Epitope screening and vaccine molecule design of PRRSV GP3 and GP5 protein based on immunoinformatics.

Porcine reproductive and respiratory syndrome (PRRS) is a respiratory disease in pigs that causes severe economic losses. Currently, live PRRSV vaccines are commonly used but fail to prevent PRRS outbreaks and reinfection. Inactivated PRRSV vaccines have poor immunogenicity, making PRRSV a significant threat to swine health globally. Therefore, there is an urgent need to develop an effective PRRSV vaccine. This study used immunoinformatics to predict, screen, design and construct a candidate vaccine that fused B-cell epitopes, CTL- and HTL-dominant protective epitopes of PRRSV strain's GP3 and GP5 proteins. The study identified 12 B-cell epitopes, 6 CTL epitopes and 5 HTL epitopes of GP3 and GP5 proteins. The candidate vaccine was constructed with 50S ribosomal protein L7/L1 molecular adjuvant, which has antigenicity, solubility, stability, non-allergenicity and a high affinity for its target receptor, TLR-3. The C-ImmSim immunostimulation results showed significant increases in cellular and humoral responses (B cells and T cells) and production of TGF-ß, IL-2, IL-10, IFN-γ and IL-12. The constructed vaccine was stable and immunogenic, and it can effectively induce strong T-cell and B-cell immune responses against PRRSV. Therefore, it is a promising candidate vaccine for controlling and preventing PRRSV outbreaks.

Effects of tracker location on the accuracy and precision of the Gazepoint GP3 HD for spectacle wearers.

Remote view eye-tracking systems are prone to errors when used on spectacle wearers due to reflections from the lenses and frame that result in inaccurate tracking. Traditionally, these trackers are situated below a computer monitor and the viewer's eye moments are recorded while they view the screen. Reflections may be influenced by the pantoscopic tilt of the spectacles, whereby the tilt angle causes incident light to be reflected to the camera. To overcome this problem, we propose mounting the tracker above the monitor to avoid these reflections and test the accuracy and precision of subjects with single vision spectacles, multifocals, and no correction, using both mounting positions. Experimental results showed that this alternate position had overall worse accuracy (4.06° ± 0.13) and precision (0.67° ± 0.05) compared to the standard configuration (2.15° ± 0.06 vs. 0.50° ± 0.03), with more invalid readings (5.91 vs. 19.19%) for single vision lens wearers. Multifocals performed better for the top-mounting position for the top portion of the monitor, suggesting higher-order aberrations from the bottom portion of the lens negatively impact data quality. Higher pantoscopic tilt angles displayed an improved accuracy for this alternate position (r(9) = - 0.69, p = 0.02), with superior accuracy for tilt angles greater than 14° compared to the standard configuration. This study quantifies the impact of spectacle wear on eye-tracking performance and suggests other alternate mounting positions may be viable in certain situations.

Validation of Gazepoint low-cost eye-tracking and psychophysiology bundle.

Eye-tracking and recording of physiological signals are increasingly used in research within cognitive science and human-computer interaction. For example, gaze position and measures of autonomic arousal, including pupil dilation, skin conductance (SC), and heart rate (HR), provide an indicator of cognitive and physiological processes. The growing popularity of these techniques is partially driven by the emergence of low-cost recording equipment and the proliferation of open-source software for data collection and analysis of such signals. However, the use of new technology requires investigation of its reliability and validation with respect to real-world usage and against established technologies. Accordingly, in two experiments (total N = 69), we assessed the Gazepoint GP3-HD eye-tracker and Gazepoint Biometrics (GPB) system from Gazepoint. We show that the accuracy, precision, and robustness of the eye-tracker are comparable to competing systems. While fixation and saccade events can be reliably extracted, the study of saccade kinematics is affected by the low sampling rate. The GP3-HD is also able to capture psychological effects on pupil dilation in addition to the well-defined pupillary light reflex. Finally, moderate-to-strong correlations between physiological recordings and derived metrics of SC and HR between the GPB and the well-established BIOPAC MP160 support its validity. However, low amplitude of the SC signal obtained from the GPB may reduce sensitivity when separating phasic and tonic components. Similarly, data loss in pulse monitoring may pose difficulties for certain HR variability analyses.

Porcine Reproductive and Respiratory Syndrome Virus Structural Protein GP3 Regulates Claudin 4 To Facilitate the Early Stages of Infection.

