The control and prevention of communicable disease is directly impacted by the genetic mutability of the underlying etiological agents. In the case of RNA viruses, genetic recombination may impact public health by facilitating the generation of new viral strains with altered phenotypes and by compromising the genetic stability of live attenuated vaccines. The landscape of homologous recombination within a given RNA viral genome is thought to be influenced by several factors; however, a complete understanding of the genetic determinants of recombination is lacking. Here, we utilize gene synthesis and deep sequencing to create a detailed recombination map of the poliovirus 1 coding region. We identified over 50 thousand breakpoints throughout the genome, and we show the majority of breakpoints to be concentrated in a small number of specific “hotspots,” including those associated with known or predicted RNA secondary structures. Nucleotide base composition was also found to be associated with recombination frequency, suggesting that recombination is modulated across the genome by predictable and alterable motifs. We tested the predictive utility of the nucleotide base composition association by generating an artificial hotspot in the poliovirus genome. Our results imply that modification of these motifs could be extended to whole genome re-designs for the development of recombination-deficient, genetically stable live vaccine strains.

Author Summary

Viral recombination is critical to understanding the evolution of viral groups and impacts vaccine design, but is poorly understood. In the poliovirus vaccine, recombination is one potential mode of failure where vaccine strains recombine to produce a pathogenic product. We combine gene synthesis and deep sequencing to generate a high-resolution recombination map of poliovirus, both as a model RNA virus and a continuing threat that has yet to be eradicated. This map shows that recombination is concentrated into hotspots and suggests that predictable and alterable motifs in the RNA sequence are associated with recombination frequency. We demonstrate the utility of these observations by re-designing a poliovirus strain to recombine more frequently than normal, facilitating future studies on the role of viral recombination during infection. This result suggests that a large-scale redesign of the entire poliovirus genome to dampen recombination may be feasible, with implications for producing safer and more stable live vaccines.

Propafenone, a class Ic antiarrythmic drug, inhibits growth of cultured Plasmodium falciparum. While the drug’s potency is significant, further development of propafenone as an antimalarial would require divorcing the antimalarial and cardiac activities as well as improving the pharmacokinetic profile of the drug. A small array of propafenone analogs was designed and synthesized to address the cardiac ion channel and PK liabilities. Testing of this array revealed potent inhibitors of the 3D7 (drug sensitive) and K1 (drug resistant) strains of P. falciparum that possessed significantly reduced ion channel effects and improved metabolic stability. Propafenone analogues are unusual among antimalarial leads in that they are more potent against the multi-drug resistant K1 strain of P. falciparum compared to the 3D7 strain.

ABSTRACT

Inclusion body disease (IBD) is an infectious fatal disease of snakes typified by behavioral abnormalities, wasting, and secondary infections. At a histopathological level, the disease is identified by the presence of large eosinophilic cytoplasmic inclusions in multiple tissues. To date, no virus or other pathogen has been definitively characterized or associated with the disease. Using a metagenomic approach to search for candidate etiologic agents in snakes with confirmed IBD, we identified and de novo assembled the complete genomic sequences of two viruses related to arenaviruses, and a third arenavirus-like sequence was discovered by screening an additional set of samples. A continuous boa constrictor cell line was established and used to propagate and isolate one of the viruses in culture. Viral nucleoprotein was localized and concentrated within large cytoplasmic inclusions in infected cells in culture and tissues from diseased snakes. In total, viral RNA was detected in 6/8 confirmed IBD cases and 0/18 controls. These viruses have a typical arenavirus genome organization but are highly divergent, belonging to a lineage separate from that of the Old and New World arenaviruses. Furthermore, these viruses encode envelope glycoproteins that are more similar to those of filoviruses than to those of other arenaviruses. These findings implicate these viruses as candidate etiologic agents of IBD. The presence of arenaviruses outside mammals reveals that these viruses infect an unexpectedly broad range of species and represent a new reservoir of potential human pathogens.

IMPORTANCE

Inclusion body disease (IBD) is a common infectious disease of captive snakes. IBD is fatal and can cause the loss of entire animal collections. The cause of the disease has remained elusive, and no treatment exists. In addition to being important to pet owners, veterinarians, breeders, zoological parks, and aquariums, the study of animal disease is significant since animals are the source of virtually every emerging infectious human disease. We searched for candidate causative agents in snakes diagnosed with IBD and found a group of novel viruses distantly related mainly to arenaviruses but also to filoviruses, both of which can cause fatal hemorrhagic fevers when transmitted from animals to humans. In addition to providing evidence that strongly suggests that these viruses cause snake IBD, this discovery reveals a new and unanticipated domain of virus biology and evolution.

