Stability and Biaxial Behavior of Fresh Cheese Coated with Nanoliposomes Encapsulating Grape Seed Tannins and Polysaccharides Using Immersion and Spray Methods.

In the food industry context, where fresh cheese stands out as a highly perishable product with a short shelf life, this study aimed to extend its preservation through multi-layer edible coatings. The overall objective was to analyze the biaxial behavior and texture of fresh cheese coated with nanoliposomes encapsulating grape seed tannins (NTs) and polysaccharides (hydroxypropyl methylcellulose; HPMC and kappa carrageenan; KC) using immersion and spray methods, establishing comparisons with uncoated cheeses and commercial samples, including an accelerated shelf-life study. NT, HPMC, and KC were employed as primary components in the multi-layer edible coatings, which were applied through immersion and spray. The results revealed significant improvements, such as a 20% reduction in weight loss and increased stability against oxidation, evidenced by a 30% lower peroxide index than the uncoated samples. These findings underscore the effectiveness of edible coatings in enhancing the quality and extending the shelf life of fresh cheese, highlighting the innovative application of nanoliposomes and polysaccharide blends and the relevance of applying this strategy in the food industry. In conclusion, this study provides a promising perspective for developing dairy products with improved properties, opening opportunities to meet market demands and enhance consumer acceptance.

Physicochemical Properties of Nanoliposomes Encapsulating Grape Seed Tannins Formed with Ultrasound Cycles.

Grape seeds are an excellent source of flavonoids and tannins with powerful antioxidant properties. However, the astringency of tannins limits their direct incorporation into food. To overcome this challenge, we investigated the encapsulation of grape seed tannins within nanoliposomes formed by ultrasound cycling. We characterized the nanoliposomes' physicochemical properties, including encapsulation efficiency, antioxidant activity, stability, microstructure, and rheological properties. Our findings reveal that the nanoliposomes exhibited excellent stability under refrigerated conditions for up to 90 days with a mean particle size of 228 ± 26 nm, a polydispersity index of 0.598 ± 0.087, and a zeta potential of -41.6 ± 1.30 mV, maintaining a spherical multilamellar microstructure. Moreover, they displayed high antioxidant activity, with encapsulation efficiencies of 79% for epicatechin and 90% for catechin. This innovative approach demonstrates the potential of using ultrasound-assisted nanoliposome encapsulation to directly incorporate grape seed tannins into food matrices, providing a sustainable and efficient method for enhancing their bioavailability and functionality.

Mechanical and Surface Properties of Edible Coatings Elaborated with Nanoliposomes Encapsulating Grape Seed Tannins and Polysaccharides.

Edible composite coatings (ECC) formulated from biopolymers that incorporate antioxidant molecules represent an innovative alternative to improve food texture and provide health benefits. Tannins have aroused great interest due to their ability to stabilize suspensions and counteract the effects of free radicals. The mechanical and surface properties are crucial to establishing its quality and applicability. In this study, the objective was to analyze the mechanical and surface properties of ECC made with nanoliposomes that encapsulate grape seed tannins (TLS) and polysaccharides such as hydroxypropylmethylcellulose (HPMC) and kappa carrageenan (KCG) for their future direct application in foods susceptible to oxidation. The inclusion of HPMC or KCG affected the density, showing values in the range of 1010 to 1050 [kg/m3], evidencing significant changes (p < 0.05) in the surface tension in the TLS/FS-HPMC and TLS/FS mixtures. KCG and in the dispersion coefficients, with values in the range of -2.9 to -17.6 [mN/m] in HPS (S1) and -17.6 to -40.9 [mN/m] in PDMS (S2). The TLS/FS-HPMC coating showed higher stiffness and elastic recovery capacity than the TLS/FS-KCG coating, suggesting that the presence of TLS influenced the stiffness of the polymer. HPMC is recommended as a suitable polymer for coating solids, while KCG is more appropriate for suspensions. These findings provide valuable information for directly applying these ECC compounds to food products, potentially offering better preservation and health benefits.

