Slov Vet Res 2024 | Vol 61 No 1 | 9 From Nature's Pharmacy to Swine Health: Harnessing Natural Compounds against PRRSV Infection Key words antiviral agents; natural compounds; PRRSV; swine industry Fredmoore L. Orosco1,2,3 1Virology and Vaccine Institute of the Philippines Program, Industrial Technology Development Institute, 2S&T Fellows Program, Department of Science and Technology, Bicutan, Taguig City, 1634, 3Department of Biology, College of Arts and Sciences, University of the Philippines Manila, Ermita, Manila, 1000, Philippines *Corresponding author: orosco.fredmoore@gmail.com Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant viral pathogen that causes substantial economic losses to the swine industry worldwide. The limited efficacy of current therapeutic approaches and emergence of new PRRSV strains highlight the urgent need for novel antiviral strategies. Natural compounds de- rived from plants, animals, bacteria, and fungi have attracted increasing attention as po- tential antiviral agents. This comprehensive review focuses on natural compounds with antiviral activity against PRRSV and explores their mechanisms of action, efficacy, and potential applications. These compounds exhibit diverse antiviral mechanisms such as viral attachment and entry inhibition, replication suppression, and modulation of host immune responses. This review also highlights challenges and future directions in this field. Research gaps include the need for further elucidation of the precise mechanisms of action, comprehensive evaluation of safety profiles, and exploration of combination therapies to enhance efficacy. Further research and translational studies are warranted to harness the full potential of these natural compounds and pave the way for the effec- tive control and management of PRRSV infections in the swine industry. Received: 11 June 2023 Accepted: 22 December 2023 Slovenian Veterinary Research DOI 10.26873/SVR-1789-2023 UDC 636.4:578.76:616.2:615.2 Pages: 9–28 Review Article Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiological agent responsible for the path- ological condition observed in swine, which was initially documented within the borders of the United States (U.S.) in 1987 and subsequently in Europe in 1990 (1). These out- breaks were characterized by detrimental effects on repro- duction, post-weaning pneumonia, and elevated mortality rates in growing swine. In the early stages, attempts to iden- tify the causative agent responsible for this novel syndrome proved to be futile, leading to its provisional designation as a mystery swine disease (MSD) in North America. However, in 1991, Koch's postulates for MSD were eventually satis- fied through the discovery of a hitherto unidentified RNA vi- rus in Europe, which was subsequently named the Lelystad virus (LV) (2). Shortly after this significant finding, the virus was successfully isolated in North America and was initially referred to as swine infertility and respiratory syndrome vi- rus (SIRSV) (3). PRRSV has emerged as a pervasive pathogen in most swine-producing nations, posing substantial economic re- percussions to the swine industry. PRRSV can infect pigs across all age groups; however, its clinical manifestations are particularly pronounced in pregnant sows and young pigs (4). In pregnant sows, PRRSV infection during the final trimester of gestation may lead to adverse outcomes, such as abortion, characterized by the delivery of stillborn, par- tially autolyzed, and mummified fetuses. Conversely, young pigs infected with PRRSV commonly display clinical signs, including elevated body temperature, severe dyspnea, di- minished appetite, lethargy, eyelid edema, and ear discolor- ation, appearing either blue or red (5). The term PRRSV encompasses two distinct genotypes: PRRSV-1, comprising genotypes initially isolated in Europe, and PRRSV-2, which consists of genotypes first identi- fied in North America (6). Presently, both virus types have 10 | Slov Vet Res 2024 | Vol 61 No 1 achieved global distribution, with PRRSV-1 primarily prev- alent in Europe, whereas PRRSV-2 exhibits a wider geo- graphic range, including North America, Asia, and South America (7). Recent investigations into multiple arteriviral nucleotide sequences in nonhuman primates have prompt- ed the reclassification of PRRSV into two separate entities: PRRSV-1 and PRRSV-2 (8). This classification was substan- tiated by the recognition of remarkable genetic variability within both PRRSV-1 and PRRSV-2, as evidenced by phylo- genetic analysis based on ORF5 (9). The extensive genetic diversity exhibited by PRRSV poses a significant challenge for the development of effective antiviral therapeutics. This review aims to discuss the molecular biology, clinical char- acteristics, transmission, and different natural compounds with antiviral activity against PRRSV, and their respective molecular mechanisms. Molecular Biology of PRRSV Taxonomy and Structure PRRSV is an enveloped RNA virus characterized by a single positive strand (Figure 1) (10). Taxonomically, it belongs to the order Nidovirales and family Arteriviridae, which also encompasses other viruses such as the lactate dehydroge- nase-elevating virus of mice, equine arteritis virus, and sim- ian hemorrhagic fever virus (11). The arterivirus genome is enclosed within a lipid envelope and associated with a sin- gular N protein comprising 110-128 amino acids, forming a core structure. Furthermore, the viral particles exhibited an approximately spherical or oval shape with diameters ranging from 50 to 60 nm. The reported buoyant densities of PRRSV virions in sucrose range from 1.13 to 1.17 g/cm3 (12). The enveloped surface of the virions appears relatively smooth, which can be attributed to the limited size of the ectodomains of the two major envelope proteins, GP5 and M. Genome Organization The complete genome of PRRSV spans approximately 15 kb, featuring a cap structure at the 5' end during mRNA pro- cessing and a poly A tail structure at the 3' end (Figure 2) (13). PRRSV isolates can be categorized into two distinct genotypes based on their genomic and antigenic varia- tions: North American (NA), PRRSV-2, and European (EU), or PRRSV-1. These genotypes exhibit an approximate se- quence identity of 65% (14). The PRRSV genome comprises 12 open reading frames (ORFs) designated as ORF1a, ORF1a', TF, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5a, and ORF5–ORF7 (15). Among these, ORF1a and ORF1b encode polyproteins pp1a and pp1ab, which undergo processing to yield 17 nonstructural proteins (NSPs) (NSP1α, NSP1β, NSP2, NSP2N, NSP2TF, and NSP3-14), which play a pivotal role in virus replication (16). Recently, a novel ORF known as the ORF trans-frame (TF) was discovered within the nsp2 region (17). This ORF, expressed through both −1 and −2 ribosomal frameshift- ing, gives rise to two additional nsps: nsp2N, a truncated version of nsp2; and nsp2TF, a fusion protein formed by the N-terminal two-thirds of nsp2 and a C-terminal region en- coded by the TF ORF spanning 169 amino acids (18). The major envelope proteins GP5 and M are encoded by ORF5 and ORF6, respectively. These proteins interact with each Figure 1: Schematic representation of PRRSV structure PRRSV mature viral particle, composed of a lipid bilayer envelope with viral receptor glycoproteins involved in infection and cell internalization. Single- stranded positive RNA is associated with nucleocapsid protein in the internal layer of the virus. Slov Vet Res 2024 | Vol 61 No 1 | 11 other to form heterodimers on the surface of viral particles. Notably, GP5 exhibits substantial variability among struc- tural proteins in the PRRSV genome, making it a commonly employed target for phylogenetic analyses (19). ORF2, ORF3, and ORF4 encode the minor envelope proteins GP2a, GP3, and GP4, respectively, forming noncovalent heterodi- mers. In addition, two small non-glycosylated proteins, E and GP5a, are encoded by ORF2b and ORF5a, respectively. The highly conserved nucleocapsid protein (N protein) is encoded by ORF7 (20). Clinical Characteristics PRRSV infections are characterized by several distinctive clinical features, including