Fuzhengjiedu San inhibits porcine reproductive and respiratory syndrome virus by activating the PI3K/AKT pathway.

BACKGROUND: Traditional Chinese medicine (TCM) has been garnering ever-increasing worldwide attention as the herbal extracts and formulas prove to have potency against disease. Fuzhengjiedu San (FZJDS), has been extensively used to treat viral diseases in pigs, but its bioactive components and therapeutic mechanisms remain unclear. METHODS: In this study, we conducted an integrative approach of network pharmacology and experimental study to elucidate the mechanisms underlying FZJDS's action in treating porcine reproductive and respiratory syndrome virus (PRRSV). We constructed PPI network and screened the core targets according to their degree of value. GO and KEGG enrichment analyses were also carried out to identify relevant pathways. Lastly, qRT-PCR, flow cytometry and western blotting were used to determine the effects of FZJDS on core gene expression in PRRSV-infected monkey kidney (MARC-145) cells to further expand the results of network pharmacological analysis. RESULTS: Network pharmacology data revealed that quercetin, kaempferol, and luteolin were the main active compounds of FZJDS. The phosphatidylinositol-3-kinase (PI3K)/Akt pathway was deemed the cellular target as it has been shown to participate most in PRRSV replication and other PRRSV-related functions. Analysis by qRT-PCR and western blotting demonstrated that FZJDS significantly reduced the expression of P65, JNK, TLR4, N protein, Bax and IĸBa in MARC-145 cells, and increased the expression of Bcl-2, consistent with network pharmacology results. This study provides that FZJDS has significant antiviral activity through its effects on the PI3K/AKT signaling pathway. CONCLUSION: We conclude that FZJDS is a promising candidate herbal formulation for treating PRRSV and deserves further investigation.

Ursonic acid from medicinal herbs inhibits PRRSV replication through activation of the innate immune response by targeting the phosphatase PTPN1.

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), has caused substantial economic losses to the global swine industry due to the lack of effective commercial vaccines and drugs. There is an urgent need to develop alternative strategies for PRRS prevention and control, such as antiviral drugs. In this study, we identified ursonic acid (UNA), a natural pentacyclic triterpenoid from medicinal herbs, as a novel drug with anti-PRRSV activity in vitro. Mechanistically, a time-of-addition assay revealed that UNA inhibited PRRSV replication when it was added before, at the same time as, and after PRRSV infection was induced. Compound target prediction and molecular docking analysis suggested that UNA interacts with the active pocket of PTPN1, which was further confirmed by a target protein interference assay and phosphatase activity assay. Furthermore, UNA inhibited PRRSV replication by targeting PTPN1, which inhibited IFN-ß production. In addition, UNA displayed antiviral activity against porcine epidemic diarrhoea virus (PEDV) and Seneca virus A (SVA) replication in vitro. These findings will be helpful for developing novel prophylactic and therapeutic agents against PRRS and other swine virus infections.

In vitro and in vivo antiviral effects of CLEVir-X against porcine reproductive and respiratory syndrome virus.

The aim of this study was to investigate the in vitro and in vivo antiviral effects of CLEVir-X, against porcine reproductive and respiratory syndrome virus (PRRSV). CLEVir-X is a nucleoside analogue and a dialdehyde form of xanthosine. CLEVir-X demonstrated antiviral action during the in vitro portion of this experiment with its inosine monophosphate dehydrogenase (IMPDH) inhibition against PRRSV. The anti-PRRSV effect of CLEVir-X was recovered through supplementation with guanosine. This suggests that PRRSV replication may be regulated through IMPDH and its guanosine biosynthetic pathway. CLEVir-X treatment in cultures resulted in mutation frequency increase of up to 7.8-fold within the viral genomes (e.g. ORF6) compared to their parallel, untreated cultures. The incorporation of CLEVir-X into the viral genome causes lethal mutagenesis and subsequent decrease in specific infectivity. During the in vivo antiviral experiment, 21-day-old pigs began oral administration of 5 mL of phosphate buffered saline containing CLEVir-X (with purity of 68 % and dosage of 40 mg/kg body weight). This treatment was provided twice daily at 9:00AM and 5:00PM for 14 days. Pigs were simultaneously intranasally inoculated with PRRSV at the beginning of CLEVir-X treatment (21 days of age). Several beneficial effects from the oral administration of CLEVir-X were observed including reduction of body temperature, alleviation of respiratory clinical signs, decreased PRRSV load in both blood and lung tissues, and mitigation of lung interstitial pneumonia lesions. The results of the present study demonstrated that CLEVir-X has mutagenic and nonmutagenic modes of antiviral action against PRRSV based on both in vitro and in vivo antiviral experiments.