A Ferritin Nanoparticle-Based Zika Virus Vaccine Candidate Induces Robust Humoral and Cellular Immune Responses and Protects Mice from Lethal Virus Challenge.

The severe consequences of the Zika virus (ZIKV) infections resulting in congenital Zika syndrome in infants and the autoimmune Guillain-Barre syndrome in adults warrant the development of safe and efficacious vaccines and therapeutics. Currently, there are no approved treatment options for ZIKV infection. Herein, we describe the development of a bacterial ferritin-based nanoparticle vaccine candidate for ZIKV. The viral envelope (E) protein domain III (DIII) was fused in-frame at the amino-terminus of ferritin. The resulting nanoparticle displaying the DIII was examined for its ability to induce immune responses and protect vaccinated animals upon lethal virus challenge. Our results show that immunization of mice with a single dose of the nanoparticle vaccine candidate (zDIII-F) resulted in the robust induction of neutralizing antibody responses that protected the animals from the lethal ZIKV challenge. The antibodies neutralized infectivity of other ZIKV lineages indicating that the zDIII-F can confer heterologous protection. The vaccine candidate also induced a significantly higher frequency of interferon (IFN)-γ positive CD4 T cells and CD8 T cells suggesting that both humoral and cell-mediated immune responses were induced by the vaccine candidate. Although our studies showed that a soluble DIII vaccine candidate could also induce humoral and cell-mediated immunity and protect from lethal ZIKV challenge, the immune responses and protection conferred by the nanoparticle vaccine candidate were superior. Further, passive transfer of neutralizing antibodies from the vaccinated animals to naïve animals protected against lethal ZIKV challenge. Since previous studies have shown that antibodies directed at the DIII region of the E protein do not to induce antibody-dependent enhancement (ADE) of ZIKV or other related flavivirus infections, our studies support the use of the zDIII-F nanoparticle vaccine candidate for safe and enhanced immunological responses against ZIKV.

Combining edible coatings technology and nanoencapsulation for food application: A brief review with an emphasis on nanoliposomes.

The use of bioactive compounds within the biopolymer-based Edible Coatings (EC) matrices has certain limitations for their application at the food industry level. Encapsulation has been considered as a strategy that enables protecting and improving the physical and chemical characteristics of the compounds; as a result, it extends the shelf life of coated foods. This review discusses recent progress in combining edible coatings with nanoencapsulation technology. We also described and discussed various works, in which nanoliposomes are used as encapsulation systems to prepare, and subsequently apply the edible coatings in plant products and meat products. The use of nanoliposomes for the encapsulation of phenolic compounds and essential oils provides an improvement in the antioxidant and antimicrobial properties of coatings by extending the shelf life of food matrices. However, when liposomes are stored for a long period of time, they may present some degree of instability manifested by an increase in size, polydispersity index, and zeta potential. This is reflected in an aggregation, fusion, and rupture of the vesicles. This investigation can help researchers and industries to select an appropriate and efficient biopolymer to form EC containing nanoencapsulated active compounds. This work also addresses the use of nanoliposomes to create EC extending markedly the shelf life of fruit, reducing the weight loss, and deterioration due to the action of microorganisms.

Effect of lyophilization on the physicochemical and rheological properties of food grade liposomes that encapsulate rutin.

