Expression profiles of immune mediators in feline infectious peritonitis virus infected cells, whole blood and peritoneal effusion fluids

Feline infectious peritonitis virus (FIPV) is the causative agent of the one of the most lethal viral diseases of the wild and domestic cats. Despite of the research on the virus and the disease it causes, the molecular pathogenesis of feline infectious peritonitis (FIP) is poorly understood. In this study, in vitro samples from FIPV infected Crandell Ress Feline Kidney (CRFK) cells and in vivo samples obtained from FIP diagnosed cats were used in an attempt to identify the involvement of different immune mediators and their associations with viral replication. Viral load in vitro showed peak at 48 hours post infection (hpi), while the increased in viral load is associated with increased in the expression of immune mediators such as MX1, RSAD2 (viperin), MCP2 (CCL8) and CXCL10 (IP10). However, most of the FIP diagnosed cats did not express or expressed very low levels of MCP2 (CCL8) and CXCL10 (IP10) in the peripheral blood mononuclear cells (PBMC). Analysis based on MILIPLEX assay detected an increased in proinflammatory related cytokines namely RANTES (CCL5), KC (CXCL1), MCP1 (CCL2) and IL8 (CXCL8) in FIPV infected CRFK cells. The increased in these immune mediators were also detected in the clinical samples such as PBMC, serum, peritoneal effusion (PE) and the supernatant of PE (PES) of cats diagnosed with FIP. However, the PE samples tend to have higher viral load with distinct expression profiles of the different immune mediators compared to the blood samples of the FIP diagnosed cats. In addition, the detection of CCL17 expression in PE but not in PBMC. No obvious variations in the expression profiles of the different immune mediators were detected among the different forms of FIP, however, the wet and mixed forms of FIP tend to have generally higher immune mediator expressions compared to dry form. In addition, the differences in expression profiles of MX1 and RSAD2 in PBMC may serve as a good indicator in distinguishing wet and dry form of FIP. Hierarchical clustering analysis based on in vivo samples indicated that MX1, CCL17 and GM-CSF have the highest correlation with viral load. In addition, the different expression profiles of cytokines such as IL1β, IL6, IL18 and TNFα between blood and PE samples, and the down regulation of SCF and Flt3L expressions in the blood samples were also detected in some of the FIP diagnosed cats. In conclusion, this study has established some insight on the differential expressions of immune mediators in FIPV infected cells and in cats diagnosed with FIP

In vitro and in vivo mechanism of immunomodulatory and antiviral activity of Edible Bird's Nest (EBN) against influenza A virus (IAV) infection

Ethnopharmacological relevance: For centuries, Edible Bird Nest (EBN) has been used in treatment of variety of respiratory diseases such as flu and cough as a Chinese natural medicine. Aim of the study: This natural remedy showed the potential to inhibit influenza A virus (IAV). However, little is known about the mechanism of this process and also the evaluation of this product in an animal model. Hence, the current study was designed to elucidate the antiviral and immunomodulatory effects of EBN against IAV strain A/Puerto Rico/8/1934 (H1N1). Materials and methods: First, influenza infected MDCK cells treated with EBNs from two locations of Malaysia (Teluk Intan and Gua Madai) that prepared with different enzymatic preparations were analyzed by RT-qPCR and ELISA for detection of viral and cytokines genes. The sialic acid composition of these EBNs was evaluated by H-NMR. Subsequently, after toxicity evaluation of EBN from Teluk Intan, antiviral and immunomodulatory effects of this natural product was evaluated in BALB/c mice by analysis of the viral NA gene and cytokine expressions in the first week of the infection. Results: EBN showed high neuraminidase inhibitory properties in both in vitro and in vivo, which was as effective as Oseltamivir phosphate. In addition, EBN decreased NS1 copy number (p<0.05) of the virus along with high immunomodulatory effects against IAV. Some of the immune changes during treatment of IAV with EBN included significant increase in IFNγ, TNFα, NFκB, IL2, some proinflammatory cytokines like IL1β, IL6, and cytokines with regulatory properties like IL10, IL27, IL12, CCL2 and IL4 depends on the stage of the infection. EBNs from two locations contained different composition of sialic acid and thymol derivatives, which gave them different antiviral properties. EBN from Gua Madai that contained more acetylated sialic acid (Neu2,4,7,8,9 Ac6) showed higher antiviral activity. Conclusion: The findings of this study support the antiviral activity of EBN against influenza virus and validate the traditional usage of this natural remedy by elucidation of toxicity and the molecular mechanism of action

