Caveolin-1 is involved in encephalomyocarditis virus replication in BHK-21 cells.

BACKGROUND: Encephalomyocarditis virus, member of Cardiovirus genus within Picornaviridae family, is an important pathogen that infects different domestic and wild animals. However, the molecular mechanism of its entry remains unclear. In this study, we investigated the mechanism of EMCV infectivity in relation to endocytic pathway using BHK-21 cells. METHODS: The function of numerous cellular key factors implicated in the various endocytic mechanisms were systematically explored using chemical inhibitors. Furthermore, RNA interference (RNAi) as well as the overexpression of dominant protein combined to virus infectivity assays, and confocal microscopy was used to examine EMCV infection in details. RESULTS: The results indicated that the EMCV entry into BHK-21 cells depends on caveolin, dynamin, and actin but not clathrin nor macropinocytosis pathways. The effects of overexpression and knockdown of caveolin-1, one components of the caveolae, was examined on EMCV infection. The results showed that EMCV infection was positive correlation with caveolin-1 expression. Confocal microscopy analysis and internalization assay showed that caveolin-1 is required at the early stage of EMCV infection. CONCLUSIONS: Caveolin-1, dynamin, and actin-dependent endocytosis pathways are necessary for EMCV infection in vitro.

Transcriptional profiling of host cell responses to encephalomyocarditis virus (EMCV).

BACKGROUD: Encephalomyocarditis virus (EMCV) has been discovered on pig farms worldwide and can cause myocarditis in piglets and reproductive failure in sows. However, little is known about the host transcriptional responses to infection and host-pathogen interactions. METHODS: In this study, transcription profiling was performed by Illumina RNA-Sequencing (RNA-seq) to identify EMCV induced differentially expressed genes in BHK-21 cells at serial time points (12, 24, and 30 h post infection (hpi)), using mock infected cells as control. RESULTS: We identified 237, 241, and 207 differentially expressed genes (DEGs) respectively, majority of which were up-regulated. A large number of DEGs clustered into host defense, cellular signaling and metabolism categories. Moreover, short time series expression analysis revealed that 12 hpi was an important time point for expression change, indicating host virus resistance. CONCLUSIONS: This RNA-seq analysis provides the first data for understanding the network of virus host interactions under EMCV infection in vitro, and for identifying host components which involved in the virus infection course.

Intracellular expression of an anti-idiotypic antibody single-chain variable fragment reduces porcine reproductive and respiratory syndrome virus infection in MARC-145 cells.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome; it is one of the most economically important viral diseases affecting the swine industry worldwide. At present, neither live-attenuated nor inactivated PRRSV vaccines can provide sustainable disease control. Our previous studies have demonstrated that PRRSV infection can produce the auto-anti-idiotypic antibodies (aAb2s) specific to the idiotypic antibodies against PRRSV GP5, which plays an important role in the host immune responses to PRRSV infection. In the present study, a single-chain variable antibody fragment (scFv) from the monoclonal anti-idiotypic antibody specific for the idiotypic antibody against GP5 was expressed in MARC-145 cells and its effect on virus infection in vitro was evaluated. METHODS: An scFv was constructed from the anti-idiotypic antibody (Mab2-5G2) and was named 5G2scFv. The lentiviral vector system was used as a vehicle to deliver 5G2scFv into MARC-145 cells. The effect of 5G2scFv expression in MARC-145 was analysed by determining the PRRSV N protein level and the virus titre in the supernatant. Virus attachment and the level of type I interferon (IFN) were determined to elucidate the mechanism of the scFv effect. RESULTS: 5G2scFv was delivered in MARC-145 cells using the lentiviral vector system as confirmed by the western blot and indirect immunofluorescence assays. The PRRSV challenge experiments demonstrated that expressed 5G2scFv in MARC-145 strongly reduced PRRSV infection and replication by inhibiting protein synthesis and progeny virus production. This effect was not due to the change of viability or virus binding, but increased IFN-α at messenger RNA and protein levels. CONCLUSIONS: The expression of the anti-idiotypic antibody 5G2scFv in MARC-145 cells has the interferential effect on PRRSV infection in the cells by induction of IFN-α, which provides a novel therapeutic approach for PRRSV infection.

Rescue and evaluation of a recombinant PRRSV expressing porcine Interleukin-4.

