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Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages.

ABSTRACT: The porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine viral pathogen that causes major economic losses worldwide. We previously reported an over-attenuated phenotype of cell-adapted PRRSV strain CA-2-P100 in vivo. In the present study, CA-2-P100 was serially propagated in cultured porcine alveolar macrophage (PAM) cells for up to 20 passages to obtain the derivative strain CA-2-MP120. Animal inoculation studies revealed that both CA-2-P100 and CA-2-MP120 had decreased virulence, eliciting weight gains, body temperatures, and histopathologic lesions similar to those in the negative control group. However, compared to CA-2-P100 infection, CA-2-MP120 yielded consistently higher viremia kinetics and enhanced antibody responses in pigs. All pigs inoculated with CA-2-MP120 developed viremia and seroconverted to PRRSV. During 20 passages in PAM cells, CA-2-MP120 acquired 15 amino acid changes that were mostly distributed in nsp2 and minor structural protein-coding regions. Among these changes, 6 mutations represented reversions to the sequences of the reference CA-2 and parental CA-2-P20 strains. These genetic drifts may be hypothetical molecular markers associated with PRRSV macrophage tropism and virulence. Our results indicate that the PAM-passaged CA-2-MP120 strain is a potential candidate for developing a live, attenuated PRRSV vaccine.

SUBMITTER: Lee SC

PROVIDER: S-EPMC5974517 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages.

Lee Seung-Chul SC Lee Sunhee S Yoo Gun-Woo GW Choi Hwan-Won HW Noh Yun-Hee YH Park Chang Eon CE Shin Jae-Ho JH Yoon In-Joong IJ Kang Shien-Young SY Lee Changhee C

Journal of veterinary science 20180501 3

The porcine reproductive and respiratory syndrome virus (PRRSV) is a globally ubiquitous swine viral pathogen that causes major economic losses worldwide. We previously reported an over-attenuated phenotype of cell-adapted PRRSV strain CA-2-P100 <i>in vivo</i>. In the present study, CA-2-P100 was serially propagated in cultured porcine alveolar macrophage (PAM) cells for up to 20 passages to obtain the derivative strain CA-2-MP120. Animal inoculation studies revealed that both CA-2-P100 and CA-2 ...[more]

PMID: 29486535

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Project description:Porcine reproductive and respiratory syndrome (PRRS) is a globally important disease threatening the pork industry, and modified live-virus (MLV) vaccines are widely used for its prevention. However, PRRS MLV shows high potential for reversion to virulence, leading to a major concern about its safety. Yet the revertant mechanism is still poorly understood. Here, attenuated virus JXwn06-P80, derived from the highly pathogenic PRRS virus (PRRSV) strain JXwn06 by serial passaging in MARC-145 cells, was reversely passaged in pigs through intranasal inoculation to mimic natural infection for 13 rounds, and the pathogenicity of viruses at the 3rd, 5th, 9th, 10th, and 11th passages was evaluated in pigs. From the 9th passage, the viruses caused mortality, which was related to their increased adaptability and replication efficiency (100 times higher than those of JXwn06-P80) in porcine alveolar macrophage (PAM) target cells. Similarly, JXwn06-P80 could also regain fatal virulence through reverse passage in PAMs for 25 or more passages, indicating that the increased adaptability in PAMs directly contributes to its regained fatal virulence. Next, the full-genome sequences were analyzed to explore the genetic evolutionary processes during adaptation both in vivo and in vitro. Finally, by a reverse genetic operation, four reverse mutation sites, NSP12-W121R, ORF2b (open reading frame 2b)-H9D, ORF5-H15L, and ORF5-V189L, were finally identified to partially contribute to the ability of the virus to adapt to PAMs, which may be related to virulence reversion during reverse passage. These findings provided direct scientific evidence for the virulence reversion of PRRS MLV and provided valuable clues for exploring its molecular mechanism. IMPORTANCE Reversion to virulence of a live attenuated vaccine is a public concern; however, direct scientific evidence is limited, and the mechanism is still poorly understood. Here, we present direct evidence for the reversion to virulence of PRRS MLV after serial passaging in pigs or target cells and found a correlation between virulence reversion and increased replication fitness in primary PAMs. The genetic evolutionary process during adaptation will provide valuable clues for exploring the molecular mechanism of PRRS MLV virulence reversion and offer important implications for understanding the reversion mechanisms of other vaccines.

