The effect of matrine and glycyrrhizic acid on porcine reproductive and respiratory syndrome virus in Vitro and in vivo.

Porcine reproductive and respiratory syndrome (PRRS) is endemic worldwide, seriously affecting the development of the pig industry, but vaccines have limited protective effects against PRRSV transmission. The aim of this study was to identify potential anti-PRRSV drugs. We examined the cytotoxicity of seven compounds formulated based on the mass ratio of glycyrrhizic acid to matrine and calculated their inhibition rates against PRRSV in vitro. The results showed that the seven compounds all had direct killing and therapeutic effects on PRRSV, and the compounds inhibited PRRSV replication in a time- and dose-dependent manner. The compound with the strongest anti-PRRSV effect was selected for subsequent in vivo experiments. Pigs were divided into a control group and a medication group for the in vivo evaluation. The results showed that pigs treated with the 4:1 compound had 100% morbidity after PRRSV challenge, and the mortality rate reached 75% on the 8th day of the virus challenge. These results suggest that this compound has no practical anti-PRRSV effect in vivo and can actually accelerate the death of infected pigs. Next, we further analyzed the pigs that exhibited semiprotective effects following vaccination with the compound to determine whether the compound can synergize with the vaccine in vivo. The results indicated that pigs treated with the compound had higher mortality rates and more severe clinical reactions after PRRSV infection (p < 0.05). The levels of proinflammatory cytokines (IL-6, IL-8, IL-1ß, IFN-γ, and TNF-α) were significantly greater in the compound-treated pigs than in the positive control-treated pigs (p < 0.05), and there was no synergistic enhancement with the live attenuated PRRSV vaccine (p < 0.05). The compound enhanced the inflammatory response, prompted the body to produce excessive levels of inflammatory cytokines and caused body damage, preventing a therapeutic effect. In conclusion, the present study revealed that the in vitro effectiveness of these agents does not indicate that they are effective in vivo or useful for developing anti-PRRSV drugs. Our findings also showed that, to identify effective anti-PRRSV drugs, comprehensive drug screening is needed, for compounds with solid anti-inflammatory effects both in vitro and in vivo. Our study may aid in the development of new anti-PRRSV drugs.

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.

Nanobody peptide conjugate: a novel CD163 based broad neutralizing strategy against porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins. RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-ß activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV. CONCLUSION: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.

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.

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.

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.

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.

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.

Xanthohumol inhibits PRRSV proliferation and alleviates oxidative stress induced by PRRSV via the Nrf2-HMOX1 axis.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent and endemic swine pathogen that causes significant economic losses in the global swine industry. Commercial vaccines provide limited protection against this virus, and no highly effective therapeutic drugs are yet available. In this study, we first screened a library of 386 natural products and found that xanthohumol (Xn), a prenylated flavonoid found in hops, displayed high anti-PRRSV activity by inhibiting PRRSV adsorption onto and internalization into cells. Transcriptome sequencing revealed that Xn treatment stimulates genes associated with the antioxidant response in the nuclear factor-erythroid 2-related factor 2 (Nrf2) signalling pathway. Xn causes increased expression of Nrf2, HMOX1, GCLC, GCLM, and NQO1 in Marc-145 cells. The action of Xn against PRRSV proliferation depends on Nrf2 in Marc-145 cells and porcine alveolar macrophages (PAMs). This finding suggests that Xn significantly inhibits PRRSV proliferation and decreases viral-induced oxidative stress by activating the Nrf2-HMOX1 pathway. This information should be helpful for developing a novel prophylactic and therapeutic strategy against PRRSV infection.

