Design of microarray probes for virus identification and detection of emerging viruses at the genus level

BACKGROUND: Most virus detection methods are geared towards the detection of specific single viruses or just a few known targets, and lack the capability to uncover the novel viruses that cause emerging viral infections. To address this issue, we developed a computational method that identifies the conserved viral sequences at the genus level for all viral genomes available in GenBank, and established a virus probe library. The virus probes are used not only to identify known viruses but also for discerning the genera of emerging or uncharacterized ones. RESULTS: Using the microarray approach, the identity of the virus in a test sample is determined by the signals of both genus and species-specific probes. The genera of emerging and uncharacterized viruses are determined based on hybridization of the viral sequences to the conserved probes for the existing viral genera. A detection and classification procedure to determine the identity of a virus directly from detection signals results in the rapid identification of the virus. CONCLUSION: We have demonstrated the validity and feasibility of the above strategy with a small number of viral samples. The probe design algorithm can be applied to any publicly available viral sequence database. The strategy of using separate genus and species probe sets enables the use of a straightforward virus identity calculation directly based on the hybridization signals. Our virus identification strategy has great potential in the diagnosis of viral infections. The virus genus and specific probe database and the associated summary tables are available a

A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples

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

A microbial detection array (MDA) for viral and bacterial detection

BACKGROUND: Identifying the bacteria and viruses present in a complex sample is useful in disease diagnostics, product safety, environmental characterization, and research. Array-based methods have proven utility to detect in a single assay at a reasonable cost any microbe from the thousands that have been sequenced. METHODS: We designed a pan-Microbial Detection Array (MDA) to detect all known viruses (including phages), bacteria and plasmids and developed a novel statistical analysis method to identify mixtures of organisms from complex samples hybridized to the array. The array has broader coverage of bacterial and viral targets and is based on more recent sequence data and more probes per target than other microbial detection/discovery arrays in the literature. Family-specific probes were selected for all sequenced viral and bacterial complete genomes, segments, and plasmids. Probes were designed to tolerate some sequence variation to enable detection of divergent species with homology to sequenced organisms, and to have no significant matches to the human genome sequence. RESULTS: In blinded testing on spiked samples with single or multiple viruses, the MDA was able to correctly identify species or strains. In clinical fecal, serum, and respiratory samples, the MDA was able to detect and characterize multiple viruses, phage, and bacteria in a sample to the family and species level, as confirmed by PCR. CONCLUSIONS: The MDA can be used to identify the suite of viruses and bacteria present in complex samples

Multiplex diagnostics : development of serological dualmode multiplex microarray immunoassay and analysis of influenza and respiratory syncytial virus disease burden in early childhood

