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Inactivation of porcine reproductive and respiratory syndrome (PRRS) virus

Inactivation of porcine reproductive and respiratory syndrome (PRRS) virus

This study addressed the third step in this process and, in particular, the specific problem of deriving dose-response curves under experimental conditions in which transmission occurs at concentrations below the threshold of quantification for culture-based methods. In this experiment, a tracer was used to model the behavior of an aerosolized pathogen in a rotating DAT. Tracers, e.g., uranine, rhodamine B, and Bacillus subtilis spores, have been used extensively in experimental aerobiology (Verreault et al. 2008). Songer (1967) aerosolized rhodamine B dye simultaneously with virus (Newcastle disease virus, infectious bovine rhinotracheitis virus, vesicular stomatitis virus, T3 bacteriophage) to track the physical loss of airborne virus within a DAT. In an experiment of similar design, Hermann et al. 2007 found no significant difference between the slopes of rhodamine B dye and porcine reproductive and respiratory syndrome virus RNA detected by quantitative PCR, i.e., the concentrations of rhodamine B and viral RNA declined in the DAT at the same rate. Under the conditions of this experiment, the fact that the theoretical line and the experimental line were not significantly different provided evidence that physical loss did affect the outcome of the tracer values. Thus, rhodamine B concentration has been shown to reflect target
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Phosphorylation of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

Phosphorylation of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

Porcine reproductive and respiratory syndrome virus (PRRSV) is a cytoplasmic RNA virus with the unique or unusual feature of having a nucleocapsid (N) protein that is specifically transported to the nucleolus of virus-infected cells. In this communication, we show that the N protein is a phosphoprotein. Phosphoamino acid analysis of authentic and recombinant N proteins demonstrated that serine residues were exclusively phosphorylated. The pattern of phosphorylated N protein cellular distribution in comparison with that of [ 35 S]methionine-labeled N protein suggested that phosphorylation does not influence subcellular localization of the protein. Time course studies showed that phosphorylation occurred during, or shortly after, synthesis of the N protein and that the protein remained stably phosphorylated throughout the life cycle of the virus to the extent that phosphorylated N protein was found in the mature virion. Two-dimensional electrophoresis and acid-urea gel electrophoresis showed that one species of the N protein is predominant in virus-infected cells, suggesting that multiple phosphorylated isoforms of N do not exist.
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Cell-Mediated Immunity in Porcine Reproductive and Respiratory Syndrome Virus

Cell-Mediated Immunity in Porcine Reproductive and Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome Virus PRRSV is a small, enveloped and cytolytic virus with a size of 50-65 nm. This positive- sense single stranded RNA virus carries a methylated cap at the 5’ end and a polyadenylated tail at the 3’ end of its genome. PRRSV belongs to order Nidovirales, family Arteriviridae, genus Arterivirus and its genomic length is about 15.4 kilobase (Cavanagh, 1997; Conzelmann et al., 1993; Wu et al., 2001). It contains 9 open reading frames (ORFs) among which ORF1a and 1b comprise about 80% of the genome and encode for two polyproteins which, after translation, are proteolytically cleaved into 14 nonstructural proteins (NSPs), e.g., NSP1α, NSP1β and NSP2 to NSP12. Both NSP 9, which encodes the viral RNA-dependent RNA polymerase (RdRp) and NSP 10, which encodes a helicase, are responsible for the viral genome replication and transcription.
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Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release

Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release

State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, People's Republic of China ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Heparan sulfate (HS) is used by PRRSV for initial attachment to target cells. However, the role of HS in the late phase of PRRSV infection and the mechanism of virus release from host cells remain largely unknown. In this study, we showed that PRRSV infection caused a decrease in HS expression and upregulated heparanase, the only known enzyme capable of degrading HS. We subsequently demonstrated that the NF- ␬B signaling pathway and cathepsin L protease were involved in regulation of PRRSV infection- induced heparanase. In addition, we found that ablation of heparanase expression using small interfering RNA duplexes increased cell surface expression of HS and suppressed PRRSV replication and release, whereas overexpression of heparanase re- duced HS surface expression and enhanced PRRSV replication and release. These data suggest that PRRSV activates NF- ␬B and cathepsin L to upregulate and process heparanase, and then the active heparanase cleaves HS, resulting in viral release.
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Exosomes Mediate Intercellular Transmission of Porcine Reproductive and Respiratory Syndrome Virus

