The M/GP5 Glycoprotein Complex of Porcine Reproductive and Respiratory Syndrome Virus Binds the Sialoadhesin Receptor in a Sialic Acid-Dependent Manner

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP3, GP4 and GP5 envelope glycoproteins, only the M/GP5 glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP5. These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines

Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages

Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners

<p>Abstract</p> <p>Background</p> <p>Enzymes in the radical SAM (rSAM) domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. Evolutionary pressures and relationships to other cellular constituents impose recognizable grammars on each class of rSAM-containing system, shaping patterns in results obtained through various comparative genomics analyses.</p> <p>Results</p> <p>An uncharacterized gene cluster found in many Actinobacteria and sporadically in Firmicutes, Chloroflexi, Deltaproteobacteria, and one Archaeal plasmid contains a PqqE-like rSAM protein family that includes Rv0693 from <it>Mycobacterium tuberculosis</it>. Members occur clustered with a strikingly well-conserved small polypeptide we designate "mycofactocin," similar in size to bacteriocins and PqqA, precursor of pyrroloquinoline quinone (PQQ). Partial Phylogenetic Profiling (PPP) based on the distribution of these markers identifies the mycofactocin cluster, but also a second tier of high-scoring proteins. This tier, strikingly, is filled with up to thirty-one members per genome from three variant subfamilies that occur, one each, in three unrelated classes of nicotinoproteins. The pattern suggests these variant enzymes require not only NAD(P), but also the novel gene cluster. Further study was conducted using SIMBAL, a PPP-like tool, to search these nicotinoproteins for subsequences best correlated across multiple genomes to the presence of mycofactocin. For both the short chain dehydrogenase/reductase (SDR) and iron-containing dehydrogenase families, aligning SIMBAL's top-scoring sequences to homologous solved crystal structures shows signals centered over NAD(P)-binding sites rather than over substrate-binding or active site residues. Previous studies on some of these proteins have revealed a non-exchangeable NAD cofactor, such that enzymatic activity <it>in vitro </it>requires an artificial electron acceptor such as N,N-dimethyl-4-nitrosoaniline (NDMA) for the enzyme to cycle.</p> <p>Conclusions</p> <p>Taken together, these findings suggest that the mycofactocin precursor is modified by the Rv0693 family rSAM protein and other enzymes in its cluster. It becomes an electron carrier molecule that serves <it>in vivo </it>as NDMA and other artificial electron acceptors do <it>in vitro</it>. Subclasses from three different nicotinoprotein families show "only-if" relationships to mycofactocin because they require its presence. This framework suggests a segregated redox pool in which mycofactocin mediates communication among enzymes with non-exchangeable cofactors.</p

Genetic Diversity of the ORF5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Isolates in Southwest China from 2007 to 2009

To gain insight into the molecular epidemiology and possible mechanisms of genetic variation of porcine reproductive and respiratory syndrome (PRRS) in Yunnan Province of China, the ORF5 gene of 32 PRRSV isolates from clinical samples collected from 2007 to 2009 were sequenced and analyzed. Nucleotide and amino acid analyses were carried out on 32 isolates and representative strains of the North American genotype, European genotype and two representative Chinese isolates. Results revealed that these isolates share 86.9–99.0% nucleotide and 87.5–98.0% amino acid identity with VR-2332 the prototypical North American PRRSV, 61.7–62.9% and 54.3–57.8% with Lelystad virus (LV) the representative strain of European genotype, 91.2–95.4% and 90.0–94.5% with CH-1a that was isolated in mainland China in 1996, 88.1–99.3% and 85.5–99.0% with JX-A1 the representative strain of High pathogenic PRRSV in China, and 86.2–99.8% and 85.5–100.0% between isolated strains of different years, respectively. Phylogenetic analysis revealed that all 32 PRRSV isolates belonged to the North American genotype and were further divided into two different subgenotypes. Subgenotype 1 comprised twenty two Yunnan isolates which divided into two branches. Subgenotype 2 comprised ten isolates which closely related to the RespPRRS vaccine and its parent strain VR-2332. The functional domains of GP5 such as the signal peptide, ectodomain, transmembrane regions and endodomain were identified and some motifs in GP5 with known functions, such as primary neutralizing epitope (PNE) and decoy epitope were also further analyzed. Our study shown the great genetic diversity of PRRSV in southwest China, rendering the guide for control and prevention of this disease