The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIβ) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIIIβ and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIIIβ by affinity purification of Strep-Tagged transiently transfected constructs followed by mass spectrometry and Western blotting for putative interacting targets. We found that the 3A proteins of Aichi virus, bovine kobuvirus, poliovirus, coxsackievirus B3, and human rhinovirus 14 all copurify with PI4KIIIβ. Furthermore, we found that multiple picornavirus 3A proteins copurify with the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), including those from Aichi virus, bovine kobuvirus, human rhinovirus 14, poliovirus, and coxsackievirus B2, B3, and B5. Affinity purification of ACBD3 confirmed interaction with multiple picornaviral 3A proteins and revealed the ability to bind PI4KIIIβ in the absence of 3A. Mass-spectrometric analysis of transiently expressed Aichi virus, bovine kobuvirus, and human klassevirus 3A proteins demonstrated that the N-terminal glycines of these 3A proteins are myristoylated. Alanine-scanning mutagenesis along the entire length of Aichi virus 3A followed by transient expression and affinity purification revealed that copurification of PI4KIIIβ could be eliminated by mutation of specific residues, with little or no effect on recruitment of ACBD3. One mutation at the N terminus, I5A, significantly reduced copurification of both ACBD3 and PI4KIIIβ. The dependence of Aichi virus replication on the activity of PI4KIIIβ was confirmed by both chemical and genetic inhibition. Knockdown of ACBD3 by small interfering RNA (siRNA) also prevented replication of both Aichi virus and poliovirus. Point mutations in 3A that eliminate PI4KIIIβ association sensitized Aichi virus to PIK93, suggesting that disruption of the 3A/ACBD3/PI4KIIIβ complex may represent a novel target for therapeutic intervention that would be complementary to the inhibition of the kinase activity itself.

Dengue virus is an emerging infectious agent that infects an estimated 50–100 million people annually worldwide, yet current diagnostic practices cannot detect an etiologic pathogen in ∼40% of dengue-like illnesses. Metagenomic approaches to pathogen detection, such as viral microarrays and deep sequencing, are promising tools to address emerging and non-diagnosable disease challenges. In this study, we used the Virochip microarray and deep sequencing to characterize the spectrum of viruses present in human sera from 123 Nicaraguan patients presenting with dengue-like symptoms but testing negative for dengue virus. We utilized a barcoding strategy to simultaneously deep sequence multiple serum specimens, generating on average over 1 million reads per sample. We then implemented a stepwise bioinformatic filtering pipeline to remove the majority of human and low-quality sequences to improve the speed and accuracy of subsequent unbiased database searches. By deep sequencing, we were able to detect virus sequence in 37% (45/123) of previously negative cases. These included 13 cases with Human Herpesvirus 6 sequences. Other samples contained sequences with similarity to sequences from viruses in the Herpesviridae, Flaviviridae, Circoviridae, Anelloviridae, Asfarviridae, and Parvoviridae families. In some cases, the putative viral sequences were virtually identical to known viruses, and in others they diverged, suggesting that they may derive from novel viruses. These results demonstrate the utility of unbiased metagenomic approaches in the detection of known and divergent viruses in the study of tropical febrile illness.

Author Summary

Dengue virus infection is a global health concern, affecting as many as 100 million people annually worldwide. A critical first step to proper treatment and control of any virus infection is a correct diagnosis. Traditional diagnostic tests for viruses depend on amplification of conserved portions of the viral genome, detection of the binding of antibodies to viral proteins, or replication of the virus in cell cultures. These methods have a major shortcoming: they are unable to detect divergent or novel viruses for which a priori sequence, serological, or cellular tropism information is not known. In our study, we use two approaches, microarrays and deep sequencing, to virus identification that are less susceptible to such shortcomings. We used these unbiased tools to search for viruses in blood collected from Nicaraguan children with clinical symptoms indicating dengue virus infection, but for whom current dengue virus detection assays yielded negative results. We were able to identify both known and divergent viruses in about one third of previously negative samples, demonstrating the utility of these approaches to detect viruses in cases of unknown dengue-like illness.

