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Engineering and characterizing porcine reproductive and respiratory syndrome virus with separated and tagged genes encoding the minor glycoproteins
Zhang M, Qian B, Veit M
Vet Microbiol2024 JulPubMed ID: 38795404Read Article
Applications: IF Reactive species: Mouse
"Abstract: 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." Article snippet: Monoclonal anti-N of PRRSV-2 (DMAB28442, Creative Diagnostics, USA, 1:3000)
Figure 1. Characterization of recombinant virus.
Development of GFP-expressing infectious clones for PRRSV using TAR cloning for antiviral drug screening
Applications: Western blotting Reactive species: PRRSV
"Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV), an Arteriviridae family enveloped RNA virus, is a major swine pathogen. Using yeast transformation-associated recombination (TAR) cloning, we efficiently generated infectious PRRSV and GFP-expressing clones, identifying transcription-regulating sequences as essential for stable foreign gene expression. Screening SARS-CoV-2 antivirals showed potent inhibition by the multitarget drug ribavirin, the polymerase inhibitors remdesivir and its metabolite GS-441524. Molnupiravir, targeting the polymerase by a different mechanism, showed reduced efficacy against PRRSV, while the protease inhibitor GC376 was ineffective. The AlphaFold-predicted structure of the PRRSV polymerase revealed conserved catalytic architecture with the SARS-CoV-2 polymerases, explaining cross-family inhibitor activity. In contrast, structural divergence in proteases correlated with GC376's inefficacy. These findings underscore the utility of the TAR cloning for arterivirus engineering, with potential applications in vector vaccine development." Article snippet: Subsequently, membranes were incubated overnight at 4 °C with primary antibodies diluted in blocking solution: a monoclonal antibody against the N protein PRRSV-1 (*, 1:1000), or a monoclonal antibody against the N protein of PRRSV-2 (DMAB28442, Creative Diagnostics, 1:3000).
Figure 1. Analysis of GFP expression relative to viral N protein
Background
Mouse anti-PRRSV monoclonal antibodies (mAbs) are a valuable tool used in various laboratory techniques, including immunofluorescence (IF) and immunoprecipitation (IP), for the detection and characterization of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is a significant viral pathogen that affects domestic pigs worldwide, causing substantial economic losses in the swine industry. Since its emergence in the late 1980s, PRRSV has become one of the most challenging diseases in the pig farming sector. PRRSV belongs to the family Arteriviridae and is an enveloped, single-stranded RNA virus. It exhibits a high degree of genetic diversity, with two major genotypes: PRRSV-1 (European type) and PRRSV-2 (North American type). PRRSV primarily targets respiratory and reproductive tissues, leading to two distinct clinical manifestations. In reproductive herds, PRRSV causes reproductive failure, including late-term abortions, stillbirths, mummification of fetuses, and reduced litter sizes. In growing pigs, the virus causes respiratory disease characterized by fever, coughing, dyspnea, and decreased growth rates. The combination of reproductive and respiratory problems contributes to significant economic losses for affected farms.
The use of mouse anti-PRRSV mAbs in IF and IP techniques provides researchers with powerful tools for PRRSV detection, characterization, and research. These antibodies offer high specificity and sensitivity, allowing for precise identification and analysis of PRRSV antigens in different sample types. They are widely utilized in both diagnostic and research laboratories to advance our understanding of PRRSV biology, pathogenesis, and immune responses.
Alternative Names
Anti-PRRSV MAb Anti-Porcine reproductive and respiratory syndrome virus monoclonal antibody Anti-Porcine reproductive and respiratory syndrome virus MAb
Q: Could you tell me the clone number of this antibody?