elevated body temperature, pro- nounced contagiousness, and substantial morbidity and fatality rates. Following infection, pigs often experience a rapid increase in body temperature, reaching 41-42°C within a span–1-2 days (21). This heightened contagious- ness results in infection spreading throughout the entire pig population within 3-5 days, with a disease duration typically lasting 1-3 weeks. Notably, the highly pathogenic PRRSV (HP-PRRSV) demonstrated a particularly high fatality rate in suckling pigs (100%) and nursery pigs (approximately 70%). Furthermore, adult pigs exhibit an increased mortal- ity rate, with finishing pigs experiencing a mortality rate of 20% and pregnant sows experiencing a minimum of 10% mortality (22). Additionally, PRRSV infections are frequently associated with an elevated incidence of abortions, ranging from 40% to 100%. Furthermore, affected pigs often display signs, such as skin erythema and severe respiratory symp- toms, including coughing, dyspnea, and tachypnea. Some pigs may also exhibit neurological signs, such as limping, and gastrointestinal manifestations, such as constipation or diarrhea (21). PRRSV infection induces a wide array of pathological changes in affected pigs. Among the prominent gross le- sions, multifocal hemorrhages are particularly notable, af- fecting various tissues and organs, such as the skin, lungs, lymph nodes, kidneys, and heart (22). Another significant observation was the presence of lymphadenopathy accom- panied by pronounced interstitial pneumonia, characterized by severe pulmonary edema and consolidation. In certain instances, edema and congestion can be observed within the brain. Additionally, severe thymus atrophy is frequently encountered, especially in piglets infected with HP-PRRSV (21). Secondary bacterial infections are a significant and fre- quently encountered issue in the context of HP-PRRSV in- fections. Among the commonly detected bacterial patho- gens, Escherichia coli, Streptococcus suis, Haemophilus parasuis, and Mycoplasma hyopneumoniae are frequently implicated (22). Furthermore, PRRSV-infected pig popula- tions often exhibit the presence of various viral pathogens, including classical swine fever virus (CSFV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2). The po- tential synergistic effects of these coinfections on PRRSV pathogenesis have garnered substantial attention within the PRRSV research community, prompting intensive in- vestigation (21). Figure 2: Genome organization of PRRSV virus Non-structural proteins are located in the 5′ end of the genome, coding for two different polyproteins pp1a and pp1ab that is cleaved into at least 14 nsps. Structural proteins near the 3′ end are associated with the viral envelope and RNA packaging. 12 | Slov Vet Res 2024 | Vol 61 No 1 Transmission of PRRSV Transmission Routes PRRSV transmission in pigs can occur through various routes, including direct contact and indirect transmission via fomites. Exposure to PRRSV primarily occurs through respiratory and oral routes as well as through mucosal or percutaneous routes. The modes of transmission encom- pass aerial transmission, either over short or longer distanc- es, as well as transmission through coitus or insemination, ingestion, contact, and occasionally through inoculation, often arising from iatrogenic factors. Vertical transmission during the later stages of gestation is particularly notewor- thy. The minimum infectious dose (MID) of PRRSV depends on the specific route of exposure. For instance, infectious dose 50 (ID50) through oral and nasal exposure has been previously evaluated. Notably, variations in infectivity have been observed among different PRRSV isolates via various transmission routes (23). In terms of sexual transmission, the ID50 for exposure via artificial insemination is 103.3 TCID50 (24). Based on available data, percutaneous exposure is asso- ciated with the lowest MID. Within the farm environment, parenteral exposure is likely to occur frequently, involving routine practices, such as ear notching, tail docking, teeth clipping, and the administration of drugs and vaccines. During the peak viremia stage, infected animals typically exhibit a viral load of at least 103–104 TCID50/mL (25). Regular pig behavior can also contribute to parenteral ex- posure, such as bites, cuts, scrapes, and abrasions, during instances of inter-pig fighting. Aggressive interactions be- tween infected sows and susceptible contacts may play a significant role in PRRSV transmission (26). PRRSV is notably susceptible to inactivation by various means, including lipid solvents, heat, desiccation, and ex- treme pH conditions (27). Notably, LV was shown to under- go inactivation after 6 min at 56°C or 3 h at 37°C. However, it displays stability for up to 140 hours at 4°C and remains viable for several months when maintained in a cell culture medium at pH 7.5 and temperatures ranging from -70°C to -20°C (28). In terms of disinfection, iodine ( 0.0075%) and quaternary ammonium compounds ( 0.0063%) achieve complete inactivation of the virus within 1 min (29). Chlorine can also completely inactivate PRRSV, although higher disinfectant concentrations (0.03%) and longer exposure times (10 min) are required. Additionally, a 10-minute ex- posure to ultraviolet light effectively leads to the complete inactivation of the virus on commonly encountered farm surfaces and materials (30). Development of Viremia and Viral Persistence Following exposure to PRRSV, viral replication initially oc- curs within permissive macrophages located in lymphoid tissues at the entry portal. Subsequently, the virus rapidly disseminates throughout the body via the lympho-haemat- ic route. In a genotype 2 model, detectable viremia was ob- served as early as 12 hours post-infection (hpi) (31). The viral load in the serum peak around 7-10 days post-infection (dpi). The duration of viremia can vary depending on factors such as the specific PRRSV strain and age of the infected animal (32). Various studies have indicated that the viremic period ranges from a few weeks, typically less than four weeks, in adult or grower-finisher pigs, to as long as three months in very young piglets (33). In the case of adult sows infected with genotype 1 PRRSV, viremia may be limited to just one week (34). During the initial phase of infection, the lungs and various lymphoid organs, including tonsils, Peyer's patches, thy- mus, and spleen, exhibit the highest viral loads (35). In the lungs, viral detection can typically be observed from 1 to 28 dpi (36). Notably, in young pigs, the virus has been reported to persist in the lungs for up to 49 dpi (37). Subsequent to the viraemic phase, viral infection enters a stage characterized by the sequestration of the virus within secondary lymphoid tissues, leading to a decline in viral rep- lication. Over time, contagiousness diminishes, although transmission remains feasible for up to three months in horizontally infected pigs. However, in congenitally infected animals, the period of contagiousness may extend beyond this time frame (38). Several situations inducing stress, such as farrowing or regrouping, can trigger reactivation of viral replication and shedding (39). Viral Shedding The presence of viremia and the distribution of susceptible macrophages within the body contribute to PRRSV shed- ding through various routes. In particular, nasal shedding appears to exhibit strain-dependent characteristics, par- ticularly in genotype 1 PRRSV. For instance, nasal shedding of genotype 1 was limited, with only four out of eight pigs showing isolation of the virus at 3 dpi and one out of eight pigs at 7 dpi, always at low titers (25). In the case of geno- type 2, nasal secretion was reported in only 1.9% (2/105) of nasal swabs collected from experimentally inoculated pigs during a 28-day observation period (40). Additionally, no vi- rus has been isolated from the nasal secretions of experi- mentally infected pigs (31). The shedding of PRRSV in oral fluids appears to be rela- tively consistent, although most of the available data focus on genotype 2 viruses. The presence of the virus in oral flu- ids and