The Antimalaria Drug Artesunate Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by Activating AMPK and Nrf2/HO-1 Signaling Pathways.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide. Currently, vaccine strategies provide limited protection against PRRSV transmission, and no effective drug is commercially available. Therefore, there is an urgent need to develop novel antiviral strategies to prevent PRRSV pandemics. This study showed that artesunate (AS), one of the antimalarial drugs, potently suppressed PRRSV replication in Marc-145 cells and ex vivo primary porcine alveolar macrophages (PAMs) at micromolar concentrations. Furthermore, we demonstrated that this suppression was closely associated with AS-activated AMPK (energy homeostasis) and Nrf2/HO-1 (inflammation) signaling pathways. AS treatment promoted p-AMPK, Nrf2, and HO-1 expression and, thus, inhibited PRRSV replication in Marc-145 and PAM cells in a time- and dose-dependent manner. These effects of AS were reversed when the AMPK or HO-1 gene was silenced by short interfering RNA. In addition, we demonstrated that AMPK works upstream of Nrf2/HO-1, as its activation by AS is AMPK dependent. Adenosine phosphate analysis showed that AS activates AMPK via improving the AMP/ADP-to-ATP ratio rather than direct interaction with AMPK. Altogether, our findings indicate that AS is a promising novel therapeutic for controlling PRRSV and that its anti-PRRSV mechanism, which involves the functional link between energy homeostasis and inflammation suppression pathways, may provide opportunities for developing novel antiviral agents. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) infections have continuously threatened the pork industry worldwide. Vaccination strategies provide very limited protection against PRRSV infection, and no effective drug is commercially available. We show that artesunate (AS), one of the antimalarial drugs, is a potent inhibitor against PRRSV replication in Marc-145 cells and ex vivo primary porcine alveolar macrophages (PAMs). Furthermore, we demonstrate that AS inhibits PRRSV replication via activation of AMPK-dependent Nrf2/HO-1 signaling pathways, revealing a novel link between energy homeostasis (AMPK) and inflammation suppression (Nrf2/HO-1) during viral infection. Therefore, we believe that AS may be a promising novel therapeutics for controlling PRRSV, and its anti-PRRSV mechanism may provide a strategy to develop novel antiviral agents.

Inhibition of endocytosis of porcine reproductive and respiratory syndrome virus by rottlerin and its potential prophylactic administration in piglets.

Owing to several limitations of porcine reproductive and respiratory syndrome virus (PRRSV) control procedures, the importance of antiviral agents is increasing; however, limited studies have been done on the development of anti-PRRSV agents. Herein, we explored the antiviral effect and mechanism of rottlerin against PRRSV. We demonstrated that treatment of rottlerin at an early stage of PRRSV infection significantly inhibited the viral replication. PRRSV infection induced protein kinase C-δ phosphorylation, which was specifically downregulated by rottlerin. The treatment of rottlerin led to disrupting the PRRSV entry pathway by blocking endocytosis of the virions. Further, to evaluate the anti-PRRSV effect of the rottlerin in vivo, we administrated rottlerin loaded liposome to pigs infected with PRRSV LMY or FL12 strain. The treatment of rottlerin-liposome reduced the blood viral load, interstitial pneumonia and clinical scores compared to untreated pigs. These results provide an evidence of anti-PRRSV effect of rottlerin in vitro via inhibiting PRRSV internalization and in vivo, all of which strongly suggest the applicability of rottlerin as a potential PRRSV prophylactic treatment.

