A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages ...of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval CI, 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi.
The characterization and use of DNA sequence polymorphisms are an important aspect of forensic analysis. A number of approaches are being explored for single nucleotide polymorphism (SNP) genotyping, ...but current detection methods are subject to limitations that adversely impact their utility for forensic analysis. We have developed a novel method for genotyping both single and multiple SNPs that uses an intercalating dye and a probe labeled with a single fluorophore to affect a fluorescence energy transfer. Melting curve analysis is then used to distinguish true alleles from mismatched alleles. We term the new method dye probe fluorescence resonance energy transfer (dpFRET). In the current work, development proceeded at first with synthetic DNA template testing to establish proof of concept for the chemistry involved, followed by the design of polymerase chain reaction (PCR)-based genomic DNA assays to demonstrate potential forensic applications. The loci chosen for testing included both nuclear (MHC DRB) and mitochondrial DNA (cytochrome
b) genes. A preliminary assessment of the sensitivity limits of the technology indicated that dpFRET was capable of accurately genotyping DNA from one single diploid cell equivalent. This technology could also potentially impact a wide range of nonforensic disciplines to aid in discovery, screening, and association of DNA sequence polymorphisms.
Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome ...this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.
: The most common markers used in forensic genetics are short tandem repeats (STRs), the alleles of which are separated and analyzed by length using capillary electrophoresis (CE). In this work, ...proof of concept of a unique STR genotyping approach has been demonstrated using asymmetric PCR and a fluorescence resonance energy transfer (FRET)‐based hybridization analysis that combines fluorophore‐labeled allele‐specific probes and a DNA intercalating dye (dpFRET) in a melt match/mismatch analysis format. The system was successfully tested against both a simple (TPOX) and a complex (D3S1358) loci, demonstrated a preliminary detection limit of <10 genomic equivalents with no allelic dropout and mixture identification in both laboratory‐generated and clinical samples. With additional development, this approach has the potential to contribute to advancing the use of STR loci for forensic applications and related fields.
Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome ...this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable.Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pnc A gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice.Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. The bbk46 gene was dispensable for B. burgdorferi infection in mice. Our findings highlight the power of the IVET-based approach for identification of B. burgdorferi in vivo-expressed genes, which might not be discovered using other genome-wide gene expression methods. Further investigation of the novel in vivo-expressed candidate genes will contribute to advancing the understanding of molecular mechanisms of B.burgdorferi survival and pathogenicity in the mammalian host.
Embryonated chicken eggs (ECE) are sometimes used for the primary isolation or passage of influenza viruses, other viruses, and certain bacteria. For small-scale experiments with pathogens that must ...be studied in biosafety level three (BSL3) facilities, inoculated ECE are sometimes manipulated and maintained in small egg incubators within a biosafety cabinet (BSC). To simplify the clean up and decontamination of an egg incubator in case of egg breakage, we explored whether ethylene breather bags could be used to encase ECE inoculated with pathogens. This concept was tested by determining embryo survival and examining virus yields in bagged ECE.
Virus yields acceptable for many applications were attained when influenza-, alpha-, flavi-, canine distemper-, and mousepox viruses were propagated in ECE sealed within ethylene breather bags.
For many small-scale applications, ethylene breather bags can be used to encase ECE inoculated with various viruses.
We are engaged in airborne transmission and epidemiology studies of respiratory pathogens, with particular interest in human parainfluenza virus type 4 (hPIV-4) and other lesser studied viruses. In ...this paper, hPIV-4 was detected in primary rhesus monkey kidney (PRMK) cells that had been inoculated with nasopharyngeal swab material obtained from a child with a mild upper respiratory tract illness. Attempts to isolate the virus in pure culture were hampered by the presence of a fast-growing simian spumavirus that was a contaminant of the PRMK cells. Total RNA was extracted from the PRMK cell culture, and PCR followed by sequencing of a subgenomic section of the fusion protein gene suggested the hPIV-4 was subtype 4B. At the time of this work, two complete but dissimilar hPIV-4B genomes had been deposited by others in GenBank. To gain better insights on hPIV-4B, and to test methods that we are developing for viral forensics, the entire genomic sequence of our virus was determined from archived RNA. The hPIV-4B genomic sequence that we determined conforms to the paramyxovirus “rule of six.” Here, we compare and contrast the genetic features of the three completely sequenced hPIV-4B genomes currently present in GenBank.
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