the steady nature of its shedding over time have important implications for PRRSV transmission. With re- gard to viral shedding in the semen of infected boars, the detection of the viral genome using RT-PCR has been re- ported as early as 3 dpi and persisted up to 92 dpi in 1 out of 4 boars inoculated with the VR-2332 isolate (41). PRRSV has also been found in the urine (40) and mammary gland secretions (42). In experimentally infected sows, genotype Slov Vet Res 2024 | Vol 61 No 1 | 13 2 PRRSV was detected using RT-PCR on the first day of lactation (42). Lastly, infected pigs have been observed to generate aerosols contaminated with the virus during re- spiratory activities, such as breathing, sneezing, or cough- ing, although the extent of aerosol transmission can vary depending on the strain (43). Anti-PRRSV Agents Natural compounds derived from plants, animals, fungi, and bacteria have received significant research attention because of their potent antiviral activities in vitro and in vivo. These compounds possess different molecular mecha- nisms, such as activation of TLR signaling, activation of in- terferon signaling, downregulation of receptors, and block- ing of virus attachment, fusion, replication, translation, assembly, maturation, and release (Figure 3). The com- pounds are discussed in detail in this section. Plant-derived Compounds Tea polyphenols Tea polyphenol (TPP) refers to the collective group of poly- phenols found in tea leaves, with catechins and their de- rivatives being the primary constituents (44). TPP exhibits a wide range of physiological activities, including antioxidant, anti-radiation, anti-aging, blood lipid-lowering, blood sug- ar-lowering, and inhibition of bacteria and enzymes (45). Structurally, TPP possesses polyphenolic characteristics such as catechins and anthocyanins (46). Green tea, which serves as a primary source of TPP, exerts notable antiviral and antifungal effects (47). Notably, epigallocatechin gal- late (EGCG) has demonstrated antiviral properties against Figure 3: Life cycle and molecular targets of antiviral compound The life cycle of PRSSV begins with binding of PRRSV with sialoadhesin (1) that will trigger clathrin-mediated endocytosis (2) to fuse with an endosome (3). The virus is then disassembled and the viral RNA is released (4) for translation in the endoplasmic reticulum (5). Translated viral proteins are assembled into new progeny virions (6) followed by maturation and budding in the Golgi body (7). This new progeny virions are released outside of the cell via exocytosis (8). 14 | Slov Vet Res 2024 | Vol 61 No 1 various viruses, including hepatitis C (HCV), chikungunya (CHIKV), hepatitis B (HBV), and Zika (ZIKV) viruses (48). TPP potently inhibited PRRSV infection in Marc-145 cells in a dose-dependent manner. TPP exerts its inhibitory ef- fects across multiple stages of the PRRSV life cycle, includ- ing attachment, internalization, replication, and release. Mechanistically, TPP impeded the transport of p65 into the nucleus, thereby suppressing the activation of the NF-κB signaling pathway, which ultimately resulted in the down- regulation of inflammatory cytokine expression induced by PRRSV infection. Additionally, TPP can interfere with the synthesis of viral nsp2, a crucial component of replication transcription complexes (RTC), leading to inhibition of viral protein translation and assembly (49). Matrine Matrine, a prominent quinolizidine alkaloid derived from the dried roots of Sophora flavescens Ait, as described in the Chinese Pharmacopeia 2005, exhibits a range of pharma- cological effects, including antiviral, anti-inflammatory, and immunoregulatory properties (50). Previous studies have revealed the inhibitory effects of matrine on PRRSV infec- tion in Marc-145 cells, suggesting that matrine can directly inactivate PRRSV and disrupt viral replication within host cells. Furthermore, an indirect immunofluorescence assay and western blot analysis demonstrated that matrine is ca- pable of inhibiting the expression of N protein in Marc-145 cells. Additionally, matrine impedes PRRSV-induced apop- tosis by inhibiting the activation of caspase-3 (51). Moreover, matrine exhibits antiviral activity against both PRRSV and PCV2 (51). The antiviral mechanisms under- lying the effects of matrine are thought to involve partial regulation of the TLR3/TLR4/NF-κB/TNF-α pathway (50). Notably, matrine treatment has been shown to improve pneumonia symptoms in PRRSV/PCV2 co-infected mice and has shown efficacy in attenuating inflammation in mice with LPS-induced acute lung injury. Furthermore, matrine directly hindered PRRSV replication by inhibiting the activ- ity of Nsp9. Recent studies have also revealed that matrine can inhibit IL-1β secretion in primary porcine alveolar mac- rophages (PAMs) by acting on the MyD88/NF-κB pathway and the NLRP3 inflammasome. This inhibition is associat- ed with downregulated expression of MyD88, NLRP3, and caspase-1 as well as suppression of ASC speck formation, IκBα phosphorylation, and hindered translocation of NF-κB from the cytoplasm to the nucleus (52). Rottlerin Rottlerin, a polyphenolic ketone compound derived from the Indian Kamala tree (Mallotus philippensis Muell. Arg), has gained prominence as a selective PKCδ inhibitor (53). Historically, rottlerin has been used in traditional Indian medicine to treat tapeworm, scabies, and herpetic ring- worm infections, implying its long-standing safety record. Notably, several prior studies have documented the antivi- ral properties of rottlerin against various viruses, including rabies, influenza, human immunodeficiency virus, and PRRSV (54). Earlier investigations have highlighted the significant im- pact of rottlerin pretreatment on diminishing both viral RNA synthesis and titer. More recently, it has been demon- strated that the administration of rottlerin during the early stages of PRRSV infection effectively impedes viral replica- tion. PRRSV infection is known to induce phosphorylation of protein kinase C-δ, a process that is specifically coun- teracted by rottlerin (55). Treatment with rottlerin disrupts the entry pathway of PRRSV by impeding endocytosis of virions. Moreover, in vivo studies involving the administra- tion of rottlerin-liposomes to PRRSV-infected pigs, specifi- cally those infected with LMY or FL12 strains, revealed a notable dose-dependent reduction in blood viral load, inter- stitial pneumonia, and clinical scores when compared to untreated pigs (55). Sanguinarine Sanguinarine, a quaternary benzo[c]phenanthridine alkaloid found in various plants such as Macleaya cordata (Wild.) R.Br., Bocconia frutescens L., Chelidonium majus L., Fumaria officinalis L., and Sanguinaria canadensis L., has been ex- tensively studied for its anti-inflammatory, anti-tumor, and antimicrobial properties (56). While its antiviral activity has rarely been reported, there have been observations of mod- erate antiviral effects of sanguinarine against tobacco mo- saic virus (57). Furthermore, a derivative of sanguinarine, 8-hydroxydihydrosanguinarine, has recently emerged as a potential drug candidate for combating COVID-19 (58). Regarding its effects on PRRSV, sanguinarine demon- strates potent antiviral activity by targeting multiple stages of the virus life cycle, including internalization, replication, and release (59). Network pharmacology and molecular docking studies identified potential anti-PRRSV targets of sanguinarine, including ALB, AR, MAPK8, MAPK14, IGF1, GSK3B, PTGS2, and NOS2. Additionally, the combination of sanguinarine and chelerythrine, another bioactive alkaloid derived from Macleaya cordata, has shown improved anti- viral effects (59). Platycodin D Platycodon grandiflorum A. DC is a widely recognized Chinese herb that is used to treat pulmonary and respira- tory diseases. Notably, saponins have been identified as the primary bioactive constituents of P. grandiflorum roots (60). Among these saponins, platycodin D (PD), an oleanane- type triterpenoid saponin with sugar chains attached at the C-3 and C-28 positions of the aglycone, is the most potent in terms of biological activity (61). Previous investigations have