BACKGROUND: The current vaccines for porcine reproductive and respiratory syndrome virus (PRRSV) have failed to provide broad protection against infection by various strains of PRRSV. Porcine Interleukin-4 (pIL-4) plays an important role in the regulation of the immune response and has been used previously as an immunological adjuvant. The objective of this study was to construct a recombinant PRRSV expressing pIL-4 and to evaluate the immune response of the recombinant virus in piglets. METHODS: The pIL-4 gene was inserted in the PRRSV (CH-1R strain) infectious clone by overlap PCR. Indirect immunofluorescence assay (IFA) and Western blotting were used to confirm the recombinant virus. The stability of the recombinant virus was assessed by DNA sequencing and IFA after 15 passages in vitro. Recombinant virus was injected into pigs and efficacy of immune protection was evaluated in comparison with the parental virus. RESULTS: The recombinant virus (CH-1R/pIL-4) was successfully rescued and shown to have similar growth kinetics as the parental virus. The recombinant virus was stable for 15 passages in cell culture. Pigs vaccinated with CH-1R/pIL-4 produced a similar humoral response to the response elicited by parental virus, but IL-4 level in the supernatant of PBMCs from pigs vaccinated with CH-1R/pIL-4 was significantly higher than the parent virus at 28 days post-immunization (DPI). Flow cytometric (FCM) analysis showed that the percentage of CD4(+)CD8(+) double positive T (DPT) cells in the CH-1R/pIL-4 vaccinated group was significantly higher than the parental virus at 3 and 7 Days Post-Challenge (DPC), and the IL-4 level in the blood significantly increased at 7 DPC. However, the viral load and histopathology did not show significant difference between the two groups. CONCLUSIONS: A recombinant PRRSV expressing porcine IL-4 was rescued and it remained genetically stable in vitro. The recombinant virus induced higher DPT ratios and IL-4 levels in the blood after HP-PRRSV challenge compared to the parental virus in piglets. However, it did not significantly improve protection efficacy of PRRSV vaccine.

An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits porcine reproductive and respiratory syndrome virus replication.

Porcine reproductive and respiratory syndrome (PRRS) is a widespread viral disease affecting the swine industry, with no cure or effective treatment. Current vaccines are inefficient mainly due to the high degree of genetic and antigenic variation within PRRS virus (PRRSV) strains. Thus, the development of novel anti-PRRSV strategies is an important area of research. The nonstructural protein 9 (Nsp9) of PRRSV is essential for viral replication, and its sequence is relatively conserved, making it a logical antiviral target for PRRSV. Camel single-domain antibodies (nanobodies) represent a promising antiviral approach because of their small size, high specificity, and solubility. However, no nanobodies against PRRSV have been reported to date. In this study, Nsp9-specific nanobodies were isolated from a phage display library of variable domains of Camellidaeheavy chain-only antibodies (VHH). One of the isolated nanobodies, Nb6, was chosen for further investigation. Co-immunoprecipitation experiments indicated that Nb6 can still maintain antigen binding capabilities when expressed in the cell cytoplasm. A MARC-145 cell line stably expressing Nb6 was established to investigate its potential antiviral activity. Our results showed that intracellularly expressed Nb6 could potently suppress PRRSV replication by inhibiting viral genome replication and transcription. More importantly, Nb6 could protect MARC-145 cells from virus-induced cytopathic effect (CPE) and fully block PRRSV replication at an MOI of 0.01 or lower. To our knowledge, this is the first report of a nanobody based antiviral strategy against PRRSV, and this finding has the potential to lead to future developments of novel antiviral treatments for PRRSV infection.

Immune responses of pigs immunized with a recombinant porcine reproductive and respiratory syndrome virus expressing porcine GM-CSF.

Porcine reproductive and respiratory syndrome virus (PRRSV) has spread worldwide, causing huge economic losses to the swine industry. The current PRRSV vaccines have failed to provide broad protection against various strains. Granulocyte macrophage colony-stimulating factor (GM-CSF), an efficacious adjuvant, has been shown to enhance the immunogenicity of various vaccines. The purpose of this study was to construct a recombinant live attenuated PRRSV that expresses porcine GM-CSF (pGM-CSF) and evaluate the immune responses of pigs immunized with the recombinant virus. The results showed that the recombinant PRRSV was successfully rescued and had similar growth properties to parental virus grown in Marc-145 cells. The recombinant virus was stable for 10 passages in cell culture. Pigs intramuscularly immunized with the recombinant virus produced a similar humoral response to that elicited using parental virus. With regard to cell-mediated immunity assessed in peripheral blood, the recombinant virus induced higher proportion of CD4(+)CD8(+) double-positive T cells (DPT), higher IFN-γ level at 0 and 7 days post-challenge (DPC), and lower viremia at 21 DPC than pigs immunized with parental virus. These results indicate that recombinant PRRSV expressing pGM-CSF can induce a significant higher cellular immune response and reduce the persistent infection compared pigs vaccinated with the parental virus. This is first report of evaluation of immune response in pigs elicited by a recombinant live attenuated PRRSV expressing porcine GM-CSF. It may represent a novel strategy for future development of genetic engineered vaccines against PRRSV infection.

