Universal sample preparation method for proteome analysis

The immune system protects the organism against infections and the damage associated with them. The first line of defense against pathogens is the innate immune response. In the case of a viral infection, it induces the interferon signaling cascade and eventually the expression of type I interferon, which then causes an antiviral state in the cells. However, many viruses have developed strategies to counteract this mechanism and prevent the production of interferon. In order to modulate or inhibit the interferon signaling cascade in their favor, viruses have found ways to interfere at every single step of the cascade, for example by inducing protein degradation or cleavage, or by mediate protein polyubiquitinylation. In this article we will review examples of viruses that modulate the interferon response and describe the mechanisms they use.

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Viral Evasion Strategies in Type I IFN Signaling - A Summary of Recent Developments.

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine diseases in the world. It is causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, with mortality varying from 2 to 100% in the most extreme cases of emergent highly pathogenic strains. PRRSV displays complex interactions with the immune system and a high mutation rate, making the development, and implementation of control strategies a major challenge. In this review, the biology of the virus will be addressed focusing on newly discovered functions of non-structural proteins and novel dissemination mechanisms. Secondly, the role of different cell types and viral proteins will be reviewed in natural and vaccine-induced immune response together with the role of different immune evasion mechanisms focusing on those gaps of knowledge that are critical to generate more efficacious vaccines. Finally, novel strategies for antigen discovery and vaccine development will be discussed, in particular the use of exosomes (extracellular vesicles of endocytic origin). As nanocarriers of lipids, proteins and nucleic acids, exosomes have potential effects on cell activation, modulation of immune responses and antigen presentation. Thus, representing a novel vaccination approach against this devastating disease.

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Key Gaps in the Knowledge of the Porcine Respiratory Reproductive Syndrome Virus (PRRSV).

In 2006, an emerging highly pathogenic strain of porcine reproductive and respiratory syndrome virus (PRRSV), which causes continuous high fever and a high proportion of deaths in vaccinated pigs of all ages, broke out in mainland China and spread rapidly to neighboring countries. To examine the epidemiology and evolutionary characteristics of Chinese PRRSV after the 2006 outbreak, we tested 2,981 clinical samples collected from 2006 to 2010 in China, determined 153 Nsp2 sequences and 249 ORF5 sequences, and analyzed the epidemiology and genetic diversity of Chinese PRRSV. Our results showed that the percentage of PRRSV-positive specimens collected from sick pigs averaged 60.85% in the past 5 years and that the highly pathogenic PRRSV has become the dominant strain in China. Furthermore, a reemerging strain which apparently evolved from the highly pathogenic PRRSV strain in 2006 appeared to be widely prevalent in China from 2009 onwards. Sequence analyses revealed that the hypervariable region of Nsp2 in most of the isolates contained a discontinuous deletion equivalent to 30 amino acids, along with other types of deletions. Extensive amino acid substitutions in the GP5 sequence translated from ORF5 were found, particularly in the potential neutralization epitope and the N-glycosylation sites. Our results suggest that Chinese PRRSV has undergone rapid evolution and can circumvent immune responses induced by currently used vaccines. Information from this study will help in understanding the evolutionary characteristics of Chinese PRRSV and assist ongoing efforts to develop and use PRRSV vaccines in the future.

Epidemiology and Evolutionary Characteristics of the Porcine Reproductive and Respiratory Syndrome Virus in China between 2006 and 2010

Background
Understanding the role of host genetics in resistance to porcine reproductive and respiratory syndrome virus (PRRSV) infection, and the effects of PRRS on pig health and related growth, are goals of the PRRS Host Genetics Consortium (PHGC).

