The mRNA Vaccine Expressing Single and Fused Structural Proteins of Porcine Reproductive and Respiratory Syndrome Induces Strong Cellular and Humoral Immune Responses in BalB/C Mice.

PRRS is a viral disease that profoundly impacts the global swine industry, causing significant economic losses. The development of a novel and effective vaccine is crucial to halt the rapid transmission of this virus. There have been several vaccination attempts against PRRSV using both traditional and alternative vaccine design development approaches. Unfortunately, there is no currently available vaccine that can completely control this disease. Thus, our study aimed to develop an mRNA vaccine using the antigens expressed by single or fused PRRSV structural proteins. In this study, the nucleotide sequence of the immunogenic mRNA was determined by considering the antigenicity of structural proteins and the stability of spatial structure. Purified GP5 protein served as the detection antigen in the immunological evaluation. Furthermore, cellular mRNA expression was detected by immunofluorescence and western blotting. In a mice experiment, the Ab titer in serum and the activation of spleen lymphocytes triggered by the antigen were detected by ELISA and ICS, respectively. Our findings demonstrated that both mRNA vaccines can significantly stimulate cellular and humoral immune responses. More specifically, the GP5-mRNA exhibited an immunological response that was similar to that of the commercially available vaccine when administered in high doses. To conclude, our vaccine may show promising results against the wild-type virus in a natural host.

Chimeric Porcine Parvovirus VP2 Virus-like Particles with Epitopes of South African Serotype 2 Foot-and-Mouth Disease Virus Elicits Specific Humoral and Cellular Responses in Mice.

Southern Africa Territories 2 (SAT2) foot-and-mouth disease (FMD) has crossed long-standing regional boundaries in recent years and entered the Middle East. However, the existing vaccines offer poor cross-protection against the circulating strains in the field. Therefore, there is an urgent need for an alternative design approach for vaccines in anticipation of a pandemic of SAT2 Foot-and-mouth disease virus (FMDV). The porcine parvovirus (PPV) VP2 protein can embed exogenous epitopes into the four loops on its surface, assemble into virus-like particles (VLPs), and induce antibodies and cytokines to PPV and the exogenous epitope. In this study, chimeric porcine parvovirus VP2 VLPs (chimeric PPV-SAT2-VLPs) expressing the T-and/or B-cell epitopes of the structural protein VP1 of FMDV SAT2 were produced using the recombinant pFastBac™ Dual vector of baculoviruses in Sf9 and HF cells We used the Bac-to-Bac system to construct the recombinant baculoviruses. The VP2-VLP--SAT2 chimeras displayed chimeric T-cell epitope (amino acids 21-40 of VP1) and/or the B-cell epitope (amino acids 135-174) of SAT FMDV VP1 by substitution of the corresponding regions at the N terminus (amino acids 2-23) and/or loop 2 and/or loop 4 of the PPV VP2 protein, respectively. In mice, the chimeric PPV-SAT2-VLPs induced specific antibodies against PPV and the VP1 protein of SAT2 FMDV. The VP2-VLP-SAT2 chimeras induced specific antibodies to PPV and the VP1 protein specific epitopes of FMDV SAT2. In this study, as a proof-of-concept, successfully generated chimeric PPV-VP2 VLPs expressing epitopes of the structural protein VP1 of FMDV SAT2 that has a potential to prevent FMDV SAT2 and PPV infection in pigs.

Stability and integrity of self-assembled bovine parvovirus virus­like particles (BPV­VLPs) of VP2 and combination of VP1VP2 assisted by baculovirus-insect cell expression: a potential logistical platform for vaccine deployment.

BACKGROUND: Bovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences. METHODS: In silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism. RESULTS: Our findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs. CONCLUSIONS: In summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.

The Accumulation of Phenyllactic Acid Impairs Host Glutamine Metabolism and Inhibits African Swine Fever Virus Replication: A Novel Target for the Development of Anti-ASFV Drugs.