Beta-papillomavirus (β-HPV) DNA is present in some cutaneous squamous cell carcinomas (cuSCC), but no mechanism of carcinogenesis has been determined. We used ultra-high throughput sequencing of the cancer transcriptome to assess whether papillomavirus transcripts are present in these cancers. Sixty-seven cuSCC samples were assayed for β-HPV DNA by PCR, and viral loads were measured with type-specific qPCR. Thirty-one SCCs were selected for whole transcriptome sequencing. Transcriptome libraries were prepared in parallel from the HPV18 positive HeLa cervical cancer cell line and HPV16 positive primary cervical and periungual SCC. Thirty percent (20/67) of the tumors were positive for β-HPV DNA, but there was no difference in β-HPV viral load between tumor and normal tissue (p=0.310). Immunosuppression and age were significantly associated with higher viral load (p=0.016 for immunosuppression; p=0.0004 for age). Transcriptome sequencing failed to identify papillomavirus expression in any of the skin tumors. In contrast, HPV 16 and 18 mRNA transcripts were readily identified in primary cervical and periungual cancers and HeLa cells. These data demonstrate that papillomavirus mRNA expression is not a factor in the maintenance of cuSCC.

Genetic manipulation of malaria parasites remains an inefficient, time-consuming and resource-intensive process. Presented here is a set of methods for 96-well plate-based transfection and culture that improve the efficiency of genetic manipulation of Plasmodium falciparum. Compared to standard protocols plate-based transfection requires 20-fold less DNA, transient transfection efficiency achieved is approximately seven-fold higher, whilst stable transfection success rate is above 90%. Furthermore the utility of this set of protocols to generate a knockout of the PfRH3 pseudogene, screened by whole-cell PCR, is demonstrated. The methods and tools presented here will facilitate genome-scale genetic manipulation of P. falciparum.

A library of diarylurea IGFR inhibitors was screened for activity against chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum. The 4-aminoquinaldine-derived diarylureas displayed promising antimalarial potency. Further exploration of the B ring of 4-aminoquinaldinyl ureas allowed identification of several quinaldin-4-yl ureas 4{13, 39} and 4{13, 58} sufficiently potent against both 3D7 and K1 strains to qualify as bone fide leads.

In meiosis, the exchange of DNA between chromosomes by homologous recombination is a critical step that ensures proper chromosome segregation and increases genetic diversity. Products of recombination include reciprocal exchanges, known as crossovers, and non-reciprocal gene conversions or non-crossovers. The mechanisms underlying meiotic recombination remain elusive, largely because of the difficulty of analyzing large numbers of recombination events by traditional genetic methods. These traditional methods are increasingly being superseded by high-throughput techniques capable of surveying meiotic recombination on a genome-wide basis. Next-generation sequencing or microarray hybridization is used to genotype thousands of polymorphic markers in the progeny of hybrid yeast strains. New computational tools are needed to perform this genotyping and to find and analyze recombination events. We have developed a suite of programs, ReCombine, for using short sequence reads from next-generation sequencing experiments to genotype yeast meiotic progeny. Upon genotyping, the program CrossOver, a component of ReCombine, then detects recombination products and classifies them into categories based on the features found at each location and their distribution among the various chromatids. CrossOver is also capable of analyzing segregation data from microarray experiments or other sources. This package of programs is designed to allow even researchers without computational expertise to use high-throughput, whole-genome methods to study the molecular mechanisms of meiotic recombination.

Objective

To assess the utility of a pan-viral DNA microarray platform (Virochip) in the detection of viruses associated with pediatric respiratory tract infections.

Study design

The Virochip was compared with conventional direct fluorescent antibody (DFA) and PCR-based testing for the detection of respiratory viruses in 278 consecutive nasopharyngeal aspirate samples from 222 children.

Results

The Virochip was superior in performance to DFA, showing a 19% increase in the detection of 7 respiratory viruses included in standard DFA panels, and was similar to virus-specific PCR (sensitivity 85–90%, specificity ≥99%, PPV 94–96%, NPV 97–98%) in the detection of respiratory syncytial virus, influenza A, and rhino-/enteroviruses. The Virochip also detected viruses not routinely tested for or missed by DFA and PCR, as well as double infections and infections in critically ill patients that DFA failed to detect.

Conclusions

Given its favorable sensitivity and specificity profile and expanded spectrum for detection, microarray-based viral testing holds promise for clinical diagnosis of pediatric respiratory tract infections.

The only essential function of a unique plastid organelle, the apicoplast, in blood-stage P. falciparum is the production of isoprenoid precursors.