Emodin from Aloe Inhibits Porcine Reproductive and Respiratory Syndrome Virus via Toll-Like Receptor 3 Activation.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe reproductive failure in sows and respiratory diseases in growing and finishing pigs and results in great economic losses to the swine industry. Although vaccines are available, PRRSV remains a major threat to the pig farms. Thus, there is an urgent need to develop antiviral drugs to compensate for vaccines. In this study, we report that Aloe extract (Ae) can strongly inhibit PRRSV in Marc-145 cells and porcine alveolar macrophages lines (iPAMs) in vitro. Furthermore, we identified a novel anti-PRRSV molecule, Emodin, from Ae by high-performance liquid chromatography (HPLC). Emodin exerted its inhibitory effect through targeting the whole stages of PRRSV infectious cycle. Moreover, we also found that Emodin can inactivate PRRSV particles directly. Notably, we confirmed that Emodin was able to significantly induce Toll-like receptor 3 (TLR3) (p < 0.01), IFN-α (p < 0.05) and IFN-ß expression in iPAMs, indicating that induction of antiviral agents via TLR3 activation by Emodin might contribute to its anti-PRRSV effect. These findings imply that the Emodin from Aloe could hamper the proliferation of PRRSV in vitro and might constitute a new approach for treating PRRSV infection.

Effects of glycyrrhizin on the growth cycle and ATPase activity of PRRSV-2-infected MARC-145 cells.

Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV) and is devastating the swine industry. MARC-145 cells, an African green monkey kidney cell line, are sensitive to PRRSV-2, and are often used for in vitro studies on PRRSV-2. Preliminary research has shown that glycyrrhizin, an important active component extracted from traditional Chinese medicinal licorice, significantly inhibits the proliferation of PRRSV-2 in MARC-145 cells; however, the in-depth molecular mechanism remains unclear. By determining the cell growth cycle, this study found that PRRSV-2 infection first increased the content of G1-phase MARC-145 cells and then decreased the content of G1-phase cells. Moreover, glycyrrhizin affected the role of PRRSV-2 in regulating the cell cycle. Furthermore, PRRSV-2 had the highest proliferation titer in G0/G1-phase MARC-145 cells, and glycyrrhizin reduced the content of PRRSV-2 in synchronized MARC-145 cells. According to the results of ATPase detection, PRRSV-2 infection weakened the Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities in MARC-145 cells, while glycyrrhizin significantly enhanced their activities in PRRSV-2-infected MARC-145 cells. The above results provide theoretical support toward clarifying the mechanism by which glycyrrhizin inhibits the proliferation of PRRSV-2 in MARC-145 cells. Moreover, these results offer references for the development and use of glycyrrhizin and the clinical treatment of PRRSV-2 infection.

Mannosylated gelatin nanoparticles enhanced inactivated PRRSV targeting dendritic cells and increased T cell immunity.

The objective of the present work was to evaluate the efficacy of a novel antigen carrier using mannosylated gelatin nanoparticles with entrapped inactivated porcine reproductive and respiratory syndrome virus (PRRSV) in inducing T cell mediated immunity in vitro. Gelatin nanoparticles (GNP) were modified with mannose to form mannosylated gelatin nanoparticles (MnGNP), which can efficiently and specifically target monocyte derived dendritic cells (MoDCs). The inactivated PRRSV was encapsulated in the MnGNP and GNP, referred to as MnGNP-PRRSV and GNP-PRRSV, respectively. All these prepared nanometer particles were characterized for size, surface charge, drug encapsulation efficiency, and drug release. The efficacy of MnGNP in targeting MoDCs was investigated, as well as the subsequent MoDCs maturation and T cell mediated cytotoxicity. The developed MnGNP-PRRSV particle was characterized with a nanometric size of 302.67 ±â€¯3.2 nm, surface charge of 23.81 ±â€¯1.26 mV, and PRRSV encapsulation efficiency of 63.2 ±â€¯1.85 %. The maximum uptake of MnGNP in MoDCs in vitro was 15.5 times higher than GNP with a shorter reaction time that peaked 4 h earlier. The uptake of MnGNP-PRRSV induced maturation of MoDCs and significantly enhanced expression of SWC-3a, CD80, CD1, SLA I, SLA II on MoDCs, compared to PRRSV (p < 0.001). The cytokine secretion of IL-1ß, IL-6, IL-10, and IL-12 was also increased in MoDCs when treated with MnGNP-PRRSV, compared to PRRSV (p < 0.05). The matured MoDCs triggered T lymphocytes in autologous peripheral blood mononuclear cells (PBMCs) activation, proliferation, and differentiation into effector cytotoxic T lymphocyte, suggesting increased amount of activated T cells after MnGNP-PRRSV treatment. Additionally, the function of T cells to kill PRRSV infected cells was 83.98 ±â€¯2.62 % when triggered by MnGNP-PRRSV, compared to 60 ±â€¯4.7 % in PRRSV group (p < 0.001). These results indicate that MnGNP with entrapped inactivated PRRSV can effectively and specifically target dendritic cells for maturation and activation, and subsequently improve T cell activation, proliferation and function to kill PRRSV infected cells.

