A murine model for neuropsychiatric disorders associated with group A β-hemolytic streptococcal infection

A syndrome of motoric and neuropsychiatric symptoms comprising various elements, including chorea, hyperactivity, tics, emotional lability, and obsessive-compulsive symptoms, can occur in association with group A β-hemolytic streptococcal (GABHS) infection. We tested the hypothesis that an immune response to GABHS can result in behavioral abnormalities. Female SJL/J mice were immunized and boosted with a GABHS homogenate in Freund's adjuvant, whereas controls received Freund's adjuvant alone. When sera from GABHS-immunized mice were tested for immunoreactivity to mouse brain, a subset was found to be immunoreactive to several brain regions, including deep cerebellar nuclei (DCN), globus pallidus, and thalamus. GABHS-immunized mice having serum immunoreactivity to DCN also had increased IgG deposits in DCN and exhibited increased rearing behavior in open-field and hole-board tests compared with controls and with GABHS-immunized mice lacking serum anti-DCN antibodies. Rearing and ambulatory behavior were correlated with IgG deposits in the DCN and with serum immunoreactivity to GABHS proteins in Western blot. In addition, serum from a GABHS mouse reacted with normal mouse cerebellum in nondenaturing Western blots and immunoprecipitated C4 complement protein and α-2-macroglobulin. These results are consistent with the hypothesis that immune response to GABHS can result in motoric and behavioral disturbances and suggest that anti-GABHS antibodies cross-reactive with brain components may play a role in their pathophysiology

Nonparametric methods for the analysis of single-color pathogen microarrays

<p>Abstract</p> <p>Background</p> <p>The analysis of oligonucleotide microarray data in pathogen surveillance and discovery is a challenging task. Target template concentration, nucleic acid integrity, and host nucleic acid composition can each have a profound effect on signal distribution. Exploratory analysis of fluorescent signal distribution in clinical samples has revealed deviations from normality, suggesting that distribution-free approaches should be applied.</p> <p>Results</p> <p>Positive predictive value and false positive rates were examined to assess the utility of three well-established nonparametric methods for the analysis of viral array hybridization data: (1) Mann-Whitney <it>U</it>, (2) the Spearman correlation coefficient and (3) the chi-square test. Of the three tests, the chi-square proved most useful.</p> <p>Conclusions</p> <p>The acceptance of microarray use for routine clinical diagnostics will require that the technology be accompanied by simple yet reliable analytic methods. We report that our implementation of the chi-square test yielded a combination of low false positive rates and a high degree of predictive accuracy.</p

An upstream open reading frame modulates ebola virus polymerase translation and virus replication

Ebolaviruses, highly lethal zoonotic pathogens, possess longer genomes than most other non-segmented negative-strand RNA viruses due in part to long 5' and 3' untranslated regions (UTRs) present in the seven viral transcriptional units. To date, specific functions have not been assigned to these UTRs. With reporter assays, we demonstrated that the Zaire ebolavirus (EBOV) 5'-UTRs lack internal ribosomal entry site function. However, the 5'-UTRs do differentially regulate cap-dependent translation when placed upstream of a GFP reporter gene. Most dramatically, the 5'-UTR derived from the viral polymerase (L) mRNA strongly suppressed translation of GFP compared to a β-actin 5'-UTR. The L 5'-UTR is one of four viral genes to possess upstream AUGs (uAUGs), and ablation of each uAUG enhanced translation of the primary ORF (pORF), most dramatically in the case of the L 5'-UTR. The L uAUG was sufficient to initiate translation, is surrounded by a "weak" Kozak sequence and suppressed pORF translation in a position-dependent manner. Under conditions where eIF2α was phosphorylated, the presence of the uORF maintained translation of the L pORF, indicating that the uORF modulates L translation in response to cellular stress. To directly address the role of the L uAUG in virus replication, a recombinant EBOV was generated in which the L uAUG was mutated to UCG. Strikingly, mutating two nucleotides outside of previously-defined protein coding and cis-acting regulatory sequences attenuated virus growth to titers 10-100-fold lower than a wild-type virus in Vero and A549 cells. The mutant virus also exhibited decreased viral RNA synthesis as early as 6 hours post-infection and enhanced sensitivity to the stress inducer thapsigargin. Cumulatively, these data identify novel mechanisms by which EBOV regulates its polymerase expression, demonstrate their relevance to virus replication and identify a potential therapeutic target