Edible bird nest (EBN) can inhibit influenza A virus by affecting autophagy pathway

Influenza A virus (IAV) is a causative agent of many worldwide epidemics with high mortality and morbidity that cause tremendous economy costs annually. This virus will invade the host cells through endocytic pathways and cause various changes in intracellular pathways. It has been seen that even a pathway like autophagy is involved in this virus life cycle to promote the virus assembly. This virus prompts the accumulation of autophagosomes by blocking the fusion of the lysosomes. Hence, this pathway can be a great target for antiviral agents. Hence, the aim of this study was to highlight inhibitory effects of Edible Bird Nest (EBN) extract against IAV infection and understand some of the molecular mechanism of action. Consequently, we have investigated the effects of four different enzyme treated EBNs against IAV and some autophagy markers. At first, the Influenza A virus (strain A/Puerto Rico/8/1934 H1N1) infected cells were treated with different concentration of these EBNs to determine the IC50 of these extracts. Afterwards, western blotting technique has been used to evaluate the autophagosome marker protein microtubule-associated protein light chain 3-II (LC3-II) in different treatments. In addition, the lysosomal activity has been determined by staining the cells with Lysotracker Red DND-99. The results demonstrated that EBN extracts in combined treatments with influenza A viruses significantly reduced the virus titer and increased the cell viability especially in post-penetration treatments (P<0.05) with IC50 range from 2.6 to 4.9 mg/ml depends on the preparation and type. Regarding the authophagy pathway, the amount of LC3-II has been significantly decreased after treatment of the infected cells with EBNs (P<0.05), which caused increasing of the lysosome activity. Accordingly, this study revealed that EBN can be an efficient antiviral agent and also showed the involvement of autophagy pathway in antiviral activity of EBN averse to influenza A virus

Induction of a robust immune response against avian influenza virus following transdermal inoculation with H5-DNA vaccine formulated in modified dendrimer-based delivery system in mouse model

This study was aimed to evaluate the immunogenicity of recombinant plasmid deoxyribonucleic acid (DNA), pBud-H5-green fluorescent protein (GFP)-interferon-regulatory factor (IRF)3 following delivery using polyamidoamine (PAMAM) dendrimer and transactivator of transcription (TAT)-conjugated PAMAM dendrimer as well as the effect of IRF3 as the genetic adjuvant. BALB/c mice were vaccinated transdermally with pBud-H5-GFP, PAMAM/pBud-H5-GFP, TAT-PAMAM/pBud-H5-GFP, and TAT-PAMAM/pBud-H5-GFP-IRF3. The expression analysis of H5 gene from the blood by using quantitative real-time reverse transcriptase polymerase chain reaction confirmed the ability of PAMAM dendrimer as a carrier for gene delivery, as well as the ability of TAT peptide to enhance the delivery efficiency of PAMAM dendrimer. Mice immunized with modified PAMAM by TAT peptide showed higher hemagglutination inhibition titer, and larger CD3+/CD4+ T cells and CD3+/CD8+ T cells population, as well as the production of cytokines, namely, interferon (IFN)-γ, interleukin (IL)-2, IL-15, IL-12, IL-6, and tumor necrosis factor-α compared with those immunized with native PAMAM. These results suggest that the function of TAT peptide as a cell-penetrating peptide is able to enhance the gene delivery, which results in rapid distribution of H5 in the tissues of the immunized mice. Furthermore, pBud-H5-GFP co-expressing IRF3 as a genetic adjuvant demonstrated the highest hemagglutination inhibition titer besides larger CD3+/CD4+ and CD3+/CD8+ T cells population, and strong Th1-like cytokine responses among all the systems tested. In conclusion, TAT-PAMAM dendrimer-based delivery system with IRF3 as a genetic adjuvant is an attractive transdermal DNA vaccine delivery system utilized to evaluate the efficacy of the developed DNA vaccine in inducing protection during challenge with virulent H5N1 virus