Rutin, α-tocopherol, and l-ascorbic acid up-regulate type I interferon-regulated gene and type I and II interferon expressions and reduce inflammatory cytokine expressions in monocyte-derived macrophages infected with highly pathogenic porcine reproductive and respiratory syndrome virus.

This study evaluated the immunomodulatory effect of two types of phytochemicals, i.e. rutin and ß-carotene, and two types of vitamins, i.e. α-tocopherol and l-ascorbic acid on improving innate immune responses to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Monocyte-derived macrophages (MDM) from eight PRRSV-seronegative pigs were inoculated with HP-PRRSV and subsequently stimulated with rutin, ß-carotene, α-tocopherol, and l-ascorbic acid in the absence or presence of either polyinosinic:polycytidylic acid or lipopolysaccharide. The mRNA expression levels of myxovirus resistance 1, interferon regulatory factor 3 (IRF3), IRF7, 2'-5'-oligoadenylatesynthetase 1, stimulator of interferon genes (STING), osteopontin (OPN), interferon alpha (IFNα), IFNß, IFNγ, interleukin-10 (IL-10), tumor necrosis factor alpha (TNFα), and transforming growth factor beta (TGFß) were evaluated by real-time PCR. Compared with control MDM, HP-PRRSV significantly suppressed mRNA expressions of all immune-related genes except IL-10 and TGFß. Compared with HP-PRRSV-inoculated MDM, stimulation with rutin, α-tocopherol, and l-ascorbic acid, but not ß-carotene significantly enhanced mRNA expression levels of IRF3, IRF7, STING, OPN, IFNα, IFNß, and IFNγ in HP-PRRSV-inoculated MDM. Stimulation with rutin also significantly reduced mRNA expression levels of TNFα and TGFß, whereas stimulation with ß-carotene and α-tocopherol significantly reduced TNFα mRNA expression in HP-PRRSV-inoculated MDM. Our findings demonstrate the potentials of rutin, α-tocopherol, and l-ascorbic acid in enhancing type I interferon-regulated genes and type I and II IFN expressions, and in reducing pro- and/or anti-inflammatory cytokine expressions in HP-PRRSV-inoculated MDM. Our findings suggest that rutin, α-tocopherol, and l-ascorbic acid may serve as effective immunomodulators for improving innate immune response to HP-PRRSV.

Inhibition of porcine reproductive and respiratory syndrome virus by PKC inhibitor dequalinium chloride in vitro.

As a severe disease characterized by reproductive failure and respiratory distress, porcine reproductive and respiratory syndrome (PRRS) is one of the most leading threats to the swine industry worldwide. Highly evolving porcine reproductive and respiratory syndrome virus (PRRSV) strains with distinct genetic diversity make the current vaccination strategy much less cost-effective and thus urge alternative protective host directed therapeutic approaches. RACK1-PKC-NF-κB signalling axis was suggested as a potential therapeutic target for PRRS control, therefore we tested the inhibitory effect of PKC inhibitor dequalinium chloride (DECA) on the PRRSV infection in vitro. RT-qPCR, western blot, Co-IP and cytopathic effect (CPE) observations revealed that DECA suppressed PRRSV infection and protected Marc-145 cells and porcine alveolar macrophages (PAMs) from severe cytopathic effects, by repressing the PKCα expression, the interaction between RACK1 and PKCα, and subsequently the NF-κB activation. In conclusion, the data presented in this study shed more light on deeper understanding of the molecular pathogenesis upon PRRSV infection and more importantly suggested DECA as a potential promising drug candidate for PRRS control.

Porcine interferon lambda 3 (IFN-λ3) shows potent anti-PRRSV activity in primary porcine alveolar macrophages (PAMs).