Carbon Monoxide Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by the Cyclic GMP/Protein Kinase G and NF-κB Signaling Pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide each year. Our previous research demonstrated that heme oxygenase-1 (HO-1) can suppress PRRSV replication via an unknown molecular mechanism. In this study, inhibition of PRRSV replication was demonstrated to be mediated by carbon monoxide (CO), a downstream metabolite of HO-1. Using several approaches, we demonstrate that CO significantly inhibited PRRSV replication in both a PRRSV permissive cell line, MARC-145, and the predominant cell type targeted during in vivo PRRSV infection, porcine alveolar macrophages (PAMs). Our results showed that CO inhibited intercellular spread of PRRSV; however, it did not affect PRRSV entry into host cells. Furthermore, CO was found to suppress PRRSV replication via the activation of the cyclic GMP/protein kinase G (cGMP/PKG) signaling pathway. CO significantly inhibits PRRSV-induced NF-κB activation, a required step for PRRSV replication. Moreover, CO significantly reduced PRRSV-induced proinflammatory cytokine mRNA levels. In conclusion, the present study demonstrates that CO exerts its anti-PRRSV effect by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-κB signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks. IMPORTANCE: PRRSV causes great economic losses each year to the swine industry worldwide. Carbon monoxide (CO), a metabolite of HO-1, has been shown to have antimicrobial and antiviral activities in infected cells. Our previous research demonstrated that HO-1 can suppress PRRSV replication. Here we show that endogenous CO produced through HO-1 catalysis mediates the antiviral effect of HO-1. CO inhibits PRRSV replication by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-κB signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks.

Griffithsin inhibits porcine reproductive and respiratory syndrome virus infection in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen that severely disrupts swine production. Despite sustained efforts, the disease is still endemic, with high mortality and morbidity. New antiviral strategies to control PRRSV are needed. Griffithsin, a red algal lectin, has potent antiviral effect on several human enveloped viruses, but this effect has not been demonstrated on PRRSV. Here, we first tested the in vitro antiviral activity of Griffithsin against PRRSV. Griffithsin exerted strong saccharide-dependent antiviral activity against PRRSV, probably through interactions with glycans on the surface of PRRSV that interfered with virus entry. Furthermore we revealed that Griffithsin's action on PRRSV involved blocking viral adsorption, and it had no effect on viral penetration. Besides Our findings also suggested that Griffithsin may interfere with cell-to-cell spread to prevent virus transmission. The remarkable potency profile of Griffithsin supports its potential value as an antiviral agent against PRRSV.

Isobavachalcone inhibits post-entry stages of the porcine reproductive and respiratory syndrome virus life cycle.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen of great economic significance that impacts the swine industry globally. Since the first report of a porcine reproductive and respiratory syndrome (PRRS) outbreak, tremendous efforts to control this disease, including various national policies and plans incorporating the use of multiple modified live-virus vaccines, have been made. However, PRRSV is still a significant threat to the swine industry, and new variants continually emerge as a result of PRRSV evolution. Several studies have shown that pandemic PRRSV strains have enormous genetic diversity and that commercial vaccines can only provide partial protection against these strains. Therefore, effective anti-PRRSV drugs may be more suitable and reliable for PRRSV control. In this study, we observed that isobavachalcone (IBC), which was first isolated from Psoralea corylifolia, had potent anti-PRRSV activity in vitro. Although many biological activities of IBC have been reported, this is the first report describing the antiviral activity of IBC. Furthermore, after a systematic investigation, we demonstrated that IBC inhibits PRRSV replication at the post-entry stage of PRRSV infection. Thus, IBC may be a candidate for further evaluation as a therapeutic agent against PRRSV infection of swine in vivo.

In vitro inhibition of porcine reproductive and respiratory syndrome virus replication by short antisense oligonucleotides with locked nucleic acid modification.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is currently insufficiently controlled. From a previous small-scale screen we identified an effective DNA-based short antisense oligonucleotide (AS-ON) targeting viral NSP9, which could inhibit PRRSV replication in both Marc-145 cells and pulmonary alveolar macrophages (PAMs). The objective of this study was to explore the strategy of incorporating locked nucleic acids (LNAs) to achieve better inhibition of PRRSV replication in vitro. METHODS: The effective DNA-based AS-ON (YN8) was modified with LNAs at both ends as gap-mer (LNA-YN8-A) or as mix-mer (LNA-YN8-B). Marc-145 cells or PAMs were infected with PRRSV and subsequently transfected. RESULTS: Compared with the DNA-based YN8 control, the two AS-ONs modified with LNAs were found to be significantly more effective in decreasing the cytopathic effect (CPE) induced by PRRSV and thus in maintaining cell viability. LNA modifications conferred longer lifetimes to the AS-ON in the cell culture model. Viral ORF7 levels were more significantly reduced at both RNA and protein levels as shown by quantitative PCR, western blot and indirect immunofluorescence staining. Moreover, transfection with LNA modified AS-ON reduced the PRRSV titer by 10-fold compared with the YN8 control. CONCLUSION: Taken together, incorporation of LNA into AS-ON technology holds higher therapeutic promise for PRRS control.

