cells treated with EPP exhibited resistance to herpes simplex virus (HSV-I) infection. However, there have been no reports on the protective effects of EPP against aquatic viruses. Currently, it is unclear whether EPP has a preventive effect on TSHSV, and further research is needed. Therefore, this study aim to evaluated the antiviral and immunomodulatory effects of EPP on TSHSV infection in T. sinensis through hematoxylin-eosin (HE) staining, qRT-PCR, and tissue immunofluorescence. This can provide a essential basis for the prevention and treatment of TSHSV.
In this study, we assessed the protective effect of EPP against TSHSV infection in T. sinensis through histopathology, qRT-PCR, and immunofluorescence techniques. For histopathological examination, tissues fixed with 4% paraformaldehyde were embedded in paraffin and sectioned at 6 μm for HE staining. The histopathological features, particularly the inflammatory response, of EPP-treated and untreated T. sinensis were observed and compared. To understand the effect of EPP on viral replication, qRT-PCR was used to detect the relative expression levels of TSHSV in different tissues across various groups. Additionally, immunofluorescence analysis was conducted to assess the accumulation of TSHSV in tissues. Fixed tissues were sectioned and embedded, followed by immunofluorescence detection using an anti-A661 polyclonal antibody as the primary antibody and a FITC-labeled goat anti-rabbit antibody as the secondary antibody to capture the enrichment of the virus. To understand the immunomodulatory effects of EPP on TSHSV infection in soft-shelled T. sinensis, the mRNA expression levels of immune-related genes rsad2 and usp18 were measured by qRT-PCR.
The results show that the mortality rate of infected T. sinensis treated with 500 mg/kg EPP was lower than those of control group. This indicates that EPP has potential protective effects in reducing the mortality rate of infected T. sinensis. When dissecting the T. sinensis three days after TSHSV infection, when they began to exhibit signs and symptoms such as difficulty breathing and neck weakness, we observed swollen lung tissue, severe congestion, obvious intestinal bleeding, and swelling without perforation. In contrast, the intestines of infected T. sinensis gavaged with EPP showed a significant improvement in congestion compared to those not gavaged with EPP. Histopathological analysis revealed that compared to the TSHSV-infected group, tissue damage was mitigated in the EPP-treated group. The livers of TSHSV-infected T. sinensis were characterized by swollen cells and condensed nuclei. There was significant blood stasis within the hepatic sinusoids. Extensive inflammatory cell infiltration was observed around the bile ducts. In the spleens of TSHSV-infected T. sinensis, lymph nodes exhibited severe atrophy, necrosis, and disintegration. In contrast, the atrophic state of the lymph nodes in the spleens of infected T. sinensis treated with EPP showed improvement. Compared to the kidney tissue of EPP-treated infected T. sinensis, kidney cells in TSHSV-infected T. sinensis exhibited karyopyknosis and karrhexis. Renal tubules were atrophied, renal corpuscles were hypertrophied and dilated, leading to increased interstitial space. Necrosis was present at sites of renal hemorrhage. Compared to the lung tissue of EPP-treated infected T. sinensis, TSHSV-infected lung tissue displayed significant structural disorder. Alveolar cells were characterized by karyorrhexis. Acute necrotizing bronchiolitis accompanied by inflammatory cell infiltration and extensive fibrin within the alveolar spaces was observed. The lung tissue showed obvious acute hemorrhage with erythrocyte extravasation. Immunofluorescence analysis of viral protein expression in various tissues revealed that on the third day of infection, EPP could suppress viral protein levels in the liver, spleen, lung, and heart. Viral load assay results showed that EPP significantly reduced the viral load in the spleen and lung. qRT-PCR was used to detect the expression of antiviral-related genes. The results showed that on the third day of infection, EPP reduced the mRNA expression of rsad2 and usp18 in various tissues of the T. sinensis, particularly in the liver, where the difference compared to the infected group was significant.
In summary, EPP has a certain protective effect on T. sinensis infected with TSHSV. EPP can effectively inhibit TSHSV proliferation and modulate the immune function of T. sinensis, showing potential as a means to prevent TSHSV. Further understanding the impact of EPP treatment on the molecular mechanisms of TSHSV will provide a basis for developing drugs to prevent TSHSV. |