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious viral disease of swine. At present, there are vaccines for the control of PRRSV infection, but the effect is not satisfactory. The recombination of attenuated vaccines causes significant difficulties with the prevention and control of PRRSV. Type III interferons (IFNs), also called IFN-λs, were newly identified and showed potent antiviral activity within the mucosal surface and immune organs. RESULTS: Therefore, primary porcine alveolar macrophages (PAMs) were used for this investigation. To this end, we found that the replication of PRRSV in PAMs was significantly reduced after pre-treatment with IFN-λ3, and such inhibition was dose- and time-dependent. The plaque formation of PRRSV abrogated entirely, and virus yields were reduced by four orders of magnitude when the primary PAMs were treated with IFN-λ3 at 1000 ng/ml. In addition, IFN-λ3 in our study was able to induce the expression of interferon-stimulated genes 15 (ISG15), 2'-5'-oligoadenylate synthase 1 (OAS1), IFN-inducible transmembrane 3 (IFITM3), and myxoma resistance protein 1(Mx1) in primary PAMs. CONCLUSIONS: IFN-λ3 had antiviral activity against PRRSV and can stimulate the expression of pivotal interferon-stimulated genes (ISGs), i.e., ISG15, Mx1, OAS1, and IFITM3. So, IFN-λ3 may serve as a useful antiviral agent.

Expression Profile of Porcine TRIM26 and Its Inhibitory Effect on Interferon-ß Production and Antiviral Response.

TRIM26, a member of the tripartite motif (TRIM) family has been shown to be involved in modulation of innate antiviral response. However, the functional characteristics of porcine TRIM26 (porTRIM26) are unclear. In this study, we used a synthesized antigen peptide to generate a polyclonal antibody against porTRIM26 with which to study the expression and function of porTRIM26. We demonstrated that polyinosinic:polycytidylic acid (poly (I:C)) stimulation and viral infection (vesicular stomatitis (VSV) or porcine reproductive and respiratory syndrome virus (PRRSV)) induce expression of porTRIM26, whereas knock-down expression of porTRIM26 promotes interferon (IFN)- production after poly (I:C) stimulation and virus infection (VSV or PRRSV). The importance of the porTRIM26-mediated modulation of the antiviral response was also shown in VSV- or PRRSV-infected cells. In summary, these findings show that porTRIM26 has an inhibitory role in IFN- expression and the antiviral response.

Antiviral activity of a novel mixture of natural antimicrobials, in vitro, and in a chicken infection model in vivo.

The aim of this study was to test in vitro the ability of a mixture of citrus extract, maltodextrin, sodium chloride, lactic acid and citric acid (AuraShield L) to inhibit the virulence of infectious bronchitis, Newcastle disease, avian influenza, porcine reproductive and respiratory syndrome (PRRS) and bovine coronavirus viruses. Secondly, in vivo, we have investigated its efficacy against infectious bronchitis using a broiler infection model. In vitro, these antimicrobials had expressed antiviral activity against all five viruses through all phases of the infection process of the host cells. In vivo, the antimicrobial mixture reduced the virus load in the tracheal and lung tissue and significantly reduced the clinical signs of infection and the mortality rate in the experimental group E2 receiving AuraShield L. All these effects were accompanied by a significant reduction in the levels of pro-inflammatory cytokines and an increase in IgA levels and short chain fatty acids (SCFAs) in both trachea and lungs. Our study demonstrated that mixtures of natural antimicrobials, such AuraShield L, can prevent in vitro viral infection of cell cultures. Secondly, in vivo, the efficiency of vaccination was improved by preventing secondary viral infections through a mechanism involving significant increases in SCFA production and increased IgA levels. As a consequence the clinical signs of secondary infections were significantly reduced resulting in recovered production performance and lower mortality rates in the experimental group E2.

Cryptotanshinone protects porcine alveolar macrophages from infection with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV) infection, imposes enormous economic impact to the world pork industry. Currently there is no effective treatment to prevent PRRSV infection in swine. We report that the natural compound cryptotanshinone (Cpt) effectively inhibits the infection of various strains of PRRSV to porcine alveolar macrophages (PAMs), the primary cell target of PRRSV in vivo. Mechanistically, Cpt inhibits the activation of signal transducer and activator of transcription 3 (STAT3), and blocks the interleukin 10 (IL-10) stimulated as well as the basal level CD163 expression in PAMs. Cpt-treatment of PAMs is effective when applied either before or after PRRSV infection, with the combined pre- and post-PRRSV infection treatment resulting in the most significant, dose-dependent inhibition of PRRSV infection. Cpt inhibited both type I/II PRRSV infection in PAMs. Our study identified a new approach to prevent/treat PRRSV infection of pigs with natural compounds.