Exosomes Mediate Intercellular Transmission of Porcine Reproductive and Respiratory Syndrome Virus

65. Verweij FJ, van Eijndhoven MA, Middeldorp J, Pegtel DM. 2013. Analysis of viral microRNA exchange via exosomes in vitro and in vivo. Methods Mol Biol 1024:53– 68. https://doi.org/10.1007/978-1-62703-453-1_5. 66. Bi J, Song S, Fang L, Wang D, Jing H, Gao L, Cai Y, Luo R, Chen H, Xiao S. 2014. Porcine reproductive and respiratory syndrome virus induces IL-1beta production depending on TLR4/MyD88 pathway and NLRP3 inflammasome in primary porcine alveolar macrophages. Mediators Inflamm 2014:403515. https://doi.org/10.1155/2014/403515.

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Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread

Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread

ABSTRACT Intercellular nanotube connections have been identified as an alternative pathway for cellular spreading of certain viruses. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nanotubes were observed connecting two dis- tant cells with contiguous membranes, with the core infectious viral machinery (viral RNA, certain replicases, and certain struc- tural proteins) present in/on the intercellular nanotubes. Live-cell movies tracked the intercellular transport of a recombinant PRRSV that expressed green fluorescent protein (GFP)-tagged nsp2. In MARC-145 cells expressing PRRSV receptors, GFP-nsp2 moved from one cell to another through nanotubes in the presence of virus-neutralizing antibodies. Intercellular transport of viral proteins did not require the PRRSV receptor as it was observed in receptor-negative HEK-293T cells after transfection with an infectious clone of GFP-PRRSV. In addition, GFP-nsp2 was detected in HEK-293T cells cocultured with recombinant PRRSV- infected MARC-145 cells. The intercellular nanotubes contained filamentous actin (F-actin) with myosin-associated motor pro- teins. The F-actin and myosin IIA were identified as coprecipitates with PRRSV nsp1 ␤, nsp2, nsp2TF, nsp4, nsp7-nsp8, GP5, and N proteins. Drugs inhibiting actin polymerization or myosin IIA activation prevented nanotube formation and viral clusters in virus-infected cells. These data lead us to propose that PRRSV utilizes the host cell cytoskeletal machinery inside nanotubes for efficient cell-to-cell spread. This form of virus transport represents an alternative pathway for virus spread, which is resistant to the host humoral immune response.
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T Cell Response to Infection by the Porcine Reproductive and Respiratory Syndrome Virus

T Cell Response to Infection by the Porcine Reproductive and Respiratory Syndrome Virus

Castro and F. Alonso. Analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection. Virus Res. 64: 33-42, 1999. 58. Sipos, W., C. Duvigneau, P. Pietschmann, K. H Ö ller, R. Hartl, K. Wahl, R. Steinborn, M.Gemeiner, M. Willheim and F. Schmoll. Parameters of humoral and cellular immunity following vaccination of pigs with a European modified-live strain of porcine reproductive and respiratory syndrome virus (PRRSV). Viral Immunol.

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Role of CD151, A tetraspanin, in porcine reproductive and respiratory syndrome virus infection

Role of CD151, A tetraspanin, in porcine reproductive and respiratory syndrome virus infection

Email: Kumar Shanmukhappa - shatm2@cchmc.org; Jeong-Ki Kim - Jeong-Ki.Kim@STJUDE.ORG; Sanjay Kapil* - sanjay.kapil@okstate.edu * Corresponding author Abstract Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is a RNA virus causing respiratory and reproductive diseases in swine. The susceptibility for PRRSV varies between the different breeds of swine. In cell culture, PRRSV virus can be propagated in primary porcine alveolar macrophages and some African green monkey kidney cell lines, such as MARC- 145 cells. Previous studies have shown that 3' untranslated region (UTR) RNAs of the arteriviruses play an important role in the replication of the virus through interactions with cellular proteins. To better understand the differences in the replication capability of PRRSV in different cell lines, we sought to identify the host cellular proteins interacting with PRRSV 3' UTR RNA. We constructed a cDNA library of MARC-145 cell line in lambda ZAP Express vector and screened the library with the positive sense 3' UTR RNA of PRRSV.
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THE NSP2 VARIATION OF VIETNAMESE PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS STRAINS