The Attenuation Phenotype of a Ribavirin-Resistant Porcine Reproductive and Respiratory Syndrome Virus Is Maintained during Sequential Passages in Pigs.

UNLABELLED: In a previous study, ribavirin-resistant porcine reproductive and respiratory syndrome virus (PRRSV) mutants (RVRp13 and RVRp22) were selected, and their resistance against random mutation was shown in cultured cells. In the present study, these ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability during three pig-to-pig passages in comparison with modified live virus (MLV) (Ingelvac PRRS MLV). Pigs challenged with RVRp22 had significantly lower (P< 0.05) viral loads in sera and tissues than pigs challenged with MLV or RVRp13 at the first passage, and the attenuated replication of RVRp22 was maintained until the third passage. Viral loads in sera and tissues dramatically increased in pigs challenged with MLV or RVRp13 during the second passage. Consistently, all five sequences associated with the attenuation of virulent PRRSV in RVRp13 and MLV quickly reverted to wild-type sequences during the passages, but two attenuation sequences were maintained in RVRp22 even after the third passage. In addition, RVRp22 showed a significantly lower (P< 0.001) mutation frequency in nsp2, which is one of the most variable regions in the PRRSV genome, than MLV. Nine unique mutations were found in open reading frames (ORFs) 1a, 2, and 6 in the RVRp22 genome based on full-length sequence comparisons with RVRp13, VR2332 (the parental virus of RVRp13 and RVRp22), and MLV. Based on these results, it was concluded that RVRp22 showed attenuated replication in pigs; further, because of the high genetic stability of RVRp22, its attenuated phenotype was stable even after three sequential passages in pigs. IMPORTANCE: PRRSV is a rapidly evolving RNA virus. MLV vaccines are widely used to control PRRS; however, there have been serious concerns regarding the use of MLV as a vaccine virus due to the rapid reversion to virulence during replication in pigs. As previously reported, ribavirin is an effective antiviral drug against many RNA viruses. Ribavirin-resistant mutants reemerged by escaping lethal mutagenesis when the treatment concentration was sublethal, and those mutants were genetically more stable than parental viruses. In a previous study, two ribavirin-resistant PRRSV mutants (RVRp13 and RVRp22) were selected, and their higher genetic stability was shown in vitro Consequently, in the present study, both of the ribavirin-resistant mutants were evaluated in terms of their genetic and phenotypic stability in vivo RVRp22 was found to exhibit higher genetic and phenotypic stability than MLV, and nine unique mutations were identified in the RVRp22 genome based on a full-length sequence comparison with the RVRp13, VR2332, and MLV genomes.

Porcine 2', 5'-oligoadenylate synthetase 2 inhibits porcine reproductive and respiratory syndrome virus replication in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) is acknowledged a fulminating infectious pathogen affecting the pig farming industry, and current vaccines and drugs could hardly inhibit this virus. The 2', 5'-oligoadenylate synthetase (OASs) have antiviral activities, but the role(s) played by porcine OAS2 in protection against PRRSV infection are unknown. Here we found that endogenous expression of the porcine OAS2 gene could be promoted by interferon (IFN)-beta or PRRSV infection in porcine alveolar macrophages. Knockdown of porcine OAS2 led to increases in PRRSV replication, and OAS2 expression suppressed replication of PRRSV in a retinoic acid inducible gene I (RIG-I)-dependent manner, anti-PRRSV activity of porcine OAS2 would be lost if RNase L and OAS2 were both silenced. This discovery illustrates a pathway that porcine OAS2 responses to host anti-PRRSV function.