A staged strategy for pathogen surveillance and discovery

Recent advances in molecular diagnostics have revolutionized microbiology by facilitating rapid, sensitive pathogen surveillance and differential diagnosis of infectious diseases. Implementation of these technologies can enable intervention when the prognosis is optimal for limiting replication, dissemination, transmission, morbidity and mortality. It may also reveal unappreciated links between infection and chronic diseases. Although new pathogens continue to emerge, we have likely collected much of the "low hanging fruit" (microbes readily associated with diseases). An important task now is to understand those disorders that reflect the interaction of microbes with other environmental factors (toxins, other stressors) and susceptibility genes in a developmental context. Here I will review the strengths and limitations of various assay platforms, describe the challenges associated with proving causation, and delineate a staged strategy for pathogen discovery focused in emerging infectious disease "hot spots," "hot hosts," and prospective birth cohorts

Molecular characterization of severe and mild cases of Influenza A (H1N1) 2009 strain from Argentina

While worldwide pandemic influenza A(H1N1) pdm case fatality rate (CFR) was 0.4%, Argentina's was 4.5%. A total of 34 strains from mild and severe cases were analyzed. A full genome sequencing was carried out on 26 of these, and a partial sequencing on the remaining eight. We observed no evidence that the high CFR can be attributed to direct virus changes. No evidence of re-assortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence was observed. Although the mutation D225G associated with severity in the latest reports from the Ukraine and Norway is not observed among the Argentine strains, an amino acid change in the area (S206T) surrounding the HA receptor binding domain was observed, the same previously established worldwide

Comprehensive viral oligonucleotide probe design using conserved protein regions

Oligonucleotide microarrays have been applied to microbial surveillance and discovery where highly multiplexed assays are required to address a wide range of genetic targets. Although printing density continues to increase, the design of comprehensive microbial probe sets remains a daunting challenge, particularly in virology where rapid sequence evolution and database expansion confound static solutions. Here, we present a strategy for probe design based on protein sequences that is responsive to the unique problems posed in virus detection and discovery. The method uses the Protein Families database (Pfam) and motif finding algorithms to identify oligonucleotide probes in conserved amino acid regions and untranslated sequences. In silico testing using an experimentally derived thermodynamic model indicated near complete coverage of the viral sequence database

Diagnostic System for Rapid and Sensitive Differential Detection of Pathogens

Naturally emerging and deliberately released pathogens demand new detection strategies to allow early recognition and containment. We describe a diagnostic system for rapid, sensitive, multiplex discrimination of microbial gene sequences and report its application for detecting 22 respiratory pathogens in clinical samples

MassTag polymerase chain reaction for differential diagnosis of viral hemorrhagic fevers

Viral hemorrhagic fevers are associated with high rates of illness and death. Although therapeutic options are limited, early differential diagnosis has implications for containment and may aid in clinical management. We describe a diagnostic system for rapid, multiplex polymerase chain reaction identification of 10 different causes of viral hemorrhagic fevers

Greene SCPrimer: a rapid comprehensive tool for designing degenerate primers from multiple sequence alignments

Polymerase chain reaction (PCR) is widely applied in clinical and environmental microbiology. Primer design is key to the development of successful assays and is often performed manually by using multiple nucleic acid alignments. Few public software tools exist that allow comprehensive design of degenerate primers for large groups of related targets based on complex multiple sequence alignments. Here we present a method for designing such primers based on tree building followed by application of a set covering algorithm, and demonstrate its utility in compiling Multiplex PCR primer panels for detection and differentiation of viral pathogens