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Probing genetic control of swine responses to PRRSV infection: current progress of the PRRS host genetics consortium

The genomic diversity of Chinese porcine reproductive and respiratory syndrome virus isolates from 1996 to 2009

Exosomes are small membrane-enclosed vesicles produced by various cells and actively released into the extracellular space. They participate in intercellular communication and transfer of biologically active proteins, lipids, and nucleic acids. Accumulating evidence suggests that exosomes derived from cells infected by some viruses selectively encapsulate viral proteins, genetic materials, or even virions to mediate cell-to-cell communication and/or virus transmission. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the global swine industry since the late 1980s. Recent studies have shown that major proteins secreted from PRRSV-infected cells are exosomal proteins and that the serum-derived exosomes from PRRSV-infected pigs contain viral proteins. However, the role of exosomes in PRRSV infection remains unclear. In this study, purified exosomes isolated from PRRSV-infected cells were shown with reverse transcription-PCR and mass spectrometry to contain viral genomic RNA and partial viral proteins. Furthermore, exosomes from PRRSV-infected cells established productive infection in both PRRSV-susceptible and -nonsusceptible cells. More importantly, exosome-mediated infection was not completely blocked by PRRSV-specific neutralizing antibodies. In summary, this study demonstrated that exosomes can mediate PRRSV transmission and are even resistant to antibody neutralization, identifying a potential immune evasion mechanism utilized by PRRSV.IMPORTANCE Exosomes have recently been characterized as bioactive vesicles that function to promote intercellular communication. The exosomes from virally infected cells containing altered compositions confer numerous novel functionalities. A study of the secretome of cells infected with PRRSV indicated that the exosomal pathway is strongly activated by PRRSV infection. Here, we demonstrate that PRRSV can utilize host exosomes to infect naive healthy cells. Furthermore, exosome-mediated viral transmission is largely resistant to PRRSV-specific neutralizing antibodies. Our study provides novel insights into an alternative mechanism of PRRSV transmission that can compromise the host's anti-PRRSV immune response.

Exosomes Mediate Intercellular Transmission of Porcine Reproductive and Respiratory Syndrome Virus.

Human interleukin-29 (IL-29) is a recently discovered cytokine displaying antiviral activity against a broad spectrum of viruses, designated as interferon (IFN)-λ1. We here report the molecular cloning, expression and antiviral activity of porcine IL-29 (poIL-29). The full-length poIL-29 cDNA sequence encoded 191 amino acids with a 19 amino acid signal peptide. Sequence alignments showed that poIL-29 had amino acid sequence similarity to wolf (76%), bear (75%), horse (75%), orangutan (73%), human (72%), cat (72%) and dog (70%) IL-29. The poIL-29 without signal anchor sequence was efficiently expressed as a 6×HIS fusion protein in Escherichia coli and the antiviral activity of the purified recombinant protein was 1.8 × 103 U/mg protein. The purified recombinant poIL-29 also exhibited significant antiviral effects against porcine reproductive and respiratory syndrome virus and pseudorabies virus in a dose-dependent manner, suggesting that poIL-29 is a potential antiviral agent against swine infectious diseases.

Molecular cloning, expression and antiviral activity of porcine interleukin-29 (poIL-29)

Metabolomic profiling of Dezhou donkey sperm associated with freezability.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which causes severe reproductive failure in sows, respiratory disease in young and growing pigs, and enormous economic losses to the global swine industry. In this study, SILAC combined with MS/MS was used to quantitatively identify the secretory proteins differentially expressed in PRRSV-infected Marc-145 cells compared with mock-infected controls. In total, we identified 204 secretory proteins showing significant differences in infected cells (163 upregulated, 41 downregulated). Intensive bioinformatic analysis of secretome data revealed that PRRSV infection strongly activated nonclassical protein secretion, especially vesicle-mediated release of exosomal proteins, including different danger-associated molecular pattern molecules and the majority of secreted proteins involved in protein binding and transport, regulation of response to stimulus, metabolic processes, and immune responses. According to the functional proteins analysis, we speculate that proteins functioning in binding, transport, and the immune response are exploited by PRRSV to facilitate virus replication and immune evasion. Our study for the first time analyzes the secretory protein profile of PRRSV-infected Marc-145 cells and provides valuable insight into the host response to PRRSV infection.

Fuwei Zhao

Papers

The genomic diversity of Chinese porcine reproductive and respiratory syndrome virus isolates from 1996 to 2009

Exosomes Mediate Intercellular Transmission of Porcine Reproductive and Respiratory Syndrome Virus.

Molecular cloning, expression and antiviral activity of porcine interleukin-29 (poIL-29)

Metabolomic profiling of Dezhou donkey sperm associated with freezability.

SILAC-based quantitative proteomic analysis of secretome of Marc-145 cells infected with porcine reproductive and respiratory syndrome virus