African swine fever (ASF) is a highly contagious and hemorrhagic disease caused by infection with the African swine fever virus (ASFV), resulting in a mortality rate of up to 100%. Currently, there are no effective treatments and commercially available vaccines for ASF. Therefore, it is crucial to identify biochemicals derived from host cells that can impede ASFV replication, with the aim of preventing and controlling ASF. The ASFV is an acellular organism that promotes self-replication by hijacking the metabolic machinery and biochemical resources of host cells. ASFV specifically alters the utilization of glucose and glutamine, which are the primary metabolic sources in mammalian cells. This study aimed to investigate the impact of glucose and glutamine metabolic dynamics on the rate of ASFV replication. Our findings demonstrate that ASFV infection favors using glutamine as a metabolic fuel to facilitate self-replication. ASFV replication can be substantially inhibited by blocking glutamine metabolism. The metabolomics analysis of the host cell after late-stage ASFV infection revealed a significant disruption of normal glutamine metabolic pathways due to the abundant expression of PLA (phenyllactic acid). Pretreatment with PLA also inhibited ASFV proliferation and glutamine consumption following infection. The metabolomic analysis also showed that PLA pretreatment greatly slowed down the metabolism of amino acids and nucleotides that depend on glutamine. The depletion of these building blocks directly hindered the replication of ASFV by decreasing the biosynthetic precursors produced during the replication of ASFV's progeny virus. These findings provide valuable insight into the possibility of pursuing the development of antiviral drugs against ASFV that selectively target metabolic pathways.

Geometric-feature-based approach to human face reconstruction with high measurement speed.

This paper presents a method based on geometry for three-dimensional (3D) face reconstruction without the need for additional images, hardware components, or objects. In our proposed method, we consider part of the nose as the feature region because its shape remains almost constant during the measurement. The geometry of this region was used to provide cues for phase unwrapping. We first spatially unwrap the phase and determine the integer multiple of 2π to be added by comparing the recovered result of the feature region and its actual shape. Then, the face can be reconstructed with the acquired absolute phase. Experimental results demonstrated that our method is capable of reconstructing a dynamic face with high measurement speed, and only three phase-shifted fringes are required per frame.

A Naked-Eye Visual Reverse Transcription Loop-Mediated Isothermal Amplification with Sharp Color Changes for Potential Pen-Side Test of Foot-and-Mouth Disease Virus.

Visual loop-mediated isothermal amplification (LAMP) is qualified to be applied in the field to detect pathogens due to its simplicity, rapidity and cost saving. However, the color changes in currently reported visual reverse transcription LAMP (RT-LAMP) for foot-and-mouth disease virus (FMDV) detection are not so obvious to the naked eye, so interpretation of results is troublesome. In this study, a new naked-eye visual RT-LAMP to detect all seven distinct serotypes of FMDV was established based on the 3D genes by using pH-sensitive neutral red as the indicator, rendering a sharp contrast of color changes between the negative (light orange) and the positive (pink). Analytical sensitivity tests showed that the detection limit of the visual RT-LAMP was 104 copies/µL while those were 103 and 104 copies/µL for the RT-qPCR and conventional RT-PCR methods, respectively. Specificity tests proved that the established visual RT-LAMP assay had no cross-reactivity with other common livestock viruses. Furthermore, the analysis of 59 clinical samples showed 98.31% and 100% concordance with the RT-qPCR and the RT-PCR, respectively. The pan-serotypic FMD visual RT-LAMP assay could be suitable for a pen-side test of all seven serotypes of FMDV because the results could be easily distinguished by the naked eye without the requirement of complicated instruments and professional technicians. Hence, the novel method may have a promising prospect in field tests which exert an important role in monitoring, preventing, and controlling FMD, especially in regions with no PCR or qPCR instrument available.

Evaluation of Four Commercial Vaccines for the Protection of Piglets against the Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (hp-PRRSV) QH-08 Strain.