Small molecules block the interaction between porcine reproductive and respiratory syndrome virus and CD163 receptor and the infection of pig cells.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the pork industry globally. PRRS is caused by PRRS virus (PRRSV). Currently there are no effective treatments against this swine disease. METHODS: Through artificial intelligence molecular screening, we obtained a set of small molecule compounds predicted to target the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163, which is a cell surface receptor specific for PRRSV infection. These compounds were screened using a cell-based bimolecular fluorescence complementation (BiFC) assay, and the function of positive hit was further evaluated and validated by PRRSV-infection assay using porcine alveolar macrophages (PAMs). RESULTS: Using the BiFC assay, we identified one compound with previously unverified function, 4-Fluoro-2-methyl-N-[3-(3-morpholin-4-ylsulfonylanilino)quinoxalin-2-yl]benzenesulfonamide (designated here as B7), that significantly inhibits the interaction between the PRRSV glycoprotein (GP2a or GP4) and the CD163-SRCR5 domain. We further demonstrated that compound B7 inhibits PRRSV infection of PAMs, the primary target of PRRSV in a dose-dependent manner. B7 significantly inhibited the infection caused by both type I and type II PRRSV strains. Further comparison and functional evaluation of chemical compounds structurally related to B7 revealed that the 3-(morpholinosulfonyl)aniline moiety of B7 or the 3-(piperidinylsulfonyl)aniline moiety in a B7 analogue is important for the inhibitory function against PRRSV infection. CONCLUSIONS: Our study identified a novel strategy to potentially prevent PRRSV infection in pigs by blocking the PRRSV-CD163 interaction with small molecules.

In vitro screening antiviral activity of Thai medicinal plants against porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) results in economic losses in the swine industry globally. Several studies have investigated the use of plant extracts in the prevention and control of PRRS outbreaks. Thai medicinal plants may be useful for treating PRRSV infection in pigs. Therefore, we investigated the in vitro anti-PRRSV and antioxidant properties of seven Thai medicinal plants: Caesalpinia sappan Linn., Garcinia mangostana Linn., Houttuynia cordata, Perilla frutescens, Clinacanthus nutans, Phyllanthus emblica, and Tiliacora triandra. RESULTS: Using antiviral screening, we observed that T. triandra extract strongly inhibited PRRSV infectivity in MARC-145 cells [virus titer 3.5 median tissue culture infective dose (TCID50)/ml (log10)] at 24 h post-infection, whereas C. sappan extract strongly inhibited PRRSV replication [virus titer 2.5 TCID50/ml (log10)] at 72 h post-infection. C. sappan extract had the highest total phenolic content [220.52 mM gallic acid equivalent/g] and lowest half-maximal inhibitory concentration [1.17 mg/ml in 2,2-diphenyl-1-picrylhydrazyl and 2.58 mg/ml in 2,2-azino-bis (3-ethylbenzothiazo-line-6-sulfonic acid) diammonium salt]. CONCLUSION: T. triandra extract could inhibit PRRSV infectivity, whereas C. sappan extract was the most effective in inhibiting PRRSV replication in MARC-145 cells. This study elucidates the antiviral activities of Thai medicinal plant extracts in vivo. The results promise that Thai medicinal plant extracts, particularly T. triandra and C. sappan extracts, can be developed into pharmaceutical drugs for the prevention of PRRS in pigs.

Yansuanmalingua inhibits replication of type 2 porcine reproductive and respiratory syndrome virus via activating the caspase-8 apoptosis pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry. The present study showed that Yansuanmalingua (YASML) can inhibit type 2 PRRSV replication using plaque assay, quantitative reverse transcriptase-polymerase chain reaction, and immunofluorescence assay. Furthermore, inhibition of PRRSV replication was shown to be related to Toll-like receptor 3 (TLR3)-dependent apoptosis-induction by YASML in the PRRSV-infected MARC-145, and TLR3-dependent apoptosis-induction by YASML was found to suppress PRRSV replication via the activation of caspase-8 and caspase-3 pathways, respectively. Meanwhile, activation of the caspase-3 pathway seemed to be related to the downregulation of myeloid cell leukemia 1 (Mcl-1) expression. Our results showed that YASML-induced TLR3-dependent apoptosis could be blocked by a pan-caspase inhibitor and small interfering RNA against TLR3. In conclusion, the present study demonstrates that YASML exerts its anti-PRRSV effect by activating the caspase-8/caspase-3 signaling pathway and by negatively regulating Mcl-1 expression. These findings not only provide new insights into the molecular mechanism of YASML inhibition of PRRSV replication via the TLR3-dependent apoptosis pathway but also suggest potential, new antiviral drugs by expressing caspase-3 or down expressing Mcl-1.