Claudins (CLDN) are a family of proteins that represent the most important components of tight junctions, where they establish the paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. Several types of viruses make full use of CLDN to facilitate entry into cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry. In this study, we found that CLDN4 functions as an anti-PRRSV factor by blocking its absorption during the early stages of infection. The small extracellular loop (ECL2) of CLDN4 restricted the viral particles outside cells by binding to GP3. A novel function of GP3-mediated regulation of CLDN4 transcription was suggested. CLDN4 can be decreased through downregulating the level of CLDN4 transcription by ubiquitinating the transcription factor, SP1. The mechanism by which highly pathogenic PRRSV infects the epithelium was proposed. Importantly, ECL2 was found to block PRRSV absorption and infection and neutralize the virus. A more in-depth understanding of PRRSV infection is described, and novel therapeutic antiviral strategies are discussed.IMPORTANCE In the present study, the role of CLDN4 in PRRSV infection was studied. The results showed that CLDN4 blocked absorption into cells and restricted extracellular viral particles via the interaction between the CLDN4 small extracellular loop, ECL2, and the viral surface protein GP3. GP3 was found to downregulate CLDN4 through ubiquitination of the transcription factor SP1 to facilitate viral entry. The mechanism by which highly pathogenic PRRSV infects the epithelium is suggested. A novel function of GP3 in regulating gene transcription was discovered. Moreover, ECL2 could block PRRSV absorption and infection, as well as neutralizing the virus in the supernatant, which may lead to the development of novel therapeutic antiviral strategies.

Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology.

Glycoprotein 3 (GP3) of the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly glycosylated domain, a short hydrophobic region, and an unglycosylated C-terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus particles, but secretion of GP3 from cells has also been reported. We analyzed the membrane topology of GP3 from various PRRSV strains. A fraction of the protein is secreted from transfected cells, GP3 from PRRSV-1 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion behavior is reversed after exchange of the variable C-terminal domain. A fluorescence protease protection assay shows that the C terminus of GP3, fused to green fluorescent protein (GFP), is resistant to proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C terminus of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region greatly enhances secretion of GP3, and fusion of this domain to GFP promotes membrane anchorage. Bioinformatics suggests that the hydrophobic region forms an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents secretion of GP3 and in its hydrophobic face enhances it. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that might explain why a fraction of the protein is secreted.IMPORTANCE PRRSV is the most important pathogen in the pork industry. It causes persistent infections that lead to reduced weight gain of piglets; highly pathogenic strains even kill 90% of an infected pig population. PRRSV cannot be eliminated from pig farms by vaccination due to the large amino acid variability between the existing strains, especially in the glycoproteins. Here, we analyzed basic structural features of GP3 from various PRRSV strains. We show that the protein exhibits an unusual hairpin-like membrane topology; membrane anchoring might occur via an amphipathic helix. This rather weak membrane anchor explains why a fraction of the protein is secreted from cells. Interestingly, PRRSV-1 strains secrete more GP3 than PRRSV-2. We speculate that secreted GP3 plays a role during PRRSV infection of pigs: it might serve as a decoy to distract antibodies away from virus particles.

Co-translational processing of glycoprotein 3 from equine arteritis virus: N-glycosylation adjacent to the signal peptide prevents cleavage.

Signal peptide cleavage and N-glycosylation of proteins are co-translational processes, but little is known about their interplay if they compete for adjacent sites. Here we report two unique findings for processing of glycoprotein 3 of equine arteritis virus. Glycoprotein 3 (Gp3) contains an N-terminal signal peptide, which is not removed, although bioinformatics predicts cleavage with high probability. There is an overlapping sequon, NNTT, adjacent to the signal peptide that we show to be glycosylated at both asparagines. Exchanging the overlapping sequon and blocking glycosylation allows signal peptide cleavage, indicating that carbohydrate attachment inhibits processing of a potentially cleavable signal peptide. Bioinformatics analyses suggest that a similar processing scheme may exist for some cellular proteins. Membrane fractionation and secretion experiments revealed that the signal peptide of Gp3 does not act as a membrane anchor, indicating that it is completely translocated into the lumen of the endoplasmic reticulum. Membrane attachment is caused by the hydrophobic C terminus of Gp3, which, however, does not span the membrane but rather attaches the protein peripherally to endoplasmic reticulum membranes.

Genetic variation and recombination analysis of PRRSV-2 GP3 gene in China from 1996 to 2023.