demonstrated the diverse pharmacological effects of PD, including its antitumor properties (62), anti-inflammato- ry effects (63), and immunological adjuvant activities (60). Moreover, PD has been shown to possess hepatoprotective properties and anti-hepatitis C virus (HCV) activity (64). Slov Vet Res 2024 | Vol 61 No 1 | 15 PD demonstrated remarkable efficacy against PRRSV in- fection in both Marc-145 cells and primary PAMs (65). Its inhibitory effects extend across various strains of PRRSV, including the highly pathogenic type 2 strains GD-HD and GD-XH, as well as the classical strains CH-1a and VR2332. Notably, PD displayed dose-dependent inhibition of PRRSV RNA synthesis, viral protein expression, and production of progeny viruses, with significant effects observed at con- centrations ranging from 1 to 4 μM. The EC50 values of PD against the four tested PRRSV strains in Marc-145 cells ranged from 0.74 to 1.76 μM (65). The antiviral mechanism of PD involves direct interaction with PRRSV virions, thereby affecting multiple stages of the viral life cycle, including viral entry and release of progeny virus. Furthermore, PD exhibited the ability decreases the production of cytokines (IFN-α, IFN-β, IL-1α, IL-6, IL-8, and TNF-α) induced by both PRRSV and LPS in PAMs (65). Cryptotanshinone Cryptotanshinone (CPT) is a natural compound derived from the roots of Salvia miltiorrhiza Bunge (Danshen). The roots of S. miltiorrhiza have long been used in traditional oriental medicine to treat various circulatory disorders, liver diseases, coronary heart disease, hepatitis, and chronic renal failure (66). Notably, CPT exhibited remarkable anti- bacterial activity, surpassing the effectiveness of the other tested tanshinones. Furthermore, its anti-inflammatory properties have been attributed to the inhibition of cyclo- oxygenase II activity and endothelin-1 expression (67). CPT has demonstrated notable efficacy in impeding the in- fection of diverse PRRSV strains in PAMs, which serve as primary targets of PRRSV in vivo. The underlying mecha- nism involves the inhibition of signal transducer and acti- vator of transcription 3 (STAT3) activation, the blockade of interleukin 10 (IL-10)-stimulated CD163 expression, and the basal level of CD163 expression in PAMs (68). Remarkably, CPT was effective in both pre- and post-PRRSV infection treatment, with the combined application resulting in sub- stantial dose-dependent inhibition of PRRSV infection. Moreover, CPT displays inhibitory effects against both type I and type II PRRSV infections in PAMs (68). Allicin Garlic (Allium sativum L.) and its organosulfur compounds (OSC) have been extensively studied for their pharmaco- logical properties, including antibacterial, antiviral, anti-in- flammatory, anticancer, and antioxidant effects (69). Allicin, an OSC present in garlic, onion, and other Allium plants, has been recognized for its significant antiviral activity against herpes simplex virus-1 and 2, parainfluenza-3, vaccinia vi- rus, vesicular stomatitis virus, and human rhinovirus-2 (70). Previous investigations have also demonstrated the an- tiviral potential of allicin against respiratory viruses, such as influenza, SARS-CoV, and rhinovirus (71). In a recent study, allicin was found to alleviate SARS-CoV-2 infection in vitro and restore host cellular pathways disrupted by viral infections (72). Furthermore, allicin has shown effi- cacy as an antiviral agent against the reticuloendotheliosis virus by reducing inflammation and oxidative damage, pri- marily through inhibition of the ERK/MAPK pathway (73). Additionally, allicin exhibits anti-inflammatory properties by inhibiting the P38 and JNK pathways as well as the TLR4/ NF-κB signaling pathway (74). Supplementation of garlic botanicals in the nursery basal diet has been shown to have beneficial effects on PRRSV- infected pigs, including reduction of viral loads and im- provement of immune responses (75). Allicin demonstrat- ed a dose-dependent inhibitory effect on both HP-PRRSV and NADC30-like PRRSV. It exerts its antiviral activity by interfering with various stages of the viral life cycle, includ- ing entry, replication, and assembly. Additionally, allicin alle- viates the expression of pro-inflammatory cytokines (such as IFN-β, IL-6, and TNFα) induced by PRRSV infection. Treatment with allicin effectively restores dysregulated pro- inflammatory signaling pathways, including the TNF and MAPK signaling pathways, which are upregulated during PRRSV infection (76). Curcumin Curcumin, a natural polyphenolic compound isolated from Curcuma longa L. rhizomes, has been extensively studied for its various biological and pharmacological effects. It is the major component of C. longa and accounts for its immunomodulatory, antitumor, anti-inflammatory, antioxi- dant, antimutagenic, antibacterial, antifungal, and antiviral activities associated with this plant (77). Among its antiviral properties, curcumin has been shown to inhibit the entry of several viruses into cells, including HCV, CHIKV, and ve- sicular stomatitis virus (VSV) (78). In the context of PRRSV infection, curcumin has been found to effectively inhibited the infection of both Marc-145 cells and PAMs by four dif- ferent genotype 2 PRRSV strains. Interestingly, curcumin did not affect the levels of major PRRSV receptor proteins on the cell surface or PRRSV binding to cells. Instead, it specifically targets two crucial steps in the PRRSV infec- tion process: virus internalization and virus-mediated cell fusion (79). Aloe vera extracts Aloe vera is known for its remarkable inhibitory effect on a wide range of viruses, including herpes simplex virus type 1, influenza virus, and pigeon paramyxovirus type 1 (80). Notably, this antiviral property has been attributed not only to the whole extract of Aloe vera but also to its isolated compounds. Emodin (1, 3, 8-trihydroxy-6-methyl- anthraquinone), an anthraquinone compound found in the roots and bark of pharmaceutical plants, such as Chinese rhubar (Rheum palmatum L.) and Aloe vera L. (81), has demonstrated significant inhibitory effects against vari- ous viruses. These include Cyprinid herpesvirus 3 (CHV3) (82), coxsackieviruses (CV) (83), ZIKV (84), enterovirus 71 (EV71) (85), Epstein-Barr virus (EBV) (86), HCoV-OC43 (87), herpes simplex virus (HSV) (88), HBV (89), and SARS-CoV 16 | Slov Vet Res 2024 | Vol 61 No 1 (90). Emodin exerts its antiviral activity through multiple mechanisms, including blockade of virus-receptor interac- tions, inhibition of viral protein translation, suppression of viral maturation, and inhibition of viral release (90). Aloe extract (Ae) has been found to exhibit potent inhibi- tory effects against PRRSV in vitro, specifically in Marc-145 cells and PAMs (91). Emodin demonstrated an inhibitory effect by targeting various stages of the PRRSV infection cycle. Emodin was able to directly inactivate PRRSV par- ticles. Additionally, emodin treatment significantly upregu- lated the expression of Toll-like receptor 3 (TLR3) (p < 0.01), IFN-α (p < 0.05), and IFN-β in iPAMs. This suggested that the anti-PRRSV effect of emodin may be attributed to the induction of antiviral agents through TLR3 activation (91). Cepharanthine Cepharanthine (CEP) is an alkaloid derived from Stephania cepharantha Hayata, which has a long history of use in Japanese medicine for various conditions, including radia- tion-induced leukopenia and certain skin and ear disorders (92). The therapeutic potential of CEP extends beyond its traditional applications, as it possesses diverse properties such as anti-inflammatory, antioxidant, immunomodula- tory, and antiparasitic effects, making it an attractive candi- date for treating viral diseases such as COVID-19 (93). Notably, CEP has demonstrated antiviral activity against HCoV-OC43, a mildly pathogenic human coronavirus (94), and severe acute respiratory syndrome coronavirus (SARS- CoV) (95). Moreover, in a comprehensive drug screen- ing study involving 2406 clinically approved drugs, CEP emerged as the most effective compound against pan- golin coronavirus closely related to SARS-CoV-2, the virus responsible for the COVID-19 pandemic (96). This