The potential use of liposomes as carriers for food active ingredients can be limited by their physical and chemical instabilities in aqueous dispersions, especially for long-term storage. Lyophilization, a process commonly used in the food industry, can also be applied to stabilize and preserve liposomes and to extend their shelf-life. In this work, liposomes with potential use for designing functional foods were prepared with soy phospholipids and rutin. Homogenization and ultrasound were used for particle size reduction. Liposomal stability was evaluated by Dynamic Light Scattering, microscopy and rheological properties. Spherical and unilamellar liposomes were obtained in this work. Zeta potential (ξ = values were around -40 mV), which indicates a great suspension stability even for more than 30 days of storage. Rutin exerted a protective effect by both preventing damage to the liposome bilayer and maintaining the spherical structure after 56 days of storage. Lyophilization caused an increase in the size of the vesicles, reaching sizes around 419 nm and aggregation of vesicles with probably structural damage after 21 storage days. However, it helped to keep the rutin encapsulated (81.9%) for longer time, when compared to refrigerated liposomes. Rheological measurements showed, in general, that the power law model fitted most of the experimental results and dynamic rheological tests showed a sol-gel phase transition between 35 and 45 °C. Lyophilization caused a significant change in all evaluated rheological parameters. For the in vitro release tests, the liposomal bilayer acted as a barrier for the rutin release to the food simulating medium; therefore, the release rate of the antioxidant from the rutin encapsulated liposome was slow compared to the free rutin release rate.

Influence of gelation on the retention of purple cactus pear extract in microencapsulated double emulsions.

A purple cactus pear (Opuntia ficus-indica) extract (CP) was encapsulated in double emulsions (DE) gelled with gelatin (DE-CP-G) and with gelatin and transglutaminase (DE-CP-GT), as well as in a DE with a liquid external aqueous phase (DE-CP), in order to study the retention of betanin as colorant agent. Both gelled DEs showed a predominantly elastic behavior, in contrast with DE-CP. The degradation rate constant of betanin was significantly higher in DE-CP-GT (90.2 x 10-3 days-1) than in DE-CP-G (11.0 x 10-3 days-1) and DE-CP (14.6 x 10-3 days-1) during cold-storage (4 °C). A shift towards yellow color was found in all the systems during cold-storage (4 °C) and after thermal treatment (70°C/30 min), especially in DE-CP-GT, denoting a higher degradation of betanin. Betalamic acid, cyclo-Dopa 5-O-ß-glucoside, 17-decarboxy-betanin and neobetanin were identified by UHPLC-MS/MS as degradation products of betanin.

Humectability and physical properties of hydroxypropyl methylcellulose coatings with liposome-cellulose nanofibers: Food application.

The objective of this study was to investigate the physical, rheological and humectability properties of edible coating forming suspensions (ECS) based on hydroxypropyl methylcellulose (HPMC) containing: liposomes that encapsulate rutin, glycerol and cellulose nanofibers on sliced surfaces of almonds and chocolate. On average, liposomes measured between 110.6 ±â€¯10.0 nm and were characterized as stable and homogeneous suspensions. Adding these liposomes to the edible coatings produced significant changes (p< 0.05) in the density and surface tension, which favor the final appearance of the coating. The presence of liposomes increased the apparent viscosity of the ECS, showing a purely viscous and fluid behavior with a good fit (R2 = 0.9996) with the Power Law model. The presence of liposomes and cellulose nanofibers decreased the value of the cohesive energy of the ECS. The studied ECS partially hydrate the surfaces of almond and chocolate as they showed contact angles under 90°.

Discovery of a non-nucleoside RNA polymerase inhibitor for blocking Zika virus replication through in silico screening.

Zika virus (ZIKV), an emerging arbovirus, has become a major human health concern globally due to its association with congenital abnormalities and neurological diseases. Licensed vaccines or antivirals against ZIKV are currently unavailable. Here, by employing a structure-based approach targeting the ZIKV RNA-dependent RNA polymerase (RdRp), we conducted in silico screening of a library of 100,000 small molecules and tested the top ten lead compounds for their ability to inhibit the virus replication in cell-based in vitro assays. One compound, 3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide (TPB), potently inhibited ZIKV replication at sub-micromolar concentrations. Molecular docking analysis suggests that TPB binds to the catalytic active site of the RdRp and therefore likely blocks the viral RNA synthesis by an allosteric effect. The IC50 and the CC50 of TPB in Vero cells were 94 nM and 19.4 µM, respectively, yielding a high selective index of 206. In in vivo studies using immunocompetent mice, TPB reduced ZIKV viremia significantly, indicating TPB as a potential drug candidate for ZIKV infections.