Plasmodium spp parasites harbor an unusual plastid organelle called the apicoplast. Due to its prokaryotic origin and essential function, the apicoplast is a key target for development of new anti-malarials. Over 500 proteins are predicted to localize to this organelle and several prokaryotic biochemical pathways have been annotated, yet the essential role of the apicoplast during human infection remains a mystery. Previous work showed that treatment with fosmidomycin, an inhibitor of non-mevalonate isoprenoid precursor biosynthesis in the apicoplast, inhibits the growth of blood-stage P. falciparum. Herein, we demonstrate that fosmidomycin inhibition can be chemically rescued by supplementation with isopentenyl pyrophosphate (IPP), the pathway product. Surprisingly, IPP supplementation also completely reverses death following treatment with antibiotics that cause loss of the apicoplast. We show that antibiotic-treated parasites rescued with IPP over multiple cycles specifically lose their apicoplast genome and fail to process or localize organelle proteins, rendering them functionally apicoplast-minus. Despite the loss of this essential organelle, these apicoplast-minus auxotrophs can be grown indefinitely in asexual blood stage culture but are entirely dependent on exogenous IPP for survival. These findings indicate that isoprenoid precursor biosynthesis is the only essential function of the apicoplast during blood-stage growth. Moreover, apicoplast-minus P. falciparum strains will be a powerful tool for further investigation of apicoplast biology as well as drug and vaccine development.

Author Summary

Malaria caused by Plasmodium spp parasites is a profound human health problem that has shaped our evolutionary past and continues to influence modern day with a disease burden that disproportionately affects the world's poorest and youngest. New anti-malarials are desperately needed in the face of existing or emerging drug resistance to available therapies, while an effective vaccine remains elusive. A plastid organelle, the apicoplast, has been hailed as Plasmodium's “Achilles' heel” because it contains bacteria-derived pathways that have no counterpart in the human host and therefore may be ideal drug targets. However, more than a decade after its discovery, the essential functions of the apicoplast remain a mystery, and without a specific pathway or function to target, development of drugs against the apicoplast has been stymied. In this study, we use a simple chemical method to generate parasites that have lost their apicoplast, normally a deadly event, but which survive—“rescued” by the addition of an essential metabolite to the culture. This chemical rescue demonstrates that the apicoplast serves only a single essential function, namely isoprenoid precursor biosynthesis during blood-stage growth, validating this metabolic function as a viable drug target. Moreover, the apicoplast-minus Plasmodium strains generated in this study will be a powerful tool for identifying apicoplast-targeted drugs and as a potential vaccine strain with significant advantages over current vaccine technologies.

Papillomaviruses have been implicated in a variety of human diseases ranging from common warts to invasive carcinoma of the anogenital mucosa. Existing assays for genotyping human papillomavirus are restricted to a small number of types. Here, we present a comprehensive, accurate microarray strategy for detection and genotyping of 102 human papillomavirus types and validate its use in a panel of 91 anal swabs. This array has equal performance to traditional dot blot analysis with the benefits of added genotype coverage and the ability to calibrate readout over a range of sensitivity or specificity values.

Among the known antimalarial drugs, chloroquine (CQ) and other 4-aminoquinolines have shown high potency and good bioavailability, yet complications associated with drug resistance necessitate the discovery of effective new antimalarial agents. ADMETa prediction studies were employed to evaluate a library of new molecules based on the 4-aminoquinolone-related structure of CQ. Extensive in vitro screening and in vivo pharmacokinetic studies in mice helped to identify two lead molecules, 18 and 4, with promising in vitro therapeutic efficacy, improved ADMET properties, low risk for drug-drug interactions, and desirable pharmacokinetic profiles. Both 18 and 4 are highly potent antimalarial compounds, with IC50 values = 5.6 nM and 17.3 nM, respectively, against the W2 (CQ-resistant) strain of Plasmodium falciparum (IC50 for CQ = 382 nM). When tested in mice, these compounds were found to have biological half-lives and plasma exposure values similar to or higher than those of CQ; they are therefore desirable candidates to pursue in future clinical trials.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Gene expression is regulated in part by protein transcription factors (TFs) that bind target regulatory DNA sequences. Predicting DNA binding sites and affinities from transcription factor sequence or structure is difficult; therefore, experimental data are required to link TFs to target sequences. We present a microfluidics-based approach for de novo discovery and quantitative biophysical characterization of DNA target sequences. We validated our technique by measuring sequence preferences for 28 S. cerevisiae TFs with a variety of DNA binding domains, including several that have proven difficult to study via other techniques. For each TF, we measured relative binding affinities to oligonucleotides covering all possible 8-bp DNA sequences to create a comprehensive map of sequence preferences; for 4 TFs, we also determined absolute affinities. We anticipate that these data and future use of this technique will provide information essential for understanding TF specificity, improving identification of regulatory sites, and reconstructing regulatory interactions.

