A panel of archived EHV-1 isolates collected (1951 to 2006) from equine abortions was analyzed using a real-time Taq-Man ® allelic discrimination PCR. Based on previous findings, isolates possessing adenine at nucleotide position 2254 (A 2254) in ORF30 were classified as having a non-neuropathogenic genotype and those with guanine at 2254 (G 2254) were designated as the neuropathogenic genotype. The resultant data demonstrated that viruses with the neuropathogenic genotype existed in the 1950s and isolates with this genotype increased from 3.3% in the 1960s to 14.4% in the 1990s. The incidence of EHV-1 isolates from 2000 to 2006 with G at position 2254 is 19.4%, suggesting that viruses with the neuropathogenic genotype are continuing to increase in prevalence within the latent reservoir of the virus, leading to greater risks for costly outbreaks of equine herpesvirus neurologic disease. Another highly significant finding was two isolates failed to react with either probe in the allelic discrimination assay. These isolates were found to possess an adenine to cytosine substitution at position 2258 (A 2258 →C 2258) in ORF30, in addition to A 2254 →G 2254. Interestingly, the nonneuropathogenic RAC-H modified live vaccine strain of EHV-1 also contains both A 2254 →G 2254 and A 2258 →C 2258 substitutions. This finding clearly suggests that additional research is required before the genetic basis of the neuropathogenic phenotype in EHV-1 is fully understood.

A single-nucleotide polymorphism (A 2254 or G 2254 ) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E 2 ) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69–72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E 1 ) was developed by redesigning primers and probes specific to ORF30. The E 1 and E 2 rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A 2254 or G 2254 ) in all archived isolates plus 168 of the clinical samples. The E 1 assay was 10 times more sensitive than E 2 , with a lower detection limit of 10 infectious virus particles. Furthermore, all A 2254 and G 2254 genotypes along with samples from three cases of dual infection (A 2254 +G 2254 ) were correctly identified by E 1 , whereas E 2 produced 20 false dual positive results with only one actual mixed A 2254 +G 2254 genotype confirmed. Based on these findings, E 1 offers greater sensitivity and accuracy for the detection and A/G 2254 genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.

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Leishmania parasites are responsible for important neglected diseases in humans and animals, ranging from self-healing cutaneous lesions to fatal visceral manifestations. During the infectious cycle, Leishmania differentiates from the extracellular flagellated promastigote to the intracellular pathogenic amastigote. Parasite differentiation is triggered by changes in environmental cues, mainly pH and temperature. In general, extracellular signals are translated into stage-specific gene expression by a cascade of reversible protein phosphorylation regulated by protein kinases and phosphatases. Though protein kinases have been actively studied as potential anti-parasitic drug targets, our understanding of the biology of protein phosphatases in Leishmania is poor. We have previously reported the principal analysis of a novel protein phosphatase 5 (PP5) in Leishmania species. Here, we assessed the role of PP5 in parasite pathogenicity, where we uncovered, using transgenic PP5 over-expressing and PP5 null-mutant parasites, its importance in metacyclogeneisis, maintaining HSP83 phosphorylation homeostasis and virulence. All together, our results indicate the importance of PP5 in regulating parasite stress and adaptation during differentiation, making this protein an attractive potential target for therapeutic intervention.

BackgroundA homeostatic relationship with the intestinal microflora is increasingly appreciated as essential for human health and wellbeing. Mutations in the leucine-rich repeat (LRR) domain of Nod2, a bacterial recognition protein, are associated with development of the inflammatory bowel disorder, Crohn's disease. We investigated the molecular mechanisms underlying disruption of intestinal symbiosis in patients carrying Nod2 mutations.Methodology/Principal FindingsIn this study, using purified recombinant LRR domains, we demonstrate that Nod2 is a direct antimicrobial agent and this activity is generally deficient in proteins carrying Crohn's-associated mutations. Wild-type, but not Crohn's-associated, Nod2 LRR domains directly interacted with bacteria in vitro, altered their metabolism and disrupted the integrity of the plasma membrane. Antibiotic activity was also expressed by the LRR domains of Nod1 and other pattern recognition receptors suggesting that the LRR domain is a conserved anti-microbial motif supporting innate cellular immunity.Conclusions/SignificanceThe lack of anti-bacterial activity demonstrated with Crohn's-associated Nod2 mutations in vitro, supports the hypothesis that a deficiency in direct bacterial killing contributes to the association of Nod2 polymorphisms with the disease.

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