Sustaining Interferon Induction by a High-Passage Atypical Porcine Reproductive and Respiratory Syndrome Virus Strain

Partial funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.Porcine reproductive and respiratory syndrome virus (PRRSV) strain A2MC2 induces type I interferons in cultured cells. The objective of this study was to attenuate this strain by serial passaging in MARC-145 cells and assess its virulence and immunogenicity in pigs. The A2MC2 serially passaged 90 times (A2MC2-P90) retains the feature of interferon induction. The A2MC2-P90 replicates faster with a higher virus yield than wild type A2MC2 virus. Infection of primary pulmonary alveolar macrophages (PAMs) also induces interferons. Sequence analysis showed that the A2MC2-P90 has genomic nucleic acid identity of 99.8% to the wild type but has a deletion of 543 nucleotides in nsp2. The deletion occurred in passage 60. The A2MC2-P90 genome has a total of 35 nucleotide variations from the wild type, leading to 26 amino acid differences. Inoculation of three-week-old piglets showed that A2MC2-P90 is avirulent and elicits immune response. Compared with Ingelvac PRRS® MLV strain, A2MC2-P90 elicits higher virus neutralizing antibodies. The attenuated IFN inducing A2MC2-P90 should be useful for development of an improved PRRSV vaccine

Porcine FcγRIIb Mediates Enhancement of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection

Antibody-dependent enhancement (ADE) of virus infection caused by the uptake of virus-antibody complexes by FcγRs is a significant obstacle to the development of effective vaccines to control certain human and animal viral diseases. The activation FcγRs, including FcγRI and FcγRIIa have been shown to mediate ADE infection of virus. In the present paper, we showed that pocine FcγRIIb, an inhibitory FcγR, mediates ADE of PRRSV infection. Stable Marc-145 cell lines expressing poFcγRIIb (Marc-poFcγRII) were established. The relative yield of progeny virus was significantly increased in the presence of sub-neutralization anti-PRRSV antibody. The Fab fragment and normal porcine sera had no effect. Anti-poFcγRII antibody inhibited the enhancement of infection when cells were infected in the presence of anti-PRRSV antibody, but not when cells were infected in the absence of antibody. These results indicate that enhancement of infection in these cells by anti-PRRSV virus antibody is FcγRII-mediated. Identification of the inhibitory FcγR mediating ADE infection should expand our understanding of the mechanisms of pathogenesis for a broad range of infectious diseases and may open many approaches for improvements to the treatment and prevention of such diseases

Do decision support systems influence variation in prescription?

<p>Abstract</p> <p>Background</p> <p>Translating scientific evidence into daily practice is problematic. All kinds of intervention strategies, using educational and/or directive strategies, aimed at modifying behavior, have evolved, but have been found only partially successful. In this article the focus is on (computerized) decision support systems (DSSs). DSSs intervene in physicians' daily routine, as opposed to interventions that aim at influencing knowledge in order to change behavior. We examined whether general practitioners (GPs) are prescribing in accordance with the advice given by the DSS and whether there is less variation in prescription when the DSS is used.</p> <p>Methods</p> <p>Data were used from the Second Dutch National Survey of General Practice (DNSGP2), collected in 2001. A total of 82 diagnoses, 749811 contacts, 133 physicians, and 85 practices was included in the analyses. GPs using the DSS daily were compared to GPs who do not use the DSS. Multilevel analyses were used to analyse the data. Two outcome measures were chosen: whether prescription was in accordance with the advice of the DSS or not, and a measure of concentration, the Herfindahl-Hirschman Index (HHI).</p> <p>Results</p> <p>GPs who use the DSS daily prescribe more according to the advice given in the DSS than GPs who do not use the DSS. Contradictory to our expectation there was no significant difference between the HHIs for both groups: variation in prescription was comparable.</p> <p>Conclusion</p> <p>We studied the use of a DSS for drug prescribing in general practice in the Netherlands. The DSS is based on guidelines developed by the Dutch College of General Practitioners and implemented in the Electronic Medical Systems of the GPs. GPs using the DSS more often prescribe in accordance with the advice given in the DSS compared to GPs not using the DSS. This finding, however, did not mean that variation is lower; variation is the same for GPs using and for GPs not using a DSS. Implications of the study are that DSSs can be used to implement guidelines, but that it should not be expected that variation is limited.</p