Development of a broadly protective modified-live virus vaccine candidate against porcine reproductive and respiratory syndrome virus.

Modified-live virus (MLV) vaccines are widely used to protect pigs against porcine reproductive and respiratory syndrome virus (PRRSV). However, current MLV vaccines do not confer adequate levels of heterologous protection, presumably due to the substantial genetic diversity of PRRSV isolates circulating in the field. To overcome this genetic variation challenge, we recently generated a synthetic PRRSV strain containing a consensus genomic sequence of PRRSV-2. We demonstrated that our synthetic PRRSV strain confers unprecedented levels of heterologous protection. However, the synthetic PRRSV strain at passage 1 (hereafter designated CON-P1) is highly virulent and therefore, is not suitable to be used as a vaccine in pigs. In the present study, we attenuated CON-P1 by continuously passaging the virus in MARC-145 cells, a non-natural host cell line. Using a young pig model, we demonstrated that the synthetic virus at passages 90 and 122 (designated as CON-P90 and CON-P122, respectively) were fully attenuated, as evidenced by the significantly reduced viral loads in serum and tissues and the absence of lung lesion in the infected pigs. Most importantly, CON-P90 confers similar levels of heterologous protection as its parental strain CON-P1. Taken together, the results indicate that CON-P90 is an excellent candidate for the formulation of next generation of PRRSV MLV vaccines with improved levels of heterologous protection.

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion.

Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

Relative contribution of porcine reproductive and respiratory syndrome virus open reading frames 2-4 to the induction of protective immunity.

The minor glycoproteins (GPs) of PRRSV, GP2, GP3, and GP4, form a heterotrimer that is required for viral infectivity, presumably due to its interaction with the key cellular receptor CD163. These 3GPs are encoded by open reading frames (ORFs) 2a, 3 and 4 (herein referred to as ORFs 2-4), respectively. The goal of this study was to investigate the immunogenicity of the PRRSV-2 minor GPs. Through the use of reverse genetics, a chimeric virus (designated SDFL24) was constructed by replacing ORFs 2-4 of the PRRSV-1 strain SD01-08 with the corresponding genes of the PRRSV-2 strain FL12. While the parental PRRSV strain SD01-08 was not neutralized by convalescent antisera raised against FL12, the chimeric virus SDFL24 gained susceptibility to neutralization by FL12-specific antisera, indicating that viral proteins encoded by ORFs 2-4 are targets of antibody neutralization. When inoculated into pigs, the chimeric virus SDFL24 elicited T-cell responses against peptides derived from FL12 minor GPs, whereas the parental virus SD01-08 did not. After challenge infection with FL12, pigs previously infected with SDFL24 developed robust kinetics of FL12-specific neutralizing antibodies as compared to those previously infected with the parental strain SD01-08. Finally, the pigs recovered from SDFL24 infection were better protected from a subsequent challenge infection with FL12 than those previously infected with SD01-08. Collectively, the results indicate that PRRSV-2 ORFs 2-4 are capable of inducing protective immunity.

Cross reactivity of immune responses to porcine reproductive and respiratory syndrome virus infection.

Because porcine reproductive and respiratory syndrome virus (PRRSV) exhibits extensive genetic variation among field isolates, characterizing the extent of cross reactivity of immune responses, and most importantly cell-mediated immunity (CMI), could help in the development of broadly cross-protective vaccines. We infected 12 PRRSV-naïve pigs with PRRSV strain FL12 and determined the number of interferon (IFN)-γ secreting cells (SC) by ELISpot assay using ten type 2 and one type 1 PRRSV isolates as recall antigens. The number of IFN-γ SC was extremely variable among animals, and with exceptions, late to appear. Cross reactivity of IFN-γ SC among type 2 isolates was broad, and we found no evidence of an association between increased genetic distance among isolates and the intensity of the CMI response. Comparable to IFN-γ SC, total antibodies evaluated by indirect immunofluorescence assay (IFA) were cross reactive, however, neutralizing antibody titers could only be detected against the strain used for infection. Finally, we observed a moderate association between homologous IFN-γ SC and neutralizing antibodies.