Vaccination is the best way to prevent economic losses from highly pathogenic porcine reproductive and respiratory syndrome virus (hp-PRRSV) disease. However, the commercially available vaccines need to periodically evaluate their efficacy against infections caused by new hp-PRRSV variants. Therefore, the objective of this study was to evaluate the efficacy of four (two modified live vaccines (MLV) and two inactivated) PRRSV commercial vaccines in piglets challenged with QH-08 and to estimate the genetic distance of the vaccine strains from recently isolated (QH-08) filed strain. Randomly, piglets (n = 5) allocated in groups 1-4 were immunized with Ingelvac PRRS MLV, CH-1a, JXA1, and JXA1-RMLV vaccines, whereas the infected and non-infected control piglets in groups 5 and 6 (n = 3), respectively, were subjected to PBS. Results indicated that JXA1 and JXA1-R MLV vaccines showed complete protection, but Ingelvac PRRS MLV and CH-1α vaccines revealed partial protection against the QH-08 PRRSV challenge. Similarly, vaccinated and challenged pigs showed lower macroscopic and microscopic lesions than the pigs in group 5. Our findings demonstrated a new insight that the variation in ORF1a and 1b coding sequence could significantly affect PRRSV vaccines efficacy. In conclusion, QH-08 is a good candidate for the design and development of an innovative PRRSV vaccine that ultimately helps in the control and prevention strategies.

Antigenicity and Immunogenicity Analysis of the E. coli Expressed FMDV Structural Proteins; VP1, VP0, VP3 of the South African Territories Type 2 Virus.

An alternative vaccine design approach and diagnostic kits are highly required against the anticipated pandemicity caused by the South African Territories type 2 (SAT2) Foot and Mouth Disease Virus (FMDV). However, the distinct antigenicity and immunogenicity of VP1, VP0, and VP3 of FMDV serotype SAT2 are poorly understood. Similarly, the particular roles of the three structural proteins in novel vaccine design and development remain unexplained. We therefore constructed VP1, VP0, and VP3 encoding gene (SAT2:JX014256 strain) separately fused with His-SUMO (histidine-small ubiquitin-related modifier) inserted into pET-32a cassette to express the three recombinant proteins and separately evaluated their antigenicity and immunogenicity in mice. The fusion protein was successfully expressed and purified by the Ni-NTA resin chromatography. The level of serum antibody, spleen lymphocyte proliferation, and cytokines against the three distinct recombinant proteins were analyzed. Results showed that the anti-FMDV humoral response was triggered by these proteins, and the fusion proteins did enhance the splenocyte immune response in the separately immunized mice. We observed low variations among the three fusion proteins in terms of the antibody and cytokine production in mice. Hence, in this study, results demonstrated that the structural proteins of SAT2 FMDV could be used for the development of immunodiagnostic kits and subunit vaccine designs.

Development of a Potential Penside Colorimetric LAMP Assay Using Neutral Red for Detection of African Swine Fever Virus.

African swine fever (ASF) has caused huge economic losses to the swine industry worldwide. Since there is no commercial ASF vaccine available, an early diagnosis is extremely important to prevent and control the disease. In this study, ASF virus (ASFV) capsid protein-encoding gene (p72) was selected and used to design primers for establishing a one-step visual loop-mediated isothermal amplification (LAMP) assay with neutral red, a pH-sensitive dye, as the color shift indicator. Neutral red exhibited a sharp contrast of color change from faint orange (negative) to pink (positive) during LAMP for detection of ASFV. The designed primer set targeting highly conserved region of the p72 gene was highly specific to ASFV and showed no cross-reactivity with other swine viruses. The detection limit for the one-step visual LAMP developed was 10 copies/reaction based on the recombinant plasmid containing the p72 gene of ASFV. More importantly, the developed one-step visual LAMP showed high consistency with the results of the real-time polymerase chain reaction (qPCR) method recommended by World Organization for Animal Health (OIE). Furthermore, the results demonstrate that the colorimetric detection with this LAMP assay could be directly applied for the whole blood and serum samples without requiring genome extraction. Based on our results, the developed one-step visual LAMP assay is a promising penside diagnostic tool for development of early and cost-effective ASF monitoring program that would greatly contribute to the prevention and control of ASF.

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

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

Review on Outbreak Dynamics, the Endemic Serotypes, and Diversified Topotypic Profiles of Foot and Mouth Disease Virus Isolates in Ethiopia from 2008 to 2018.