Inactivation of airborne porcine reproductive and respiratory syndrome virus (PRRSv) by a packed bed dielectric barrier discharge non-thermal plasma.

Porcine reproductive and respiratory syndrome virus (PRRSv) is one of the most significant airborne viruses impacting the pork industry in the US. Non-thermal plasmas (NTPs) are electrical discharges comprised of reactive radicals and excited species that inactivate viruses and bacteria. Our previous experiments using a packed bed NTP reactor demonstrated effective inactivation of bacteriophage MS2 as a function of applied voltage and power. The present study examined the effectiveness of the same reactor in inactivating aerosolized PRRSv. A PRRSv solution containing ∼105 TCID50/ml of PRRSv VR2332 strain was aerosolized at 3 ml/min by an air-jet nebulizer and introduced into 5 or 12 cfm air flow followed by NTP exposure in the reactor. Twin impingers upstream and downstream of the reactor collected samples of the virus-laden air flow for subsequent TCID50 assay and qPCR analyses. An optical particle sizer measured upstream and downstream aerosol size distributions, giving estimates of aerosol filtration by the reactor. The results showed that PRRSv was inactivated to a similar degree as MS2 at the same conditions, with the maximum 1.3-log inactivation of PRRSv achieved at 20 kV and 12 cfm air flow rate. The results demonstrate the potential of properly optimized NTPs in controlling PRRSv transmission.

Novel Lentivirus-Based Method for Rapid Selection of Inhibitory Nanobody against PRRSV.

The emergence and re-emergence of porcine reproductive and respiratory syndrome virus (PRRSV) has resulted in huge economic losses for the swine industry. Current vaccines are of limited efficacy against endemic circulating PRRSV variants. New strategies against PRRSV infection are in urgent need. Here, a nanobody library in Marc-145 cells is constructed for antiviral nanobodies. Nanobody encoding sequences from two non-immunized llamas were cloned to generate a pseudotyped lentiviral library. Several candidates were selected from survival cells post-PRRSV inoculation and further characterized. Nb9 was identified with strong antiviral activity. Moreover, Nb9 exerted antiviral activity via its interaction with PRRSV viral proteins, as revealed by immunofluorescence assay and Western blot. Taken together, the novel function-based screen of the lentivirus nanobody library, instead of the conventional affinity-based screen, offers an alternative strategy for antiviral reagents against PRRSV and other pathogens.

Glycyrrhizic-Acid-Based Carbon Dots with High Antiviral Activity by Multisite Inhibition Mechanisms.

With the gradual usage of carbon dots (CDs) in the area of antiviral research, attempts have been stepped up to develop new antiviral CDs with high biocompatibility and antiviral effects. In this study, a kind of highly biocompatible CDs (Gly-CDs) is synthesized from active ingredient (glycyrrhizic acid) of Chinese herbal medicine by a hydrothermal method. Using the porcine reproductive and respiratory syndrome virus (PRRSV) as a model, it is found that the Gly-CDs inhibit PRRSV proliferation by up to 5 orders of viral titers. Detailed investigations reveal that Gly-CDs can inhibit PRRSV invasion and replication, stimulate antiviral innate immune responses, and inhibit the accumulation of intracellular reactive oxygen species (ROS) caused by PRRSV infection. Proteomics analysis demonstrates that Gly-CDs can stimulate cells to regulate the expression of some host restriction factors, including DDX53 and NOS3, which are directly related to PRRSV proliferation. Moreover, it is found that Gly-CDs also remarkably suppress the propagation of other viruses, such as pseudorabies virus (PRV) and porcine epidemic diarrhea virus (PEDV), suggesting the broad antiviral activity of Gly-CDs. The integrated results demonstrate that Gly-CDs possess extraordinary antiviral activity with multisite inhibition mechanisms, providing a promising candidate for alternative therapy for PRRSV infection.