Phonon Raman spectra of colloidal CdTe nanocrystals: effect of size, non-stoichiometry and ligand exchange

Resonant Raman study reveals the noticeable effect of the ligand exchange on the nanocrystal (NC) surface onto the phonon spectra of colloidal CdTe NC of different size and composition. The oleic acid ligand exchange for pyridine ones was found to change noticeably the position and width of the longitudinal optical (LO) phonon mode, as well as its intensity ratio to overtones. The broad shoulder above the LO peak frequency was enhanced and sharpened after pyridine treatment, as well as with decreasing NC size. The low-frequency mode around 100 cm-1 which is commonly related with the disorder-activated acoustical phonons appears in smaller NCs but is not enhanced after pyridine treatment. Surprisingly, the feature at low-frequency shoulder of the LO peak, commonly assigned to the surface optical phonon mode, was not sensitive to ligand exchange and concomitant close packing of the NCs. An increased structural disorder on the NC surface, strain and modified electron-phonon coupling is discussed as the possible reason of the observed changes in the phonon spectrum of ligand-exchanged CdTe NCs

Expensive Egos: Narcissistic Males Have Higher Cortisol

Background: Narcissism is characterized by grandiosity, low empathy, and entitlement. There has been limited research regarding the hormonal correlates of narcissism, despite the potential health implications. This study examined the role of participant narcissism and sex on basal cortisol concentrations in an undergraduate population. Methods and Findings: Participants were 106 undergraduate students (79 females, 27 males, mean age 20.1 years) from one Midwestern and one Southwestern American university. Narcissism was assessed using the Narcissistic Personality Inventory, and basal cortisol concentrations were collected from saliva samples in a laboratory setting. Regression analyses examined the effect of narcissism and sex on cortisol (log). There were no sex differences in basal cortisol, F(1,97) =.20, p =.65, and narcissism scores, F(1,97) =.00, p =.99. Stepwise linear regression models of sex and narcissism and their interaction predicting cortisol concentrations showed no main effects when including covariates, but a significant interaction, b =.27, p =.04. Narcissism was not related to cortisol in females, but significantly predicted cortisol in males. Examining the effect of unhealthy versus healthy narcissism on cortisol found that unhealthy narcissism was marginally related to cortisol in females, b =.27, p =.06, but significantly predicted higher basal cortisol in males, b =.72, p =.01, even when controlling for potential confounds. No relationship was found between sex, narcissism, or their interaction on selfreported stress

Emergence of Fatal PRRSV Variants: Unparalleled Outbreaks of Atypical PRRS in China and Molecular Dissection of the Unique Hallmark

Porcine reproductive and respiratory syndrome (PRRS) is a severe viral disease in pigs, causing great economic losses worldwide each year. The causative agent of the disease, PRRS virus (PRRSV), is a member of the family Arteriviridae. Here we report our investigation of the unparalleled large-scale outbreaks of an originally unknown, but so-called “high fever” disease in China in 2006 with the essence of PRRS, which spread to more than 10 provinces (autonomous cities or regions) and affected over 2,000,000 pigs with about 400,000 fatal cases. Different from the typical PRRS, numerous adult sows were also infected by the “high fever” disease. This atypical PRRS pandemic was initially identified as a hog cholera-like disease manifesting neurological symptoms (e.g., shivering), high fever (40–42°C), erythematous blanching rash, etc. Autopsies combined with immunological analyses clearly showed that multiple organs were infected by highly pathogenic PRRSVs with severe pathological changes observed. Whole-genome analysis of the isolated viruses revealed that these PRRSV isolates are grouped into Type II and are highly homologous to HB-1, a Chinese strain of PRRSV (96.5% nucleotide identity). More importantly, we observed a unique molecular hallmark in these viral isolates, namely a discontinuous deletion of 30 amino acids in nonstructural protein 2 (NSP2). Taken together, this is the first comprehensive report documenting the 2006 epidemic of atypical PRRS outbreak in China and identifying the 30 amino-acid deletion in NSP2, a novel determining factor for virulence which may be implicated in the high pathogenicity of PRRSV, and will stimulate further study by using the infectious cDNA clone technique