Porcine reproductive and respiratory syndrome virus (PRRSV) has become widespread in China particularly the highly pathogenic porcine reproductive and respiratory syndromes (HP-PRRSV), NADC30, and NADC34 strains, and has posed a threat to the swine industry for over 20 years. To monitor genetic variation in PRRSV-2 GP3 strains in China, we analyzed 618 strains isolated between 1996 to 2023 and constructed phylogenetic trees. Additionally, 60 selected strains were used to analyze nucleotide and amino acid homology. PRRSV GP3 gene exhibited nucleotide identity ranging from 78.2% to 100.0% and amino acid similarity ranging from 74.9% to 99.6%. The GP3 gene in the 60 selected strains consisted of 254 amino acids, and amino acid mutations in the strains primarily occurred in B-cell epitopes, T-cell epitopes, and highly variable regions. The glycosylation sites of the strains used for amino acid sequence comparisons remained unaltered, except for the N29 site in the GD20220303-2022 strain. PRRSV-2 strains in China belong to lineages 1, 3, 5, and 8. Recombination analysis detected two recombination events, involving lineages 1 and 8. In conclusion, this study investigated multiple strains of the PRRSV-2 GP3 gene to explore the prevalence and genetic diversity of the GP3 gene in China from a gene family perspective. The results of the analyses provide a basis for clinical prevention strategies and vaccine development.

Engineering and characterizing porcine reproductive and respiratory syndrome virus with separated and tagged genes encoding the minor glycoproteins.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen affecting pigs and belongs to the enveloped plus-stranded RNA virus family Arteriviridae. A unique feature of Arteriviruses is that the genes encoding the structural proteins overlap at their 3` and 5` ends. This impedes mutagenesis opportunities and precludes the binding of short peptides for antibody detection, as this would alter the amino acids encoded by the overlapping gene. In this study, we aimed to generate infectious PRRSV variants with separated genes encoding the minor glycoproteins Gp2, Gp3, and Gp4, accompanied by appended tags for detection. All recombinant genomes facilitate the release of infectious virus particles into the supernatant of transfected 293 T cells, as evidenced by immunofluorescence of infected MARC-145 cells using anti-nucleocapsid antibodies. Furthermore, expression of Gp2-Myc and Gp3-HA was confirmed through immunofluorescence and western blot analysis with tag-specific antibodies. However, after two passages of Gp2-Myc and Gp3-HA viruses, the appended tags were completely removed as indicated by sequencing the viral genome. Recombinant viruses with separated Gp2 and Gp3 genes remained stable for at least nine passages, while those with Gp3 and Gp4 genes separated reverted to wild type after only four passages. Notably, this virus exhibited significantly reduced titers in growth assays. Furthermore, we introduced a tag to the C-terminus of Gp4. The Gp4-HA virus was consistently stable for at least 10 passages, and the HA-tag was detectable by western blotting and immunofluorescence.

Immunological evaluation of recombination PRRSV GP3 and GP5 DNA vaccines in vivo.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a threat to the health of pigs worldwide, but commercially available vaccines offer limited protection against PRRSV infection. It is necessary to develop a more effective DNA vaccine. The immunological effects of DNA vaccines with three adjuvants were examined in pigs (Susscrofa domestica) challenged with PRRSV. These DNA vaccines, which encoded PRRSV GP3 and GP5, were formulated with A1, A2, and A3. Serum specific and neutralizing antibodies, IL-4, IFN-γ, IL-2, IL-10, CD4+ and CD8+T-lymphocytes, health status, histopathology, and viral loads were determined. The results showed that the use of adjuvant A3 led to higher levels of neutralizing antibodies and a lower viral load in pigs compared to the other adjuvants. The neutralizing antibody titers of the pVAX-GP35+A1 and pVAX-GP35+A3 groups reached a peak of 1:19 at 35 dpi. The maximum concentration of IL-4 was 136.77 pg/mL in the pVAX-GP35+A3 group. At 35 dpi, the IFN-γ concentration in the pVAX-GP35+A1 group was 227.4 pg/mL. pVAX-GP35+A3 group shows the highest IL-2 and IL-10 expression to the peak of 597.6 pg/mL and 189.1 pg/mL, respectively. We found a formulation demonstrated beneficial immune outcomes. This study provides an alternative vaccine to protect pigs from PRRSV.