Single-chain anti-idiotypic antibody retains its specificity to porcine reproductive and respiratory syndrome virus GP5.

Monoclonal anti-idiotypic antibody (Mab2-5G2) raised against idiotypic antibodies to membrane glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV). The variable regions of the heavy chain (VH) and light chain (VL) of Mab2-5G2 were cloned and connected with a (Gly4Ser)3 linker. The recombinant scFv gene was cloned into the pEasy-E1 vector and expressed in E. coli as inclusion bodies. The expressed scFv-His proteins renatured in a pH and urea gradient buffer retained the same immunological properties as that of Mab2-5G2. Renatured scFv-His protein retained the same characteristics as that of Mab2-5G2 by recognizing and binding to Marc-145 cells. Furthermore, renatured scFv-His along with Mab2-5G2 were used to immunize rabbits to produce anti-anti-idiotypic antibodies (Ab3) that neutralized PRRSV infection of Marc-145 cells. These results demonstrated that the expressed scFv-His protein possessed the same characteristics of Mab2-5G2 and will be suitable for future investigations of Mab2-5G2 antibody structure and its ability to interact with potential PRRSV cellular receptor as well as immunological properties against PRRSV infection.

Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold.

The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells following induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined specific neuronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuronal-specific proteins, including ßIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differentiation medium differentiated into a multilayered neural network-like structure with long nerve fibers that was composed of several parallel microfibers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sectioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.

A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit.

Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (PRRSV) carrying an enhanced green fluorescent protein (EGFP) reporter gene as a separate transcription unit. A copy of the transcription regulatory sequence for ORF6 (TRS6) was inserted between the N protein and 3'-UTR to drive the transcription of the EGFP gene and yield a general purpose expression vector. Successful recovery of PRRSV was obtained using an RNA polymerase II promoter to drive transcription of the full-length virus genome, which was assembled in a bacterial artificial chromosome (BAC). The recombinant virus showed growth replication characteristics similar to those of the wild-type virus in the infected cells. In addition, the recombinant virus stably expressed EGFP for at least 10 passages. EGFP expression was detected at approximately 10 h post infection by live-cell imaging to follow the virus spread in real time and the infection of neighbouring cells occurred predominantly through cell-to-cell-contact. Finally, the recombinant virus generated was found to be an excellent tool for neutralising antibodies and antiviral compound screening. The newly established reverse genetics system for PRRSV could be a useful tool not only to monitor virus spread and screen for neutralising antibodies and antiviral compounds, but also for fundamental research on the biology of the virus.

PK-15 cells transfected with porcine CD163 by PiggyBac transposon system are susceptible to porcine reproductive and respiratory syndrome virus.

The PiggyBac (PB) transposon system is a non-viral DNA-transfer system in which a transposase directs integration of a PB transposon into a TTAA site in the genome. Transgenic expression of porcine CD163 is necessary and sufficient to confer non-permissive cells susceptible to infection with porcine reproductive and respiratory syndrome virus (PRRSV). Such permissive cells can be used as a tool for PRRSV cellular receptor and other studies. One of the problems in studying PRRSV is the lack of porcine cell lines. In this study, efficient transfection and expression of porcine CD163 in PK-15 cells by PB transposition was demonstrated. The stable PK-15CD163 cell line was used in PRRSV infection assays. The data indicated that the average PB transgene copy number per genome was approximately 10. In line with previous literature the integration of PB into the genome had a bias toward the TTAA chromosomal site. The PK-15CD163 cell line was susceptible to infection by different PRRSV strains and the virus grew to similar titers compared to the Marc-145 cell line. This simplification of PK-15CD163 cell line production will provide a valuable tool to facilitate PRRSV cellular receptor studies and to accelerate existing vectors for PK-15 cell-based gene transfer and expression.

Complete genome sequence of a highly pathogenic porcine reproductive and respiratory syndrome virus variant.

Following the 2006 outbreaks of the highly pathogenic porcine reproductive and respiratory syndrome, the causative agent was identified as the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). To investigate whether the HP-PRRSV variant continues circulating and accelerating evolution, we sequenced and analyzed the complete genome of the identified HP-PRRSV field strain SD16. The sequence data indicate that the HP-PRRSV variant continues to prevail and accelerate evolution, especially in the nonstructural protein.