Respiratory syncytial virus (RSV) and influenza A (IAV) and B (IBV) viruses infect humans throughout their life with exceptionally high rates of re-infection. Serological assays are commonly used to diagnose and characterize host immune responses against microbial pathogens acquired by natural infection or vaccination. The current methods are mostly based on enzyme immunoassay (EIA) or other conventional methods in a single analyte format. Presently, there is a need to shift from single analytical methods to microarray format which enables the detection of antibodies against multiple targets in a rapid and cost-effective manner. In this study, a highly sensitive single analyte EIA method was used for serological analyses in children’s sera. In addition, multiplex microarray immunoassay (MAIA) methods were developed for rapid and simultaneous detection of IgG antibodies against seven viral antigens (H1N1pdm09 vaccine ag, IAV H1N1, IAV H3N2, IBV Victoria, IBV Yamagata, RSV and adenovirus hexon protein). We found out that MAIA is well suitable for large-scale serosurveillance and vaccine immunity studies. We followed-up virus-specific immunity by MAIA in response to natural infection and vaccination in a large cohort of 0-2 year old children. Our serological findings showed a high rate of respiratory virus infections and reinfections in young children. The applicability of MAIA in vaccine immunity studies was also analysed. We developed a specific, sensitive, sample and antigen saving assay for simultaneous detection of IgM and IgG antibodies against H1N1pdm09 vaccine ag and IBV Yamagata. MAIA showed excellent correlation with EIA and a good correlation with hemagglutination inhibition assay in measurement of vaccine-induced antibodies in sera of Pandemrix-vaccinated adults.Respiratory syncytial virus (RSV) ja influenssa A- (IAV) ja B(IBV)-virukset ovat merkittäviä ylähengitystieinfektioiden aiheuttajia koko ihmisen elinkaaren ajan. RSV:n ja influenssan aiheuttamat infektiot voivat joskus olla vakavia tai jopa henkeä uhkaavia erityisesti pienillä lapsilla ja ikäihmisillä. Nykyiset serodiagnostiset menetelmät perustuvat entsyymi-immunologisiin (EIA) ja muihin määritysmenetelmiin, joissa immuunivastetta voidaan tutkia kerrallaan vain yhtä taudinaiheuttajaa kohtaa. Tällä hetkellä on seerumin vasta-ainetutkimusten osalta suuri tarve siirtyä monianalyyttisiin menetelmiin, jotka olisivat nopeampia ja kustannustehokkaampia kuin nykyiset tutkimusmenetelmät. Tässä tutkimuksessa on käytetty herkkiä EIA-menetelmiä pienten lasten ja influenssarokotettujen henkilöiden seerumien virusspesifisen immuunivasteen tutkimiseen. Työssä on myös kehitetty monianalyyttinen mikrosiruperusteinen immunomääritysmenetelmä (MAIA) samanaikaiseen seerumin IgG-luokan vastaaineiden mittaamiseen eri influenssavirusantigeeneja, RSV:tä ja adenoviruksen heksoniproteiinia kohtaan. Tutkimuksemme osoittivat, että hengitysteiden virusinfektiot ja yllämainittujen virusten aiheuttamat uusintainfektiot ovat erittäin tavallisia pienillä lapsilla. MAIA-menetelmää sovellettiin myös aikuisten influenssarokotevasteiden analyysiin. Menetelmää kehitettiin edelleen siten, että näytteestä voitiin samalla kertaa mitata seerumin IgG- ja IgM-luokan vasta-aineita eri IAV- ja IBV-virusantigeeneja kohtaan. Menetelmä osoittautui erittäin spesifiseksi, herkäksi ja näytettä ja antigeeneja säästäväksi ja tulokset olivat hyvin yhtenevät muiden perinteisten vasta-ainetutkimusmenetelmien kanssa. Kehittämämme MAIA-menetelmä toimi erittäin hyvin ja sen voitiin osoittaa sopivan erinomaisesti laajojen väestön seerumiaineistojen ja virusrokotevasteiden analyysiin