THE NSP2 VARIATION OF VIETNAMESE PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS STRAINS

2. Cavanagh, D. (1997) Nidovirales: a new order comprising Coronaviridae and Arteriviridae, Arch Virol 142:629–633. 3. Chen, Z., Zhou, X., Lunney, J.K., Lawson, S., Sun, Z., Brown, E., Hennings, J.C., Knudsen, D., Nelson, E. and Fang, Y. (2010), Immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response, Journal of General Virology 91:

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Biosecurity protocols for the prevention of spread of porcine reproductive and respiratory syndrome virus

Biosecurity protocols for the prevention of spread of porcine reproductive and respiratory syndrome virus

Introduction Porcine reproductive and respiratory syndrome (PRRS) is an economically significant disease of swine that has been estimated to cost the US industry approximately $560 million a year. Preventing the spread of PRRSV within and between pig populations is a critical component of a farm’s disease control program. To aid in controlling the spread of this agent, this manual provides a summary of data from experiments conducted from our group at the University of Minnesota that were specifically designed to identify the routes of PRRSV transmission and to develop protocols of biosecurity to reduce this risk. All protocols have been, and continue to be validated during an on­going experiment that has been in process over the past 2 years at our Swine Disease Eradication Center (SDEC) production region model farm. The authors of this manual continue to practice these protocols and procedures on a daily basis; therefore, as of this writing our confidence in their ability to prevent PRRSV spread is very high. We hope that swine veterinarians can utilize this information to help their clients develop effective biosecurity programs for sustainable PRRS control.
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A synthetic porcine reproductive and respiratory syndrome unprecedented levels of heterologous protection

A synthetic porcine reproductive and respiratory syndrome unprecedented levels of heterologous protection

A synthetic porcine reproductive and respiratory syndrome A synthetic porcine reproductive and respiratory syndrome unprecedented levels of heterologous protection.. unprecedented leve[r]

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Heterogeneous antigenic properties of the porcine reproductive and respiratory syndrome virus nucleocapsid

Heterogeneous antigenic properties of the porcine reproductive and respiratory syndrome virus nucleocapsid

2. Nieuwenhuis N, Duinhof TF, van Nes A (2012) Economic analysis of outbreaks of porcine reproductive and respiratory syndrome virus in nine sow herds. Vet Rec 170:225 3. Wensvoort G, Terpstra C, Pol JM, ter Laak EA, Bloemraad M, de Kluyver EP, Kragten C, van Buiten L, den Besten A, Wagenaar F, Broekhuijsen JM, Moonen PLJM, Zetstra T, de Boer EA, Tibben HJ, de Jong MF, van ‘t Veld P, Greenland GJR, van Gennep JA, Voets MTH, Verheijden JHM, Braamskamp J (1991) Mystery swine disease in The Netherlands: the isolation of Lelys‑

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Control of the PI3K/Akt pathway by porcine reproductive and respiratory syndrome virus