Over 50% of genes in Plasmodium falciparum, the deadliest human malaria parasite, contain predicted introns, yet experimental characterization of splicing in this organism remains incomplete. We present here a transcriptome-wide characterization of intraerythrocytic splicing events, as captured by RNA-Seq data from four timepoints of a single highly synchronous culture. Gene model-independent analysis of these data in conjunction with publically available RNA-Seq data with HMMSplicer, an in-house developed splice site detection algorithm, revealed a total of 977 new 5′ GU-AG 3′ and 5 new 5′ GC-AG 3′ junctions absent from gene models and ESTs (11% increase to the current annotation). In addition, 310 alternative splicing events were detected in 254 (4.5%) genes, most of which truncate open reading frames. Splicing events antisense to gene models were also detected, revealing complex transcriptional arrangements within the parasite’s transcriptome. Interestingly, antisense introns overlap sense introns more than would be expected by chance, perhaps indicating a functional relationship between overlapping transcripts or an inherent organizational property of the transcriptome. Independent experimental validation confirmed over 30 new antisense and alternative junctions. Thus, this largest assemblage of new and alternative splicing events to date in Plasmodium falciparum provides a more precise, dynamic view of the parasite’s transcriptome.

Background

High-throughput sequencing of an organism's transcriptome, or RNA-Seq, is a valuable and versatile new strategy for capturing snapshots of gene expression. However, transcriptome sequencing creates a new class of alignment problem: mapping short reads that span exon-exon junctions back to the reference genome, especially in the case where a splice junction is previously unknown.

Methodology/Principal Findings

Here we introduce HMMSplicer, an accurate and efficient algorithm for discovering canonical and non-canonical splice junctions in short read datasets. HMMSplicer identifies more splice junctions than currently available algorithms when tested on publicly available A. thaliana, P. falciparum, and H. sapiens datasets without a reduction in specificity.

Conclusions/Significance

HMMSplicer was found to perform especially well in compact genomes and on genes with low expression levels, alternative splice isoforms, or non-canonical splice junctions. Because HHMSplicer does not rely on pre-built gene models, the products of inexact splicing are also detected. For H. sapiens, we find 3.6% of 3′ splice sites and 1.4% of 5′ splice sites are inexact, typically differing by 3 bases in either direction. In addition, HMMSplicer provides a score for every predicted junction allowing the user to set a threshold to tune false positive rates depending on the needs of the experiment. HMMSplicer is implemented in Python. Code and documentation are freely available at http://derisilab.ucsf.edu/software/hmmsplicer.

Fumagillin, an irreversible inhibitor of MetAP2, has been shown to potently inhibit growth of malaria parasites in vitro. Here, we demonstrate activity of fumagillin analogs with an improved pharmacokinetic profile against malaria parasites, trypanosomes, and amoebas. A subset of the compounds showed efficacy in a murine malaria model. The observed SAR forms a basis for further optimization of fumagillin based inhibitors against parasitic targets by inhibition of MetAP2.

Klassevirus is a proposed new genus of picornavirus that has been associated with pediatric diarrhea. In this study, we used recombinant klassevirus 3C protease as the capture antigen for an indirect serological enzyme-linked immunosorbent assay (ELISA). Four of six klassevirus reverse transcription (RT)-PCR-positive individuals demonstrated seroconversion against the 3C protease, suggesting that klassevirus infection and replication occur in humans. Additional screening of 353 samples from an age-banded serological cohort from two St. Louis hospitals indicated a seroprevalence of 6.8%.

Malaria parasites utilize a short N-terminal amino acid motif termed the Plasmodium export element (PEXEL) to export an array of proteins to the host erythrocyte during blood stage infection. Using immunoaffinity chromatography and mass spectrometry, insight into this signal-mediated trafficking mechanism was gained by discovering that the PEXEL motif is cleaved and N-acetylated. PfHRPII and PfEMP2 are two soluble proteins exported by Plasmodium falciparum that were demonstrated to undergo PEXEL cleavage and N-acetylation, thus indicating that this N-terminal processing may be general to many exported soluble proteins. It was established that PEXEL processing occurs upstream of the brefeldin A-sensitive trafficking step in the P. falciparum secretory pathway, therefore cleavage and N-acetylation of the PEXEL motif occurs in the endoplasmic reticulum (ER) of the parasite. Furthermore, it was shown that the recognition of the processed N-terminus of exported proteins within the parasitophorous vacuole may be crucial for protein transport to the host erythrocyte. It appears that the PEXEL may be defined as a novel ER peptidase cleavage site and a classical N-acetyltransferase substrate sequence.