The chemoprevention of spirulina platensis and garlic against diethylnitrosamine induced liver cancer in rats via amelioration of inflammatory cytokines expression and oxidative stress.

Natural antioxidant products play a vital role in the treatment and prevention of cancer disease because they have no side effects. This study aimed to compare the chemoprotective effect of Spirulina platensis (SP) and garlic against hepatocellular carcinoma (HCC) in rats. This study was being done by using 60 male Wistar rats and divided into four groups. Group (I): normal group. Group (II): HCC group induced by injection of a single dose of DEN (200 mg/kg/I.P) and after 14 days injected CCl4 (1 mg/kg/I.P) 3 times/week/six weeks. Group (III): HCC group received SP orally at a dose (500 mg/kg). Group (IV): HCC group received garlic (250 mg/kg) orally. The results revealed that the Spirulina and garlic treatment have a significant decrease in Glutamate pyruvate transaminase, Glutamate oxaloacetate transaminase, GGT, LDH, and the Malondialdehyde (MDA) activity, and furthermore, a significant increase in the total protein level, the superoxide dismutase (SOD), and Catalase (CAT) activity nearly to normal activity. Furthermore, the hepatic expression of tumor necrosis factor (TNF-α), interleukin-6 (IL-6), inducible nitric oxide synthase, transforming growth factor-beta (TGF-ß1), Heat Shock Protein glycoprotein 96 (HSPgp96), and Glypican 3 (GP3) were down regulated by the Spirulina and garlic treatment in comparison with those in HCC group. All findings reported that the chemoprotective of both Spirulina and garlic that have nearly the same effect may be due to antioxidant activity and inhibition of lipid peroxidation, amelioration of pro-inflammatory cytokine, HSPgp96, and GP3.

Expression of the Heterotrimeric GP2/GP3/GP4 Spike of an Arterivirus in Mammalian Cells.

Equine arteritis virus (EAV), an enveloped positive-strand RNA virus, is an important pathogen of horses and the prototype member of the Arteiviridae family. Unlike many other enveloped viruses, which possess homotrimeric spikes, the spike responsible for cellular tropism in Arteriviruses is a heterotrimer composed of 3 glycoproteins: GP2, GP3, and GP4. Together with the hydrophobic protein E they are the minor components of virus particles. We describe the expression of all 3 minor glycoproteins, each equipped with a different tag, from a multi-cassette system in mammalian BHK-21 cells. Coprecipitation studies suggest that a rather small faction of GP2, GP3, and GP4 form dimeric or trimeric complexes. GP2, GP3, and GP4 co-localize with each other and also, albeit weaker, with the E-protein. The co-localization of GP3-HA and GP2-myc was tested with markers for ER, ERGIC, and cis-Golgi. The co-localization of GP3-HA was the same regardless of whether it was expressed alone or as a complex, whereas the transport of GP2-myc to cis-Golgi was higher when this protein was expressed as a complex. The glycosylation pattern was also independent of whether the proteins were expressed alone or together. The recombinant spike might be a tool for basic research but might also be used as a subunit vaccine for horses.

Development of a monoclonal antibody against PRRSV glycoprotein 3 using an immuodominant peptide as immunogen.

Glycoprotein 3 (GP3), a highly glycosylated membrane protein, is a protective antigen and minor structural protein of porcine reproductive and respiratory syndrome virus (PRRSV), and plays a crucial role in virus assembly and infection. In the present study, we synthesized 23 overlapping peptides span GP3 protein sequence and used pig anti-PRRSV serums to identify immunodominant peptides by indirect ELISA. Five immunodominant peptides GP3-P3, P4, P5, P6 and P7 were identified and GP3-P4 (P55LCPTRQAAAEILEPGKS72) was conjugated to carrier protein BSA. One mAb 1E5 against GP3 was generated from BALB/c mice immunized with the conjugates BSA-P4. The Characterization of mAb was identified by Western blot, Dot-ELISA, IPMA and IFA. We found that mAb 1E5 can specifically react with HP-PRRSV strains but not C-PRRSV or NADC30-like PRRSV strains tested in this study. Site-directed alanine substitution analysis revealed that 8 amino acid residues were involved in antibody binding, among them E65, L67 and P69 were critical residue recognized by mAb 1E5. Taken together, this study provided a novel strategy for generating specific mAbs against virus proteins by using immunodominant peptides as targets, and the mAb 1E5 may be useful for development of rapid differential detection method differentiating HP-PRRSV from C-PRRSV and NADC30-like PRRSV.

Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells.

Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorporated into the virion particles as monomers and as a Gp2/Gp3-HA/Gp4 trimer. Gp3-HA localized in ER and, to a lesser extent, in the Golgi, it also co-localized with the E protein but not with the N protein. The co-localization of Gp3-HA and the E protein with ERGIC was reduced. Moreover, EAV with Gp3-HA could become a valuable research tool for identifying host cell factors during infection and the role of Gp3 in virus attachment and entry.

Isolation, Cloning and Co-Expression of Lipase and Foldase Genes of Burkholderia territorii GP3 from Mount Papandayan Soil.

Lipases are industrial enzymes that catalyze both triglyceride hydrolysis and ester synthesis. The overexpression of lipase genes is considered one of the best approaches to increase the enzymatic production for industrial applications. Subfamily I.2. lipases require a chaperone or foldase in order to become a fully-activated enzyme. The goal of this research was to isolate, clone, and co-express genes that encode lipase and foldase from Burkholderia territorii GP3, a lipolytic bacterial isolate obtained from Mount Papandayan soil via growth on Soil Extract Rhodamine Agar. Genes that encode for lipase (lipBT) and foldase (lifBT) were successfully cloned from this isolate and co-expressed in the E. coli BL21 background. The highest expression was shown in E. coli BL21 (DE3) pLysS, using pET15b expression vector. LipBT was particulary unique as it showed highest activity with optimum temperature of 80°C at pH 11.0. The optimum substrate for enzyme activity was C10, which is highly stable in methanol solvent. The enzyme was strongly activated by Ca2+, Mg2+, and strongly inhibited by Fe2+ and Zn2+. In addition, the enzyme was stable and compatible in non-ionic surfactant, and was strongly incompatible in ionic surfactant.

Construction and immunological evaluation of recombinant Newcastle disease virus vaccines expressing highly pathogenic porcine reproductive and respiratory syndrome virus GP3/GP5 proteins in pigs.

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) poses a significant threat to the pig industry, for which vaccination is considered to be an effective means of prevention and control. Here, we developed two recombinant Newcastle disease virus (NDV) LaSota-vectored PRRS candidate vaccines, rLaSota-GP5 and rLaSota-GP3-GP5, using reverse genetic techniques. The two recombinant viruses exhibited a high degree of genetic stability after 10 successive generations in chicken embryos. There was no significant difference in pathogenicity compared with the rLaSota parent strain in poultry, mice and pigs. The recombinant viruses could not be detected in the feeding environment of immunized pigs, but could be detected in the organs and tissues of pigs for no more than 10 days after immunization. Importantly, in contrast to rLaSota-GP5, rLaSota-GP3-GP5 elicited both significant humoral and cellular immune responses in pigs. In particular, the neutralizing antibody titer in the rLaSota-GP3-GP5 group was 1.51 times significantly higher than that of the commercial vaccine group at 42 days post-immunization. At the same time, there was significant difference in the level of IFN-γ between the rLaSota-GP3-GP5 group and the commercial vaccine group. Furthermore, the viral load in the organs and tissues of rLaSota-GP3-GP5-immunized pigs was substantially lower than that of unimmunized pigs after being challenged with HP-PRRS virus GD strain. These results suggest that rLaSota-GP3-GP5 is a safe and promising candidate vaccine, and there is potential for further development of a recombinant virus vaccine for PRRS using NDV.

Adaptions of field PRRSVs in Marc-145 cells were determined by variations in the minor envelope proteins GP2a-GP3.

The recent rapid evolution of PRRSVs has resulted in certain biological characteristic changes, such as the fact that an increasing number of field PRRSVs can be isolated from PAMs but not from Marc-145 cells. In this study, we first isolated Marc-145-unadaptive field PRRSV strains from PAMs; sequence analysis showed that these PRRSVs belong to the HP-PRRSV (lineage 8) branch or NADC30-Like (lineage 1) branch. We further found major variations in ORF2-4 regions. To explore the viral adaptation mechanisms in detail, we constructed a full-length cDNA clone of MY-376, a Marc-145-unadaptive PRRSV. Construction of serially chimeric viruses of HuN4-F112 (a Marc-145-adaptive strain) and MY-376 demonstrated that variation in the minor envelope protein (GP2a and GP3) complex is a main determinant of PRRSV tropism for Marc-145 cells.