Curcumin is a promising inhibitor of genotype 2 porcine reproductive and respiratory syndrome virus infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) could lead to pandemic diseases and huge financial losses to the swine industry worldwide. Curcumin, a natural compound, has been reported to serve as an entry inhibitor of hepatitis C virus, chikungunya virus and vesicular stomatitis virus. In this study, we investigated the potential effect of curcumin on early stages of PRRSV infection. RESULTS: Curcumin inhibited infection of Marc-145 cells and porcine alveolar macrophages (PAMs) by four different genotype 2 PRRSV strains, but had no effect on the levels of major PRRSV receptor proteins on Marc-145 cells and PAMs or on PRRSV binding to Marc-145 cells. However, curcumin did block two steps of the PRRSV infection process: virus internalization and virus-mediated cell fusion. CONCLUSIONS: Our results suggested that an inhibition of genotype 2 PRRSV infection by curcumin is virus strain-independent, and mainly inhibited by virus internalization and cell fusion mediated by virus. Collectively, these results demonstrate that curcumin holds promise as a new anti-PRRSV drug.

Porcine Interferon Stimulated Gene 12a Restricts Porcine Reproductive and Respiratory Syndrome Virus Replication in MARC-145 Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe losses in the global pig industry. In the present study, we investigated the molecular characterization of porcine interferon stimulated gene 12a (ISG12A) and confirmed its anti-PRRSV ability for the first time. We found that porcine ISG12A was localized in mitochondria and significantly decreased the number of cells in G2/S phase. Porcine ISG12A mRNA was up-regulated in cells/tissues of Tongcheng (TC) pigs and Large White (LW) pigs after PRRSV challenge. More importantly, the ectopic overexpression of ISG12A could significantly suppress PRRSV replication at 24, 36 and 48 h post challenge (hpc), which was confirmed by detecting PRRSV ORF7 mRNA with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and PRRSV N protein with indirect immunofluorescence assay (IFA) in MARC-145 cells. Meanwhile, knockdown of endogenic ISG12A could obviously facilitate PRRSV replication in MARC-145 cells at 36 hpc. The results will lead to a better understanding of the interaction between host immune system and PRRSV, which may help us develop novel therapeutic tools to control PRRSV.

Ivermectin inhibits porcine reproductive and respiratory syndrome virus in cultured porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating viral pathogen of swine that causes huge financial losses in the pig industry worldwide. Ivermectin is known to be a potent inhibitor of importin α/ß-mediated nuclear transport and exhibits antiviral activity towards several RNA viruses by blocking the nuclear trafficking of viral proteins. Although PRRSV replication occurs exclusively in the cytoplasm of infected cells, the nucleocapsid (N) protein has been shown to distinctly localize in the nucleus and nucleolus throughout infection. Here, we sought to assess whether ivermectin suppresses PRRSV replication in cultured porcine alveolar macrophage (PAM) cells and to investigate the effect of ivermectin on the subcellular localization of the PRRSV N protein. Our data demonstrate that ivermectin treatment inhibits PRRSV infection in PAM-pCD163 cells in a dose-dependent manner. The antiviral activity of ivermectin on PRRSV replication was most effective when cells were treated during the early stage of infection. Treatment of PRRSV-infected cells with ivermectin significantly suppressed viral RNA synthesis, viral protein expression, and progeny virus production. However, immunofluorescence and cell fractionation assays revealed that ivermectin was incapable of disrupting the nuclear localization of the N protein, both in PRRSV-infected PAM-pCD163 cells and in PAM cells stably expressing the PRRSV N protein. This finding suggests that an alternative mechanism of action accounts for the ability of ivermectin to diminish PRRSV replication. Taken together, our results suggest that ivermectin is an invaluable therapeutic or preventative agent against PRRSV infection.

In Vitro Antiviral Activity of Germacrone Against Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious diseases affecting the swine industry worldwide; however, there is no efficient control strategies against PRRSV at present. Therefore, development of new antiviral treatment strategies is urgently needed. As reported, germacrone can efficiently impair influenza virus replication. In this study, we exploited whether germacrone has the potential to inhibit PRRSV infection. Our results showed that the germacrone significantly inhibited replication of PRRSV in vitro and repressed the synthesis of viral RNA and protein. However, it did not block PRRSV binding and entry. Further studies confirmed that germacrone impaired PRRSV replication at an early stage, and inhibited infection of both classic and highly pathogenic type II PRRSV strains. Collectively, our findings imply that the germacrone has the potential to be used as an anti-PRRSV drug.