discov- ery is particularly significant considering the high genomic similarity between SARS-CoV and SARS-CoV-2 (97). Given these promising findings, CEP has garnered significant at- tention as a potential therapeutic option for the treatment of COVID-19 by exploiting its established antiviral properties and favorable activity against related coronaviruses (96). In recent investigations, CEP has demonstrated superior in- hibitory effects on PRRSV infection compared to tilmicosin, as evidenced by reductions in both RNA and protein levels. Notably, CEP treatment led to a 5.6-fold decrease in TCID50, providing substantial protection against PRRSV infection in Marc-145 cells (98). Mechanistically, detailed analyses involving western blot assessments of Marc-145 cells and PAMs subjected to CEP treatment and PRRSV infection at various time points revealed the ability of CEP to sup- press the expression of integrins β1 and β3, integrin-linked kinase (ILK), RACK1, and PKCα. These effects culminated in the suppression of NF-κB signaling, ultimately alleviating PRRSV infection. These findings underscore the potential of CEP as a valuable intervention strategy against PRRSV infection, offering new insights into its antiviral mecha- nisms and therapeutic implications (98). Glycyrrhizin Glycyrrhizin, a triterpene saponin found in licorice root (Glycyrrhiza glabra L.), possesses a diverse range of bio- logical activities, including antibacterial, antiviral, anti- inflammatory, anticancer, antioxidant, liver protection, neuroprotection, skin whitening, hypoglycemic, and mem- ory-enhancing properties (99). These characteristics high- light the promising potential of licorice in cosmetic produc- tion and therapeutic applications for various conditions such as liver disease, diabetes, ischemia-reperfusion injury, Alzheimer's disease, Parkinson's disease, epilepsy, depres- sion, and cancer (100). Numerous studies have documented the potent antiviral effects of glycyrrhizin against a range of viruses, including the hepatitis B virus (HBV) (101), HCV (102), herpes simplex virus (HSV) (103), SARS coronavirus (104), and influenza viruses (105). Recent studies have revealed dose-depen- dent inhibitory effects of glycyrrhizin on the proliferation of PRRSV. Treatment with glycyrrhizin effectively reduced PRRSV proliferation and PRRSV-encoded protein expres- sion, which primarily targeted the penetration stage of the PRRSV life cycle, exerting minimal influence on the pro- cesses of viral adsorption or release (105). Flavaspidic acid AB Flavaspidic acid AB (FA-AB) is a naturally occurring com- pound derived from Dryopteris crassirhizoma Nakai, a semi-evergreen fern with a rich history in traditional Chinese medicine (106). The rhizome of D. crassirhizoma has traditionally been employed as an anti-infection agent, particularly for respiratory ailments such as the common cold and flu. Notably, it has been utilized in combination with other Chinese herbal medicines, including Astragalus, Atractylodes, Red Atractylodes, Pogostemon, Adenophora, and Lonicera, in a prescription formula to prevent SARS (106). FA-AB belongs to the phloroglucinol derivative family (107). Extensive investigations have demonstrated the antibacte- rial, antitumor, and antioxidant properties of phloroglucinol derivatives (107). Additionally, dimeric phloroglucinols have shown inhibitory effects against HIV-1 reverse transcrip- tase, highlighting their potential for antiviral intervention (108). FA-AB inhibits the internalization and intercellular transmis- sion of PRRSV, although it does not interfere with the initial binding of PRRSV to host cells (109). Remarkably, when FA-AB treatment was initiated 24 hours after viral infection, it effectively suppressed PRRSV replication, as evidenced by kinetic analysis of viral replication. Moreover, FA-AB can induce the expression of important antiviral cytokines, in- cluding IFN-α, IFN-β, and IL1-β, in PAMs (109). Caesalpinia sappan (CS) heartwood Caesalpinia sappan L. 1753 (CS), derived from the Leguminosae family, is a renowned medicinal plant that is Slov Vet Res 2024 | Vol 61 No 1 | 17 widely distributed and cultivated in various tropical Asian regions such as Southern China, India, Myanmar, Vietnam, Sri Lanka, and Thailand (110). CS dried heartwood has been utilized in traditional medicine practices, including Indian Ayurveda and Traditional Chinese Medicine (111). CS heart- wood exhibits a diverse range of biological activities, includ- ing antioxidant (112), antibacterial (113), anti-inflammatory (114), hypoglycemic (115), and hepatoprotective (116) prop- erties, as reported in previous studies. Moreover, CS extract constituents have demonstrated significant activity against the H3N2 strain of influenza virus (117). Additionally, CS showed promising antiviral activity against PRRSV replica- tion in MARC-145 cells, with a significant reduction in the viral titer observed at 72 hpi. Notably, this antiviral effect was attributed to the presence of specific compounds such as byakangelicin, brazilin, naringenin, and brazilein (118). Saponin Components Saikosaponin A (SSA), Saikosaponin D (SSD), Panax no- toginseng saponins (PNS), Notoginsenoside R1 (SR1), and Anemoside B4 (AB4) have gained significant attention in recent research because of their diverse bioactivity (119). Specifically, its antiviral potential against PRRSV was in- vestigated. In a study involving 132 healthy piglets, saponin components were evaluated for their effects on PRRSV- induced immunopathological damages (120). Piglets were divided into 22 groups, with each group consisting of six ani- mals. The control group received an intramuscular injection of PRRSV solution, while the low-, middle-, and high-dose treatment groups were administered PRRSV solution fol- lowed by intraperitoneal injections of AB4, PNS, SR1, SSA, or SSD at varying doses. The results demonstrated that all five saponin components reduced the incidence and sever- ity of PRRSV-induced immunopathological damage, includ- ing symptoms, such as elevated body temperature, weight loss, anemia, and internal inflammation. Furthermore, these saponin components exhibited the ability to enhance protein absorption and immune responses (120). Isobavachalcone Isobavachalcone (IBC) is a prenylated chalcone compound belonging to the flavonoid subclass that was originally de- rived from Psoralea corylifolia L. (121). Extensive research has revealed that IBC exhibits a broad range of biological activities, including antibacterial, antifungal, anticancer, antireverse transcriptase, antitubercular, and antioxidant properties (121). Notably, IBC demonstrated inhibitory ef- fects on PRRSV replication at the post-entry stage of in- fection. This suggests that IBC may serve as a promising therapeutic candidate for the treatment of PRRSV infection in swine (122). Ursolic acid derivatives Ursolic acid (UA) and its derivatives are widely recognized as prominent examples of pentacyclic triterpenoids (PTs), which possess diverse biological activities, including anti- viral and antibacterial properties. UA has exceptional anti- HIV activity, which is attributed to its ability to inhibit HIV-1 proteases (123). Both oleanolic acid (OA) and UA possess anti-HCV activity by suppressing the enzymatic activity of HCV NS5B RNA-dependent RNA polymerase, acting as non-competitive inhibitors (124). Recent investigations have revealed that the amidation of the 17-carboxylic acid group of UA yields notable improve- ments in both anti-PRRSV efficacy and cytotoxicity at- tenuation in MARC-145 cells. This modified derivative po- tently inhibited PRRSV infection not only in MARC-145 cells but also in PAMs and PRRSV-infected cells in vivo (125). Moreover, it displayed broad-spectrum inhibitory activities against various PRRSV strains, including the highly patho- genic NADC30-like and GD-XH strains, as well as the clas- sical CH-1a and VR2332 strains in vitro. Mechanistically, the compound exerted its antiviral effects by directly inactivat- ing PRRSV virions, thereby disrupting multiple stages of the viral life cycle, including viral entry, replication, and release, while leaving