Control of the PI3K/Akt pathway by porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is a small, enveloped, positive-strand RNA virus belonging to the family Arteriviridae [ 9 , 22 ]. Since it emerged in late 1980s in Europe and North America, it has caused significant economic losses to the pork industry worldwide [ 1 , 26 ]. In 2006, an outbreak of HP-PRRSV described as ‘‘pig high fever disease’’ occurred in China and caused disastrous loses to the farmers [ 31 , 39 ]. This disease is currently a major concern for the swine industry worldwide. Phosphatidylinositol-3-kinase (PI3K)/Akt is a key sig- naling transduction pathway in the regulation of cell sur- vival, cell proliferation and differentiation, and cell apoptosis [ 6 – 8 , 10 ]. The activation of PI3K-Akt pathway promotes cell survival by the phosphorylation of numerous substrates such as glycogen synthase kinase-3 (GSK-3), FoxO1, Bad and mTOR [ 5 , 11 , 24 , 36 , 38 ]. Many viruses are known to manipulate this pathway in favor of their replication, such as influenza A virus [ 29 ], hepatitis B virus [ 16 ], hepatitis C virus [ 17 ], severe acute respiratory syn- drome coronavirus (SARS-CoV) [ 23 ], Junı´n virus [ 19 ], human immunodeficiency virus type 1 [ 20 ], bovine her- pesvirus type 1 [ 40 ] and infectious bursal disease virus [ 34 ]. Previous studies have shown that during PRRSV infection of porcine monocyte-derived dendritic cells (Mo-DCs), the PI3K/Akt pathway is activated at 90 min and 4 h postinfection (h p.i.), and it is inhibited at 12 h p.i. [ 37 ]. In vivo, the virus shows a very narrow cell tropism and infects a specific subpopulation of porcine macrophages L. Zhu and S. Yang contributed equally to this work
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Porcine reproductive and respiratory syndrome virus: understanding and managing persistent infection

Porcine reproductive and respiratory syndrome virus: understanding and managing persistent infection

Diagnostic performance of assays for the detection of anti-porcine reproductive and respiratory syndrome virus antibodies in serum and muscle transudate (“meat juice”) based on samples[r]

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Adaptable inactivated vaccine for combating Porcine Reproductive and Respiratory Syndrome Virus

Adaptable inactivated vaccine for combating Porcine Reproductive and Respiratory Syndrome Virus

SUMMARY Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus that is involved with reproductive failure (weak- and stillborn piglets, premature farrowing, late-term abortions) in sows and respiratory disease in pigs of all ages, resulting in huge economic losses to the swine industry. A high genetic variability has been demonstrated within PRRSV variants and the genetic differences between virus variants are mirrored in different virulence, pathogenicity, immunogenicity, … This high variability of the virus represents a major hurdle for effective PRRSV prevention and control. Broad application of current available PRRSV vaccines is the most commonly used strategy to combat the clinical and economical impact of PRRSV infections. It is generally accepted that there is a need for new and safe vaccines that can protect against infection with those virus variants that escape immunity induced by the currently available commercial vaccines. The general goal of this work was to evaluate autogenous inactivated PRRSV vaccines – prepared according to a previously optimized in-house protocol – with the capacity of commercially available attenuated/inactivated PRRSV vaccines in naïve and PRRSV-immune animals.
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Pathogenesis of the respiratory form of porcine reproductive and respiratory syndrome: modulatory role of cytokines in the immune response

Pathogenesis of the respiratory form of porcine reproductive and respiratory syndrome: modulatory role of cytokines in the immune response

I I NT N TR RO OD DU UC CT TI IO ON N Porcine Reproductive and Respiratory Syndrome (PRRS) is worldwide spread and constitute one of the most significant diseases in the swine industry. This syndrome is characterised by inducing reproductive failure in sows and respiratory symptoms in growing and finishing pigs. Although there is some controversy, PRRS virus (PRRSV) is considered as able to modulate the immune response, making easier the concomitant infection with secondary bacteria, and playing a significant role in the onset of the Porcine Respiratory Disease Complex (PRDC) (Rossow, 1998). Although several studies have been carried out to elucidate the host immune response evoked against PRRSV, there are still a lot of aspects which still remain unclear.
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Role of Toll-Like Receptors in Activation of Porcine Alveolar Macrophages by Porcine Reproductive and Respiratory Syndrome Virus

Role of Toll-Like Receptors in Activation of Porcine Alveolar Macrophages by Porcine Reproductive and Respiratory Syndrome Virus

Control of virus replication initially depends on rapid activation of the innate immune response. Toll-like receptor (TLR) ligands are potent inducers of innate immunity against viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV), a positive-sense RNA virus, initiates infection in porcine alveolar macrophages (PAMs), elicits weak immune responses, and establishes a persistent infection. To understand the role of single-stranded RNA and double-stranded RNA (dsRNA) intermediates in eliciting host immunity, we sought to determine if TLRs, particularly those that respond to viral molecular patterns, are involved in PRRSV infection. Activation of TLR3 in PAMs with dsRNA increased gene expression for alpha interferon and suppressed PRRSV infectivity. In contrast, TLR4 activation by the treatment of PAMs with lipopolysaccharide did not influence PRRSV infectivity.
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Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus

Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus

a Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, People’s Republic of China ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive- stranded RNA virus belonging to the family Arteriviridae. Synthesis of the viral RNA is di- rected by replication/transcription complexes (RTC) that are mainly composed of a net- work of PRRSV nonstructural proteins (nsps) and likely cellular proteins. Here, we mapped the interaction network among PRRSV nsps by using yeast two-hybrid screen- ing in conjunction with coimmunoprecipitation (co-IP) and cotransfection assays. We identified a total of 24 novel interactions and found that the interactions were centered on open reading frame 1b (ORF1b)-encoded nsps that were mainly connected by the transmembrane proteins nsp2, nsp3, and nsp5. Interestingly, the interactions of the core enzymes nsp9 and nsp10 with transmembrane proteins did not occur in a straightfor- ward manner, as they worked in the co-IP assay but were poorly capable of finding each other within intact mammalian cells. Further proof that they can interact within cells required the engineering of N-terminal truncations of both nsp9 and nsp10. How- ever, despite the poor colocalization relationship in cotransfected cells, both nsp9 and nsp10 came together with membrane proteins (e.g., nsp2) at the viral replication and transcription complexes (RTC) in PRRSV-infected cells. Thus, our results indicate the exis- tence of a complex interaction network among PRRSV nsps and raise the possibility that the recruitment of key replicase proteins to membrane-associated nsps may involve some regulatory mechanisms during infection.
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Crystal Structure of Porcine Reproductive and Respiratory Syndrome Virus Leader Protease Nsp1α

Crystal Structure of Porcine Reproductive and Respiratory Syndrome Virus Leader Protease Nsp1α

Tianjin State Laboratory of Protein Science, Nankai University, Tianjin 300071, China 3 Received 15 December 2008/Accepted 4 August 2009 Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV), a positive-strand RNA virus that belongs to the Arteriviridae family of Nidovirales, has been identified as the causative agent of PRRS. Nsp1 ␣ is the amino (N)-terminal protein in a polyprotein encoded by the PRRSV genome and is reported to be crucial for subgenomic mRNA synthesis, presumably by serving as a transcription factor. Before functioning in transcription, nsp1 ␣ proteolytically releases itself from nsp1␤. However, the structural basis for the self- releasing and biological functions of nsp1 ␣ remains elusive. Here we report the crystal structure of nsp1␣ of PRRSV (strain XH-GD) in its naturally self-processed form. Nsp1 ␣ contains a ZF domain (which may be required for its biological function), a papain-like cysteine protease (PCP) domain with a zinc ion unexpectedly bound at the active site (which is essential for proteolytic self-release of nsp1 ␣), and a carboxyl-terminal extension (which occupies the substrate binding site of the PCP domain). Furthermore, we determined the exact location of the nsp1 ␣ self-processing site at Cys-Ala-Met1802Ala-Asp-Val by use of crystallographic data and N-terminal amino acid sequencing. The crystal structure also suggested an in cis self-processing mechanism for nsp1 ␣. Furthermore, nsp1␣ appears to have a dimeric architecture both in solution and as a crystal, with a hydrophilic groove on the molecular surface that may be related to nsp1 ␣’s biological function.
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Evolution of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Passages in Pigs

Evolution of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Passages in Pigs

Department of Veterinary Diagnostic and Production Animal Medicine 1 and Department of Statistics, 2 Iowa State University, Ames, Iowa 50010, and Department of Veterinary PathoBiology, University of Minnesota, St. Paul, Minnesota 55108 3 Received 6 November 2001/Accepted 12 February 2002 Porcine reproductive and respiratory syndrome (PRRS) viruses are recognized as possessing a high degree of genetic and antigenic variability. Viral diversity has led to questions regarding the association of virus mutation and persistent infection in the host and has raised concerns vis-à-vis protective immunity, the ability of diagnostic assays to detect novel variants, and the possible emergence of virulent strains. The purpose of this study was to describe ongoing changes in PRRS virus during replication in pigs under experimental conditions.
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