Strategies to broaden the cross-protective efficacy of vaccines against porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viral pathogens currently affecting swine production worldwide. Although PRRS vaccines have been commercially available for over 20 years, the available vaccines are considered inadequately effective for control and eradication of the virus. Major obstacles for the development of a highly effective PRRS vaccine include the highly variable nature of the viral genome, the viral ability to subvert the host immune system, and the incomplete understanding of the immune protection against PRRSV infection. This article summarizes the impediments for the development of a highly protective PRRS vaccine and reviews the vaccinology approaches that have been attempted to overcome one of the most formidable challenges, which is the substantial genetic variation among PRRSV isolates, to broaden the antigenic coverage of PRRS vaccines.

Identification of viral genes associated with the interferon-inducing phenotype of a synthetic porcine reproductive and respiratory syndrome virus strain.

We recently generated a fully synthetic porcine reproductive and respiratory syndrome virus strain (designated as PRRSV-CON), which confers unprecedented levels of heterologous protection. We report herein that the synthetic PRRSV-CON possesses a unique phenotype in that it induces type-I interferons (IFNs) instead of suppressing these cytokines as most of the naturally occurring PRRSV isolates do. Through gain- and loss- of-function studies, the IFN-inducing phenotype of PRRSV-CON was mapped to the 3.3kb genomic fragment encoding three viral nonstructural proteins: nsp1α, nsp1ß and the N-terminal part of nsp2. Further studies indicated that a cooperation among these 3 proteins was required for effective induction of IFNs. Collectively, this study constitutes the first step toward understanding the mechanisms by which the synthetic PRRSV-CON confers heterologous protection.

Complete Genome Sequence of Highly Virulent Porcine Reproductive and Respiratory Syndrome Virus Variants That Recently Emerged in the United States.

A recent outbreak of particularly virulent disease caused by porcine reproductive and respiratory syndrome virus has occurred in swine herds across the United States. We report here the complete genome sequence of eight viral isolates from four Nebraska herds experiencing an outbreak of severe disease in 2016.

A Synthetic Porcine Reproductive and Respiratory Syndrome Virus Strain Confers Unprecedented Levels of Heterologous Protection.

UNLABELLED: Current vaccines do not provide sufficient levels of protection against divergent porcine reproductive and respiratory syndrome virus (PRRSV) strains circulating in the field, mainly due to the substantial variation of the viral genome. We describe here a novel approach to generate a PRRSV vaccine candidate that could confer unprecedented levels of heterologous protection against divergent PRRSV isolates. By using a set of 59 nonredundant, full-genome sequences of type 2 PRRSVs, a consensus genome (designated PRRSV-CON) was generated by aligning these 59 PRRSV full-genome sequences, followed by selecting the most common nucleotide found at each position of the alignment. Next, the synthetic PRRSV-CON strain was generated through the use of reverse genetics. PRRSV-CON replicates as efficiently as our prototype PRRSV strain FL12, both in vitro and in vivo. Importantly, when inoculated into pigs, PRRSV-CON confers significantly broader levels of heterologous protection than does wild-type PRRSV. Collectively, our data demonstrate that PRRSV-CON can serve as an excellent candidate for the development of a broadly protective PRRSV vaccine. IMPORTANCE: The extraordinary genetic variation of RNA viruses poses a monumental challenge for the development of broadly protective vaccines against these viruses. To minimize the genetic dissimilarity between vaccine immunogens and contemporary circulating viruses, computational strategies have been developed for the generation of artificial immunogen sequences (so-called "centralized" sequences) that have equal genetic distances to the circulating viruses. Thus far, the generation of centralized vaccine immunogens has been carried out at the level of individual viral proteins. We expand this concept to PRRSV, a highly variable RNA virus, by creating a synthetic PRRSV strain based on a centralized PRRSV genome sequence. This study provides the first example of centralizing the whole genome of an RNA virus to improve vaccine coverage. This concept may be significant for the development of vaccines against genetically variable viruses that require active viral replication in order to achieve complete immune protection.