cellular susceptibility to PRRSV unaffected (125). Xanthohumol Xanthohumol (Xn), a prenylated flavonoid originating from the hop plant Humulus lupulus L., emerges as a natural compound with diverse bioactive properties, (126). Notably, Xn has garnered attention for its anti-inflammatory poten- tial, as demonstrated by its ability to counteract lipopoly- saccharide (LPS)-induced acute lung injury and ischemia reperfusion-induced liver injury in murine models (127). Additionally, Xn exhibits anti-proliferative effects in vari- ous cancer cell lines, including breast, colon, and ovarian cancers (128). The antiviral activity of Xn has also been documented against human immunodeficiency virus (HIV), bovine viral diarrhea virus (BVDV), and HSV-1 and -2 (129). Xn, a prenylated flavonoid compound, displays potent in- hibitory effects against various sub-genotype strains of PRRSV when tested on PAMs (130). Notably, Xn exhibited a low half-maximal inhibitory concentration (IC50), empha- sizing its efficacy in combating PRRSV infections in vitro. Furthermore, Xn treatment led to a reduction in the expres- sion levels of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, and TNF-α, in PAMs infected with PRRSV and those treated with LPS. Animal challenge experiments us- ing highly pathogenic PRRSV infections have shown that Xn effectively mitigates clinical signs, lung pathology, and inflammatory responses in the lung tissues of infected pigs (130). Toosendanin Toosendanin (TSN) is a tetracyclic triterpene derived from the bark and fruit of Melia toosendans Sieb. et Zucc. Traditionally, it has been used as an agricultural insecti- cide and digestive tract parasiticide in China (131). Notably, TSN has demonstrated significant efficacy in combating botulism, as evidenced by in vivo and in vitro studies (132). Subsequent studies have highlighted its potential as an an- ticancer agent with the ability to induce apoptosis in diverse 18 | Slov Vet Res 2024 | Vol 61 No 1 cancer cell types (133). Recently, TSN has garnered atten- tion for its antiviral properties, exhibiting activity against influenza A virus (IAV) (134), HCV (135), severe fever with thrombocytopenia syndrome virus (SFTSV), and SARS- CoV-2 (136). TSN exhibited robust inhibitory effects on the replication of type 2 PRRSV both in vitro, using Marc-145 cells, and ex vivo, using PAMs, even at sub-micromolar concentrations (137). Transcriptomic analyses further elucidated that TSN treatment upregulated IFI16 expression in Marc-145 cells. Additionally, we demonstrated that TSN induces the activa- tion of caspase-1 and maturation of IL-1β through an IFI16- dependent pathway (137). Iota-Carrageenan Carrageenan (CG), a sulfated galactan derived from marine red algae (Rhodophyta), has garnered significant attention because of its various biological activities (138). It is widely recognized as a safe compound by regulatory authorities and has extensive applications in the food, cosmetic, and pharmaceutical industries as a stabilizer, emulsifier, or thickener (139). Previous studies have demonstrated the anticoagulant, antitumor, and immunomodulatory proper- ties of carrageenan (140). Notably, carrageenan exhibits potent inhibitory effects against a range of viruses includ- ing IAV, dengue virus-2 (DENV-2), human rhinovirus (HRV), and HSV-1 (141). Recent investigations have revealed the effectiveness of CG in inhibiting replication of the CH-1a strain of PRRSV at both the mRNA and protein levels in Marc-145 cells and PAMs (142). The antiviral mechanism of CG primarily occurs during viral attachment and entry into the viral life cycle. Moreover, CG hampered viral release in Marc-145 cells and mitigated CH-1a-induced apoptosis during the late stages of infection. Furthermore, CG inhibits CH-1a-induced NF- κB activation, thereby interfering with cytokine production in both Marc-145 cells and PAMs (142). Griffithsin Griffithsin, a lectin derived from marine red algae of Griffithsia spp., is a small protein consisting of 121 amino acids (143). Griffithsin effectively inhibits viral infectiv- ity through its interaction with glycan moieties associated with the glycoproteins of various enveloped viruses (144). Extensive studies have demonstrated the remarkable an- tiviral activity of Griffithsin against several human envel- oped viruses, including HIV (143), Middle East respiratory syndrome coronavirus (MERS-CoV) (145),SARS-CoV (146), HCV (147), HSV-2 (148), and Japanese encephalitis virus (JEV) (149). An exceptional characteristic of Griffithsin is its impressive thermostability, as it remains stable even at temperatures as high as 80°C (150). Griffithsin displays resistance to or- ganic solvents (143) and protease degradation (151), further emphasizing its potential as a therapeutic agent. Moreover, extensive cytotoxicity studies have revealed the superior safety profile of Griffithsin (152). No cytotoxic effects were observed against various cell types, and it demonstrated minimal impact on peripheral blood mononuclear cell acti- vation as well as cytokine and chemokine production (152). Griffithsin demonstrated potent antiviral activity against PRRSV, which was likely mediated by its specific inter- actions with glycans present on the surface of the virus, thereby impeding viral entry. Notably, Griffithsin effectively blocked viral adsorption while leaving viral penetration un- affected. Additionally, Griffithsin exhibited the ability to hin- der cell-to-cell spread, thereby interrupting virus transmis- sion (153). Proanthocyanidin A2 Proanthocyanidins, a class of naturally occurring polyphe- nolic bioflavonoids abundant in various plant sources, such as fruits, vegetables, nuts, seeds, and bark, including grape seeds, have garnered attention for their diverse array of bio- active properties, including antioxidant, cardioprotective, anticancer, antibacterial, antiviral, and anti-inflammatory activities (154). Notably, grape seed-derived proanthocyani- dins have demonstrated significant bioactivity in vitro (155). Of particular interest is Proanthocyanidin A2 (PA2), a dimer- ic form of proanthocyanidin that results from the conden- sation of catechins (156). The antiviral potential of PA2 and its analogs has been highlighted against various viruses, including HSV, Coxsackie B virus (CBV), and canine distem- per virus (CDV) (157). PA2 showed remarkable antiviral activity against PRRSV infection both in vitro (158). Notably, PA2 exhibited broad- spectrum inhibitory effects against traditional genotype II PRRSV strains, such as CH-1a, GD-XH, and GD-HD strains, with comparable potency and EC50 values ranging from 2.2 to 3.2 μg/ml. Treatment with PA2 results in a dose-de- pendent reduction in viral RNA synthesis, viral protein ex- pression, and progeny virus production in PRRSV-infected Marc-145 cells (158). Furthermore, PA2 exerted immunomodulatory effects by suppressing the expression of key cytokines (TNF-α, IFN-α, IL-1β, and IL-6) induced by PRRSV infection in PAMs. This highlights the potential of PA2 in mitigating the in- flammatory response associated with PRRSV infection. Mechanistically, PA2 exhibits multifaceted antiviral mecha- nisms by targeting various pathways, including inhibition of viral entry and blocking progeny virus release (158). Bacterial Compounds Tilmicosin Tilmicosin, a chemically modified macrolide antibiotic derived from tylosin, is an essential veterinary antimicro- bial agent used to treat bacterial infections in animals. Originally synthesized in Streptomyces fradiae, tilmicosin Slov Vet Res 2024 | Vol 61 No 1 | 19 has specifically been formulated for veterinary use in cattle, sheep, and swine. It is available in injectable form for cattle and sheep, whereas a premix feed formulation is utilized for swine (159). The antimicrobial activity of tilmicosin is effective against a wide range of gram-positive and gram- negative bacteria. Additionally, tilmicosin demonstrates ef- ficacy against intracellular bacteria such as Rhodococcus sp. and Mycoplasma sp., making it a valuable therapeutic option in veterinary medicine (160). Limited research has explored the antiviral properties of tilmicosin against