A: clone 3E7
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References
Genetic signatures of the immune-escaping type 2 porcine reproductive and respiratory syndrome virus in farms with a robust vaccination program
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important porcine viruses worldwide. Recently, severe PRRS outbreaks had occurred in two farms located in eastern and southern Thailand where stringent vaccination had been routinely practiced. Genetic analysis of GP5 identified two highly virulent PRRSVs designated as NA/TH/S001/2015 and NA/TH/E001/2016 from the southern and eastern farms, respectively. Both incidences were the first outbreaks of severe PRRSV since the implementation of the modified live virus (MLV) vaccine, indicating the concurrent emergence of immune-escape viruses. The genetics of the two PRRSV variants, the previous studied sequences from Thailand, and the reference strains were characterized with a focus on the GP5 and NSP2 genes. The results indicated that NA/TH/S001/2015 and NA/TH/E001/2016 shared less than 87% nucleotide similarity to the MLV and PRRSV type 2, lineages 1 and 8.7 (NA), respectively. A comparative analysis of the retrospective GP5 sequences categorized the PRRSVs into five groups based on the clinical outcomes, and both of the novel PRRSV strains were in the same group. Epitope A, T cell epitope, and Nlinked glycosylation patterns within GP5 of both PRRSV variants were highly variable and significantly differed from those of MLV. As observed in highly virulent type 2 strains, NA/TH/S001/2015 contained a single amino acid deletion at position 33 in the hypervariable region 1 (HV-1) of GP5. Amino acid analysis of the hypervariable region of NSP2 revealed that NA/TH/E001/2016 had a unique deletion pattern that included two discontinuous deletions: a 127-amino acid deletion from residues 301 to 427 and a single amino acid deletion at position 470. These results indicate the emergence of two novel PRRSV strains and highlight the common genetic characteristics of the immune-escaping PRRSV variants.
Porcine Fc gamma RIIb mediated PRRSV ADE infection through inhibiting IFN-beta by cytoplasmic inhibitory signal transduction
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Antibody-dependent enhancement (ADE) in porcine reproductive and respiratory syndrome virus (PRRSV) infection is a significant obstacle to the development of effective vaccines for controlling PRRS. Our previous results have demonstrated that porcine Fc gamma RIIb (poFc gamma RIIb) play an important role in mediating ADE of PRRSV infection in vitro. However, the underlying mechanisms involved in poFc gamma RIIb mediated-ADE are still not clear. In this study, MARC-145 cel1 lines stably expressing mutated poFc gamma RIIb (MARC-poFc gamma RIIb-T and MARC-poFc gamma RIIb-CT) in cytoplasm were established and the capacity of poFc RIIb mutants in mediating ADE of PRRSV was investigated. Our results showed that removal of cytoplasmic domain or disruption the tyrosine residue within ITIM (immunoreceptor tyrosine-based inhibition motif) of the poFc gamma RIIb abolished the ability of poFc gamma RIIb to mediate ADE of PRRSV. Furthermore, we found that SHIP1 and TBK1 were involved in poFc RIIb-mediated ADE of PRRSV infection. Taken together, our findings indicated that poFc RIIb mediated the ADE pathway of PRRSV infection through recruiting SHIP-1, which further inhibited of TBK-1-IRF3-IFN-beta signaling pathway to enhance PRRSV infection. These findings will contribute to the molecular mechanism of ADE infection and provide some implications for vaccine development. (C) 2019 Elsevier B.V. All rights reserved.
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV): Pathogenesis and interaction with the immune system
Annual Review of Animal Biosciences
Authors: Lunney, J. K., Fang, Y., Ladinig, A., Chen, N., Li, Y., Rowland, B., Renukaradhya, G. J.
This review addresses important issues of porcine reproductive and respiratory syndrome virus (PRRSV) infection, immunity, pathogenesis, and control. Worldwide, PRRS is the most economically important infectious disease of pigs. We highlight the latest information on viral genome structure, pathogenic mechanisms, and host immunity, with a special focus on immune factors that modulate PRRSV infections during the acute and chronic/persistent disease phases. We address genetic control of host resistance and probe effects of PRRSV infection on reproductive traits. A major goal is to identify cellular/viral targets and pathways for designing more effective vaccines and therapeutics. Based on progress in viral reverse genetics, host transcriptomics and genomics, and vaccinology and adjuvant technologies, we have identified new areas for PRRS control and prevention. Finally, we highlight the gaps in our knowledge base and the need for advanced molecular and immune tools to stimulate PRRS research and field applications.
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Figure 1. Characterization of recombinant virus. 
Figure 1. Analysis of GFP expression relative to viral N protein 