Innate and adaptive immunity against Porcine Reproductive and Respiratory Syndrome Virus.

Many highly effective vaccines have been produced against viruses whose virulent infection elicits strong and durable protective immunity. In these cases, characterization of immune effector mechanisms and identification of protective epitopes/immunogens has been informative for the development of successful vaccine programs. Diseases in which the immune system does not rapidly clear the acute infection and/or convalescent immunity does not provide highly effective protection against secondary challenge pose a major hurdle for clinicians and scientists. Porcine reproductive and respiratory syndrome virus (PRRSV) falls primarily into this category, though not entirely. PRRSV causes a prolonged infection, though the host eventually clears the virus. Neutralizing antibodies can provide passive protection when present prior to challenge, though infection can be controlled in the absence of detectable neutralizing antibodies. In addition, primed pigs (through natural exposure or vaccination with a modified-live vaccine) show some protection against secondary challenge. While peripheral PRRSV-specific T cell responses have been examined, their direct contribution to antibody-mediated immunity and viral clearance have not been fully elucidated. The innate immune response following PRRSV infection, particularly the antiviral type I interferon response, is meager, but when provided exogenously, IFN-α enhances PRRSV immunity and viral control. Overall, the quality of immunity induced by natural PRRSV infection is not ideal for informing vaccine development programs. The epitopes necessary for protection may be identified through natural exposure or modified-live vaccines and subsequently applied to vaccine delivery platforms to accelerate induction of protective immunity following vaccination. Collectively, further work to identify protective B and T cell epitopes and mechanisms by which PRRSV eludes innate immunity will enhance our ability to develop more effective methods to control and eliminate PRRS disease.

Protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein 5.

Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses to the swine industry worldwide. Although inactivated and live vaccines are commercially available for the control of PRRS, both types of vaccine have not always proven successful in terms of generating a protective immune response, particularly in the case of inactivated vaccines. In this study, we tested whether an inactivated vaccine could induce a humoral immune response to PRRS during a homologous challenge. Amino acid substitutions were introduced into glycoprotein (GP) 5 of the FL12 strain of the PRRS virus (PRRSV) using site-directed mutagenesis with a pFL12 infectious clone. The substitutions led to double deglycosylation in the putative glycosylation moieties on GP5. The mutant virus was subsequently inactivated with binary ethylenimine. The efficacy of the inactivated mutant virus was compared with that of the inactivated wild-type PRRSV. Only the inactivated mutant PRRSV induced serum neutralizing antibodies at six weeks post-vaccination. The group that was administered the inactivated mutant virus twice exhibited a significantly increased neutralizing antibody titer after a challenge with the virulent homologous strain and exhibited more rapid clearing of viremia compared to other groups, including the groups that were administered either the inactivated mutant or wild-type virus only once and the group that was administered the inactivated wild-type virus twice. Histopathological examination of lung tissue sections revealed that the group that was administered the inactivated mutant virus twice exhibited significantly thinner alveolar septa, whereas the thickness of the alveolar septa of the other groups were markedly increased due to lymphocyte infiltration. These results indicated that the deglycosylation of GP5 enhanced the immunogenicity of the inactivated mutant PRRSV and that twice administrations of the inactivated mutant virus conferred better protection against the homologous challenge. These findings suggest that the inactivated PRRSV that expresses a hypo-glycosylated GP5 is a potential inactivated vaccine candidate and a valuable tool for controlling PRRS for the swine industry.