Project description:Porcine reproductive and respiratory syndrome (PRRS) has devastated pig industries worldwide for many years. It is caused by a small RNA virus (PRRSV), which targets almost exclusively pig monocytes or macrophages. In the present study, five SAGE (serial analysis of gene expression) libraries derived from 0 hour mock-infected and 6, 12, 16 and 24 hours PRRSV-infected porcine alveolar macrophages (PAMs) produced a total 643,255 sequenced tags with 91,807 unique tags. Differentially expressed (DE) tags were then detected using the Bayesian framework followed by gene/mRNA assignment, arbitrary selection and manual annotation, which determined 699 DE genes for reactome analysis. The DAVID, KEGG and REACTOME databases assigned 573 of the DE genes into six biological systems, 60 functional categories and 504 pathways. The six systems are: cellular processes, genetic information processing, environmental information processing, metabolism, organismal systems and human diseases as defined by KEGG with modification. Self-organizing map (SOM) analysis further grouped these 699 DE genes into ten clusters, reflecting their expression trends along these five time points. Based on the number one functional category in each system, cell growth and death, transcription processes, signal transductions, energy metabolism, immune system and infectious diseases formed the major reactomes of PAMs responding to PRRSV infection. Our investigation also focused on dominant pathways that had at least 20 DE genes identified, multi-pathway genes that were involved in 10 or more pathways and exclusively-expressed genes that were included in one system. Overall, our present study reported a large set of DE genes, compiled a comprehensive coverage of pathways, and revealed system-based reactomes of PAMs infected with PRRSV. We believe that our reactome data provides new insight into molecular mechanisms involved in host genetic complexity of antiviral activities against PRRSV and lays a strong foundation for vaccine development to control PRRS incidence in pigs.

Project description:BackgroundLong noncoding RNA (lncRNA) is highly associated with inflammatory response and virus-induced interferon production. By far the majority of studies have focused on the immune-related lncRNAs of mice and humans, but the function of lncRNAs in porcine immune cells are poorly understood. Porcine reproductive and respiratory syndrome virus (PRRSV) impairs local immune responses in the lungs of nursery and growing pigs, whereas the virus triggers the inflammatory responses. Porcine alveolar macrophage (PAM) is the primary target cell of PRRSV, thus PRRSV is used as an in vitro model of inflammation. Here, we profiled lncRNA and mRNA repertories from PRRSV-infected PAMs to explore the underlying mechanism of porcine lncRNAs in regulating host immune responses.ResultsIn this study, a total of 350 annotated lncRNAs and 1792 novel lncRNAs in PAMs were identified through RNA-seq analysis. Among them 86 differentially expressed (DE) lncRNAs and 406 DE protein-coding mRNAs were identified upon PRRSV incubation. GO category and KEGG pathway enrichment analyses revealed that these DE lncRNAs and mRNAs were mainly involved in inflammation- and pathogen infection-induced pathways. The results of dynamic correlated expression networks between lncRNAs and their predicted target genes uncovered that numerous lncRNAs, such as XLOC-022175, XLOC-019295, and XLOC-017089, were correlated with innate immune genes. Further analysis validated that these three lncRNAs were positively correlated with their predicted target genes including CXCL2, IFI6, and CD163. This study suggests that porcine lncRNAs affect immune responses against PRRSV infection through regulating their target genes in PAMs.ConclusionThis study provides both transcriptomic and epigenetic status of porcine macrophages. In response to PRRSV infection, comprehensive DE lncRNAs and mRNAs were profiled from PAMs. Co-expression analysis demonstrated that lncRNAs are emerging as the important modulators of immune gene activities through their critical influence upon PRRSV infection in porcine macrophages.

Dataset Information

Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages.

Publications

Phenotypic and genotypic analyses of an attenuated porcine reproductive and respiratory syndrome virus strain after serial passages in cultured porcine alveolar macrophages.

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