Enteroviruses (Picornaviridae family) are a common cause of human illness worldwide and are associated with diverse clinical syndromes, including asymptomatic infection, respiratory illness, gastroenteritis, and meningitis. In this study, we report the identification and complete genome sequence of a novel enterovirus isolated from a case of acute respiratory illness in a Nicaraguan child. Unbiased deep sequencing of nucleic acids from a nose and throat swab sample enabled rapid recovery of the full-genome sequence. Phylogenetic analysis revealed that human enterovirus 109 (EV109) is most closely related to serotypes of human enterovirus species C (HEV-C) in all genomic regions except the 5′ untranslated region (5′ UTR). Bootstrap analysis indicates that the 5′ UTR of EV109 is likely the product of an interspecies recombination event between ancestral members of the HEV-A and HEV-C groups. Overall, the EV109 coding region shares 67 to 72% nucleotide sequence identity with its nearest relatives. EV109 isolates were detected in 5/310 (1.6%) of nose and throat swab samples collected from children in a pediatric cohort study of influenza-like illness in Managua, Nicaragua, between June 2007 and June 2008. Further experimentation is required to more fully characterize the pathogenic role, disease associations, and global distribution of EV109.

Background

Malaria parasites generate free haem upon catabolism of host haemoglobin during their intraerythrocytic growth cycle. In order to minimize oxidative toxicity of the ferric iron, the free haem molecules are polymerized into the biomineral beta-haematin (commonly referred to as haemozoin). Haemozoin crystals are paramagnetic, and this property can be exploited for the purification of late stage parasites as they contain larger haemozoin crystals than early stage parasites and uninfected cells. Commercially available magnets that were originally developed for the purpose of antibody-mediated cell purification are widely used for this purpose. As these methods are not necessarily optimized for parasite purification, the relationship between magnetic field strength and the quantity and quality of yield during parasite purification was explored.

Methods

Inexpensive rare-earth neodymium magnets with commercially available disposable columns were employed to explore the relationship between magnetic field strength and recovery of free haemozoin and infected erythrocytes (iRBCs).

Results

Yields of free haemozoin increased nearly linearly with increasing magnetic field strength to the strongest fields tested (8,500 Gauss). Stronger magnetic fields also improved the recovery of iRBCs with no detrimental effects on parasite viability. An in-house constructed magnetic stand, built for $75 in materials, produced superior results when compared with much more expensive commercial products.

Conclusions

Existing protocols for the magnetic purification of free haemozoin and iRBCs result in sub-optimal yields. Inexpensive high-strength neodymium magnets offer a better option, resulting in higher yields with no detrimental effects on parasite viability.

Background

The manual counting of cells by microscopy is a commonly used technique across biological disciplines. Traditionally, hand tally counters have been used to track event counts. Although this method is adequate, there are a number of inefficiencies which arise when managing large numbers of samples or large sample sizes.

Results

We describe software that mimics a traditional multi-register tally counter. Full customizability allows operation on any computer with minimal hardware requirements. The efficiency of counting large numbers of samples and/or large sample sizes is improved through the use of a "multi-count" register that allows single keystrokes to correspond to multiple events. Automatically updated multi-parameter values are implemented as user-specified equations, reducing errors and time required for manual calculations. The user interface was optimized for use with a touch screen and numeric keypad, eliminating the need for a full keyboard and mouse.

Conclusions

Our software provides an inexpensive, flexible, and productivity-enhancing alternative to manual hand tally counters.

A proventricular dilatation disease (PDD) outbreak provided the opportunity to investigate the transmissibility of avian Bornavirus (ABV) and its linkage to PDD under natural conditions. Upon exposure to a bird with a fatal case of PDD, 10 birds became symptomatic and died. ABV2 RNA was recovered from available tissues. Further screening revealed that 12/46 exposed birds were ABV2+. Three chicks boarded at this aviary developed PDD. They harbored the same ABV2 isolate and transmitted it to five of eight chicks in their home aviary. These findings demonstrate that ABV infection precedes the development of PDD. ABV-specific Western blotting and reverse transcription-PCR indicate that ABV2 is not strictly neurotropic.