PRRSV; however, promising findings have emerged. Previous investigations have demonstrat- ed the dose-dependent inhibitory effects of tilmicosin on PRRSV replication in cultured PAMs (160). Additionally, tylvalosin, a macrolide derivative, exhibits inhibitory activ- ity against both European and North American strains of PRRSV in cultured cells (161). In an experimental setting us- ing PRRSV-infected pigs, the administration of tilmicosin as a feed additive resulted in noticeable reductions in lymph node hypertrophy, lung lesions, and viremia compared with non-medicated infected controls (162). Tilmicosin has demonstrated significant potential for mitigating the severity of PRRSV infections in various ex- perimental settings. In a study involving experimentally PRRSV-infected nursery pigs, tilmicosin treatment yielded notable improvements in disease outcomes, as evidenced by reduced clinical signs, improved feed consumption, and enhanced weight gain, compared to non-medicated chal- lenged pigs. Furthermore, there was a tendency towards lower virus titers in the lungs and serum of tilmicosin-treat- ed pigs (163). Field evaluations of tilmicosin in sows have yielded prom- ising results (164). In one study, the administration of an aqueous form of tilmicosin to nursery pigs in a controlled environment resulted in a 50% reduction in mortality, lower body temperature, a significant increase in average daily gain, and reduced lung lesions in the medicated group compared to the non-treated group (165,166). These find- ings highlight the potential of tilmicosin in improving both clinical outcomes and performance indicators in PRRSV- infected pigs. The accumulation of tilmicosin in macro- phages, the primary target cells for PRRSV replication, may provide a mechanistic explanation for the observed reduc- tion in clinical severity (167). Tulathromycin Tulathromycin (TUL), a triamide compound, possesses unique structural features characterized by a lactone ring containing three polar amine groups. This antimicrobial agent is commonly employed for the treatment and pre- vention of swine respiratory diseases associated with Actinobacillus pleuropneumoniae (App), a gram-negative bacterium frequently found in PRRSV-infected pigs (168). Recent investigations have revealed additional properties of tulathromycin beyond its antimicrobial effects. Studies have demonstrated its ability to inhibit the production of CXCL-8 and LTB4, the key mediators of inflammation, in stimulated neutrophils and macrophages (169). Moreover, tulathromycin promotes apoptotic death of neutrophils and facilitates their phagocytic clearance by macrophages, a crucial process known as efferocytosis, which contributes to the resolution of inflammation (170). TUL also exhibited potent immunomodulatory properties in the absence of any direct antiviral effects against PRRSV. TUL has exhibited an additive effect with PRRSV in inducing macrophage apop- tosis and effectively inhibiting virus-induced necrosis (171). Actinobacillus pleuropneumoniae Porcine pleuropneumonia, a significant disease affecting the swine industry worldwide, is caused by Actinobacillus pleuropneumoniae (App). In recent years, in vitro models using St. Jude Porcine Lung (SJPL) cell line, an immortal- ized epithelial cell line, have been developed to investigate host-pathogen interactions (172). These models have been instrumental in studying co-infections involving App and porcine viral pathogens. Interestingly, during App-PRRSV coinfection of SJPL cells, it was unexpectedly observed that App culture supernatants exhibited robust antiviral activity against PRRSV (173). This finding was further supported by another study that confirmed the antiviral effect of App culture supernatant (174). Antiviral activity of App against PRRSV has also been observed in PAMs (174). Moreover, App inhibits PRRSV replication by inducing cell cycle arrest in the G2/M phase of SJPL cells (175). Fungal Compounds Cryptoporus volvatus The utilization of mushrooms in medical applications has a rich history in Asian countries, and its usage has slightly increased in the Western hemisphere over the past few decades (176). Antiviral properties have been attributed not only to whole mushroom extracts but also to isolated compounds (177). C. volvatus, a member of the Order Aphyllophorales and genus Cryptoporus (178), is found in specific regions of China. The fruiting body of this mush- room has been traditionally employed in the treatment of asthma and bronchitis, with references dating back to the 15th century in the "Materia Medica of Yunnan" (178). Extracts of C. volvatus obtained from various separation processes exhibit differing degrees of inhibitory activity against PRRSV (179). A specific anti-PRRSV component, CM-H-L-5, was isolated from a water-soluble fraction of C. volvatus. The inhibitory effect of CM-H-L-5 against PRRSV was dose-dependent. Chemical analysis revealed that CM- H-L-5 is a low-molecular-weight polyol fragment containing amide and carboxylic acid groups (179). 20 | Slov Vet Res 2024 | Vol 61 No 1 Deoxynivalenol (DON) Mycotoxin Deoxynivalenol (DON), a trichothecene mycotoxin, is pro- duced by various Fusarium spp. molds that are commonly found in feed and other organic substrates. Cereal grains such as wheat, barley, and corn are major sources of DON contamination (180). Pigs, owing to their high grain-based diets, are particularly susceptible to DON toxicity, making them frequently exposed to this mycotoxin (181). In the context of PRRSV infection, it has been observed that DON concentrations ranging from 140 to 280 exert a significant impact on cell survival (182). Specifically, these DON concentrations remarkably increased the survival rate of PRRSV-infected cells. Furthermore, DON at these con- centrations led to a substantial reduction in PRRSV repli- cation. This inhibitory effect is attributed to the induction of pro-inflammatory cytokines and the early activation of apoptosis. These mechanisms appear to interrupt the viral replication cycle and impede PRRSV propagation within the host (182). Animal-Derived Compounds Honeybee Venom Honeybee (Apis mellifera Linnaeus 1758) venom (HBV) is recognized as an alternative medicine owing to its thera- peutic properties, particularly in the management of pain, inflammation, and immune-related conditions such as rheumatoid arthritis and multiple sclerosis (183). Notably, HBV has demonstrated immunomodulatory effects on the Th1 immune response. Administration of HBV leads to the differentiation of CD4+ T lymphocytes into Th1 cells, there- by enhancing the production of interferon-gamma (IFN-γ) in mouse models (184). Furthermore, HBV phospholipase plays an important role in the maturation of dendritic cells and subsequent activation of dendritic cell-associated im- mune responses (185,186). Investigations have been conducted to explore the potential antiviral activity of HBV against PRRSV. In a recent study, HBV was administered to healthy pigs via nasal, neck, and rectal routes, followed by intranasal inoculation with PRRSV (187). Significantly increased levels of CD4+/CD8+ cell ratio, IFN-γ, and IL-12 were observed in HBV-administered pigs via nasal and rectal administration. In pigs experimentally challenged with PRRSV, the viral genome load in the serum, lung, bronchial lymph nodes, and tonsils was significantly reduced, accompanied by mitigation of interstitial pneumo- nia severity in the nasal and rectal administration groups. Moreover, HBV administration leads to a substantial eleva- tion in the levels of Th1 cytokines (IFN-γ and IL-12) and up- regulation of pro-inflammatory cytokines (TNF-α and IL-1β) (187). Caprylic Monoglyceride Medium-chain fatty acids (MCFAs), including caprylic monoglycerides (CMG), are a class of fatty acids with carbon chain lengths ranging from 8 to 10 carbon atoms. While MCFAs are present in small quantities in nature, they are primarily derived from milk and breast milk and can also be found in palm kernel oil and coconut oil (188). Notably, MCFAs possess antimicrobial properties and their effects on animal productivity vary depending on the dosage em- ployed (189). One