Differences in signal peptide processing between GP3 glycoproteins of Arteriviridae.

We reported previously that carbohydrate attachment to an overlapping glycosylation site adjacent to the signal peptide of GP3 from equine arteritis virus (EAV) prevents cleavage. Here we investigated whether this unusual processing scheme is a feature of GP3s of other Arteriviridae, which all contain a glycosylation site at a similar position. Expression of GP3 from type-1 and type-2 porcine reproductive and respiratory syndrome virus (PRRSV) and from lactate dehydrogenase-elevating virus (LDV) revealed that the first glycosylation site is used, but has no effect on signal peptide cleavage. Comparison of the SDS-PAGE mobility of deglycosylated GP3 from PRRSV and LDV with mutants having or not having a signal peptide showed that GP3´s signal peptide is cleaved. Swapping the signal peptides between GP3 of EAV and PRRSV revealed that the information for co-translational processing is not encoded in the signal peptide, but in the remaining part of GP3.

Characterisation of different forms of the accessory gp3 canine coronavirus type I protein identified in cats.

ORF3 is a supplemental open reading frame coding for an accessory glycoprotein gp3 of unknown function, only present in genotype I canine strain (CCoV-I) and some atypical feline FCoV strains. In these latter hosts, the ORF3 gene systematically displays one or two identical deletions leading to the synthesis of truncated proteins gp3-Δ1 and gp3-Δ2. As deletions in CoV accessory proteins have already been involved in tissue or host switch, studies of these different gp3 proteins were conducted in canine and feline cell. All proteins oligomerise through covalent bonds, are N-glycosylated and are maintained in the ER in non-infected but also in CCoV-II infected cells, without any specific retention signal. However, deletions influence their level of expression. In canine cells, all proteins are expressed with similar level whereas in feline cells, the expression of gp3-Δ1 is higher than the two other forms of gp3. None of the gp3 proteins modulate the viral replication cycle of heterologous genotype II CCoV in canine cell line, leading to the conclusion that the gp3 proteins are probably advantageous only for CCoV-I and atypical FCoV strains.

Antigenic characteristics of glycosylated protein 3 of highly pathogenic porcine reproductive and respiratory syndrome virus.

Highly pathogenic (HP)-porcine reproductive and respiratory syndrome virus (PRRSV) emerged in 2006 and has now become a global threat to pig farms. Despite extensive characterization of HP-PRRSV proteins by direct analysis and comparison with typical PRRSV, immune recognition remain poorly understood. Glycosylated protein 3 (GP3) has an important function in inducing protective immune response. To analyze the antigenic character of HP-PRRSV GP3, a total of 217 peptides were printed on a chip and used to react with HP-PRRSV specific serum. The reactions of these peptides to HP-PRRSV specific pig serum were scanned and quantified using the software PepSlide Analyzer by fluorescence intensity. The intensity plots showed various reactions in different parts of GP3. The highest reaction intensity value reached 29,184.5 with the peptide sequence of CSENDHDELGFMVPP. Conversely, 88 peptides showed no reaction with 0 florescence intensity. A further analysis based on the result of the peptide microarray revealed an antigen reaction active region (AR) from Y(51) to S(106) in GP3. The AR had four parts of variation that may be a significant mutation of the typical PRRSV to HP-PRRSV. Acquired data may be useful for understanding HP-PRRSV variation and its GP3 immune recognition.

Identification of two dominant linear epitopes on the GP3 protein of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV).

Glycosylated protein 3 (GP3) of PRRSV is variable between different PRRSV strains, so it is helpful for subtype classifying by using distinct epitopes. In this study, two dominant linear GP3 epitopes that were recognized by highly dilute serum in an enzyme-linked immunosorbent assay (ELISA) were identified. Sequence alignments of 36 North American (NA) PRRSV isolates revealed that the epitope H(87)DELGFMV(94) is well conserved, whereas the epitope T(59)RQAAAEILE(68) differs in other low-virulence NA-type strains, which have at least one amino acid mutation in this region. A mutational analysis revealed that none of these mutations could be recognized by the purified antibodies directed against the corresponding epitope, indicating that the genetic variations altered the antigenicity of the antigenic region. Using ELISA, we also found that antibodies directed against the two epitopes were present in more than 45 of 50 HP-PRRS-positive pig sera, suggesting that their antigenicity is excellent in vivo.