Foot and mouth disease (FMD) endemicity in Ethiopia's livestock remains an ongoing cause for economic concern, with new topotypes still arising even in previously unaffected areas. FMD outbreaks occur every year almost throughout the country. Understanding the outbreak dynamics, endemic serotypes, and lineage profiles of FMD in this country is very critical in designing control and prevention programs. For this, detailed information on outbreak dynamics in Ethiopia needs to be understood clearly. In this article, therefore, we review the spatial and temporal patterns and dynamics of FMD outbreaks from 2008 to 2018. The circulating serotypes and the topotypic profiles of the virus are also discussed. FMD outbreak data were obtained from; reports of MoARD (Ministry of Agriculture and Rural Development)/MoLF (Ministry of livestock and Fishery, NVI (National Veterinary Institute), and NAHDIC (National Animal Health Diagnostic and Investigation Center); published articles; MSc works; PhD theses; and documents from international organizations. To effectively control and prevent FMD outbreaks, animal health agencies should focus on building surveillance systems that can quickly identify and control ongoing outbreaks and implement efficient preventive measures.

Development of a New RT-PCR with Multiple Primers for Detecting Southern African Territories Foot-and-mouth Disease Viruses.

INTRODUCTION: The extremely high genetic variation and the continuously emerging variants of foot-and-mouth disease virus (FMDV) of Southern African Territory (SAT) serotypes including SAT1, SAT2, and SAT3 make it necessary to develop a new RT-PCR for general use for monitoring viruses based on the updated genome information. MATERIAL AND METHODS: A FMDV SAT-D8 one-step RT-PCR was established based on the 1D2A2B genes of the SAT serotype viruses with a multiplex primer set. FMDV A, O, C, and Asia 1 serotypes, other vesicular disease viruses, inactivated SAT viruses, and 125 bovine, ovine, caprine and porcine tissue samples collected from the Chinese mainland were included for evaluating the assay. RESULTS: The new RT-PCR was proven to be specific without cross-reactions with Eurasian FMDV, swine vesicular disease virus (SVDV), Seneca valley virus (SVV), or other common viral pathogens of cattle, sheep, goat, and pig. An around 257 bp-sized amplicon clearly appeared when the inactivated SAT viruses were detected. However, all 125 samples collected from FMDV-susceptible animals from the Chinese mainland which has not known SAT epidemics showed negative results. CONCLUSIONS: A FMDV SAT-D8 one-step RT-PCR is a promising method for primary screening for FMDV SAT serotypes.

Absolute three-dimensional shape measurement with a known object.

This paper presents a novel method for absolute three-dimensional (3D) shape measurement that does not require conventional temporal phase unwrapping. Our proposed method uses a known object (i.e., a ping-pong ball) to provide cues for absolute phase unwrapping. During the measurement, the ping-pong ball is positioned to be close to the nearest point from the scene to the camera. We first segment ping-pong ball and spatially unwrap its phase, and then determine the integer multiple of 2π to be added such that the recovered shape matches its actual geometry. The nearest point of the ball provides zmin to generate the minimum phase Φmin that is then used to unwrap phase of the entire scene pixel by pixel. Experiments demonstrated that only three phase-shifted fringe patterns are required to measure absolute shapes of objects moving along depth z direction.

Phase error compensation for three-dimensional shape measurement with projector defocusing.

This paper analyzes the phase error for a three-dimensional (3D) shape measurement system that utilizes our recently proposed projector defocusing technique. This technique generates seemingly sinusoidal structured patterns by defocusing binary structured patterns and then uses these patterns to perform 3D shape measurement by fringe analysis. However, significant errors may still exist if an object is within a certain depth range, where the defocused fringe patterns retain binary structure. In this research, we experimentally studied a large depth range of defocused fringe patterns, from near-binary to near-sinusoidal, and analyzed the associated phase errors. We established a mathematical phase error function in terms of the wrapped phase and the depth z. Finally, we calibrated and used the mathematical function to compensate for the phase error at arbitrary depth ranges within the calibration volume. Experimental results will be presented to demonstrate the success of this proposed technique.