significant application of MCFAs is their ability to miti- gate the transmission of the porcine epidemic diarrhea vi- rus (PEDV) through feed and ingredients (190). Additionally, MCFAs have been demonstrated to influence the growth performance of animals by serving as readily available en- ergy substrates, modulating gastrointestinal morphology, and exerting antimicrobial effects (191). MCFAs as a feed additive can also suppress African swine fever virus (ASFV) infection(192). In light of the potential antiviral and antimicrobial effects of MCFAs, we investigated their antiviral activity against PRRSV. Recently, a study evaluated the cytotoxicity of four MCFAs, namely caprylic acid, CMG, decanoic monoglycer- ide, and monolaurin, along with their inhibitory effects on PRRSV. The results demonstrated that CMG exhibited the lowest toxicity towards cells among the four MCFAs, while displaying the highest inhibition rate against PRRSV (193). To further assess the impact of CMG on PRRSV infection, piglets were treated with varying concentrations of CMG, revealing a significant decrease in mortality and viral load following PRRSV infection in piglets administered higher CMG concentrations (p < 0.05). Additionally, the pulmonary pathology in piglets was ameliorated by CMG treatment. Notably, CMG administration resulted in a significant down- regulation of pro-inflammatory cytokines, including IL-6, IL- 8, IL-1β, IFN-γ, and TNF-α, while upregulating the levels of the anti-inflammatory cytokine IL-10 in comparison to the positive control group (p < 0.05) (193). Protegrin-1 Antimicrobial peptides (AMPs), including protegrin-1 (PG-1), are polypeptides of less than 100 amino acids (194). AMPs are found in both plant and animal kingdoms and exhibit broad-spectrum antimicrobial activity against bacteria, fun- gi, and viruses involved in the innate immune response to infection (195). PG-1, originally isolated from porcine leuko- cytes (196), is considered to be an antibiotic agent against Gram-positive and Gram-negative bacteria and fungi in vi- tro (197). Furthermore, previous studies have shown that PG-1 inhibits dengue NS2B-NS3 serine protease and viral replication in MK2 cells (198). PG-1 also strongly inhibits PRRSV infection and replication by suppressing viral RNA and protein synthesis, virus prog- eny production, and viral particle release. Furthermore, dur- ing the PRRSV life cycle, PG-1 mainly blocked viral attach- ment in Marc-145 cells. However, in PAMs, PG-1 neither inhibits PRRSV replication nor elevates antiviral cytokine expression (199). Slov Vet Res 2024 | Vol 61 No 1 | 21 Porcine Plasma Ficoline Ficolins are proteins that activate the complement system and exhibit the ability to bind N-acetyl groups in various saccharides, particularly N-acetylglucosamine (GlcNAc) (200). This suggests that ficolins may also have the capac- ity to bind certain viruses that display host glycans on their surfaces (201). Viral glycoproteins often possess complex-type oligosac- charides that are characterized by two terminal GlcNAc res- idues (202). Similar collagenous lectins have been shown to bind glycoproteins in IAV, HIV, HSV, and non-enveloped rotavirus (RV) (203). In a recent study, the antiviral activity of plasma-purified and recombinant ficolin α was assessed against PRRSV. The results revealed a reduction in the cy- topathic effect of PRRSV-infected Marc-145 cells and inhi- bition of viral replication in the presence of ficolin α, which is dependent on GlcNAc recognition. Additionally, plasma ficolin α and recombinant ficolin α bind to PRRSV-coated wells in a GlcNAc-dependent manner (204). Cecropin P1 Cecropin P1 (CP1) is a small antimicrobial peptide originally derived from the intestine of pigs, and it has demonstrated antiviral activity against various viruses, including infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, snakehead rhabdovirus, and infectious pancreatic necrosis virus, in in vitro studies (205). CP1 exhibits signifi- cant antiviral effects against PRRSV, both as an extracel- lular virucidal agent and as an inhibitor, when administered prior to, simultaneously with, or following viral inoculation. The inhibitory mechanism of CP1 primarily targets viral at- tachment rather than viral entry into Marc-145 cells (206). Moreover, CP1 effectively impeded viral particle release and mitigated virus-induced apoptosis during the late stages of infection. The inhibitory action of CP1 against PRRSV was also extended to PAMs in vivo. Additionally, CP1 upregu- lates the expression of IL6 in PAMs, which could potentially contribute to its ability to inhibit PRRSV infection (206). Cecropin D Cecropin D (CD) is an antimicrobial peptide originally de- rived from Hyalophora cecropia Linnaeus 1758 pupae, and it has been previously demonstrated to possess antibacte- rial activity against both Gram-positive and Gram-negative bacteria, including Escherichia coli DH5α, K88, K99, Streptococcus zooepidemicus C55138, and Staphylococcus aureus Cowan I (207). In the context of PRRSV infection, CD exerted inhibitory effects during viral attachment and the early stages of viral entry into Marc-145 cells. Furthermore, CD effectively suppressed virus-induced apoptosis during the late phase of PRRSV infection and attenuated viral re- lease within cells. These observations collectively contrib- ute to the inhibition of PRRSV infection by CD. Importantly, similar inhibitory effects against PRRSV infection are evi- dent when CD is utilized in PAMs during in vivo infection in pigs (208). Conclusions This comprehensive review highlights the potential of natu- ral compounds derived from plants, animals, bacteria, and fungi as effective antiviral agents against PRRSV. These compounds exhibit diverse mechanisms of action target- ing various stages of the PRRSV replication cycle (attach- ment, entry, fusion, replication, translation, maturation, and release). These compounds have shown promising broad- spectrum antiviral activities both in vitro and in vivo. Although significant progress has been made in the field of natural compounds with antiviral activity against PRRSV, several research gaps still need to be addressed. First, fur- ther studies are needed to elucidate the precise mecha- nisms by which these natural compounds exert their an- tiviral effects. Understanding the molecular interactions between these compounds and PRRSV components will provide valuable insights for the development of more targeted interventions. Second, comprehensive investiga- tions on the safety, pharmacokinetics, and toxicity profiles of these natural compounds are essential. These studies will help determine the optimal dosage and administration routes as well as evaluate potential side effects, ensur- ing their safe use in veterinary medicine. Finally, there is a need for more comprehensive studies to evaluate the ef- ficacy of combination therapies using natural compounds. Investigating the synergistic effects of combining differ- ent compounds or combining them with existing antiviral drugs may enhance overall antiviral efficacy and reduce the emergence of drug-resistant viral strains. Future research should explore the application of advanced technologies, such as nanotechnology and targeted deliv- ery systems, to enhance the bioavailability and therapeutic potential of these natural compounds. These innovative ap- proaches may improve compound stability, increase tissue specificity, and enhance the antiviral efficacy. Further re- search addressing the aforementioned research gaps and exploring new avenues, combined with rigorous preclinical and clinical trials, will accelerate the translation of these natural compounds into effective antiviral therapies for the control and prevention of PRRSV infections in swine. Acknowledgments The author would like to thank the DOST S&T Fellows Program, the Philippine Council for Agriculture, Aquatic and Natural Resources Research and Development (DOST- PCAARRD) for funding this research project, and the Industrial Technology Development Institute (DOST-ITDI) for hosting this research project. Conflict of Interest. 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