Microarrays in the Diagnosis of Human Herpesvirus infections

Currently, there are nine known human herpesviruses and these viruses appear to have been a very common companion of humans throughout the millenia. Of human herpesviruses, herpes simplex viruses 1 and 2 (HSV-1, HSV-2), causative agents of herpes labialis and genital herpes, and varicella-zoster virus (VZV), causative agent of chicken pox, are also common causes of central nervous system (CNS) infections. In addition, human cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7), all members of the herpesvirus family, can also be associated with encephalitis and meningitis. Accurate diagnostics and fast treatment are essential for patient recovery in CNS infections and therefore sensitive and effective diagnostic methods are needed. The aim of this thesis was to develop new potential detection methods for diagnosing of human herpesvirus infections, especially in immunocompetent patients, using the microarray technique. Therefore, methods based on microarrays were developed for simultaneous detection of HSV-1, HSV-2, VZV, CMV, EBV, HHV-6A, HHV-6B, and HHV-7 nucleic acids, and for HSV-1, HSV-2, VZV, and CMV antibodies from various clinical samples. The microarray methods developed showed potential for efficiently and accurately detecting human herpesvirus DNAs, especially in CNS infections, and for simultaneous detection of DNAs or antibodies for multiple different human herpesviruses from clinical samples. In fact, the microarray method revealed several previously unrecognized co-infections. The microarray methods developed were sensitive and provided rapid detection of human herpesvirus DNA, and therefore the method could be applied to routine diagnostics. The microarrays might also be considered as an economical tool for diagnosing human herpesvirus infections.Herpesvirukset ovat kulkeneet ihmisen kumppaneina vuosituhat toisensa jälkeen ja tällä hetkellä tunnetaan yhdeksän ihmiselle patogeenista herpesvirusta. Huuli- ja genitaaliherpestä aiheuttavat herpes simplex virus 1 ja 2 (HSV-1, HSV-2) sekä vesirokkoa aiheuttava varicalla-zoster virus (VZV) ovat ihmisen herpesviruksia, jotka voivat aiheuttaa myös vakavia keskushermostoperäisiä infektioita. Ihmisen herpesviruksista myös ihmisen cytomegalovirus (CMV), Epstein-Barr virus (EBV), ihmisen herpesvirukset 6 ja 7 (HHV-6, HHV-7), voidaan liittää aivokuumeeseen ja aivokalvontulehdukseen. Näiden virusten nopea ja tarkka diagnostiikka on oleellista oikean hoidon aloittamiselle sekä potilaan toipumiselle varsinkin keskushermostoperäisissä infektioissa. Taudinaiheuttajien diagnosoimiseksi tarvitaan herkkiä ja tehokkaita menetelmiä. Tämän väitöskirjan tavoitteina oli kehittää uusia mikrosirupohjaisia menetelmiä ihmisen herpesvirusten aiheuttamien infektioiden diagnostiikkaan. Kehitetyillä mikrosiruilla tunnistettiin yhtä aikaa kahdeksan eri herpesviruksen nukleiinihappoa erilaisista kliinisistä näytteistä. Toisella serologisella mikrosirulla detektoitiin HSV-1, HSV-2, VZV ja CMV:lle spesifisiä vasta-aineita. Herpesvirusten nukleiinihappojen ja vasta-aineiden tunnistamiseen kehitetyt mikrosirumenetelmät osoittautuivat soveltuviksi usean herpesviruksen samanaikaiseen diagnosointiin. Varsinkin keskushermostopohjaisten infektioiden tunnistamisessa mikrosiru osoittautui tehokkaaksi. Mikrosirujen avulla tunnistettiin myös mahdollisia useamman viruksen samanaikaisia infektioita

Bioinformatic applications for virology research

Ph.DDOCTOR OF PHILOSOPH

Developing a sensitive, high-throughput tool for rapid detection of agronomically important seed-borne pathogens of tomato

The limited specificity, sensitivity and multiplex capacity of detection techniques currently available for important seed-borne pathogens of tomato is a significant risk for the global tomato trade and production industry. These pathogens can be associated with seed at low concentrations but, due to their highly virulent nature, these low levels can be sufficient to infect germinating seedlings and spread to neighbouring plants and fields, potentially causing epidemics and economic losses. In this study, detection techniques currently available for phytodiagnostics were evaluated for the capacity to accurately detect and identify five agronomically important seed-borne pathogens of tomato: Pepino mosaic virus (PepMV), Tomato mosaic virus (ToMV), Clavibacter michiganensis subsp. michiganensis (Cmm), Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato. A prototype diagnostic microarray was also designed in an attempt to develop a tool that could simultaneously detect these five seed-borne pathogens from a single sample. Viral detection based on serological techniques was rapid, accurate and reliable but only detected a single pathogen per assay and required supplementary bioassays to indicate the viability of detected viral pathogens. Selective media plating for bacterial detection demonstrated unreliable recovery of targeted bacteria from infected seed and leaf samples and required supplementary tests to validate the identity of presumptive positives. Assays were lengthy, laborious and sometimes too ambiguous for accurate diagnosis of bacterial pathogens. Nucleic acid-based technologies demonstrated improved sensitivity and specificity for detection of targets from pure culture, leaf and seed extracts, compared to conventional and serological methods, yet also required supplementary bioassays or media assays to validate the viability of detected pathogens. Amplification efficiency however, was affected by the presence of PCR inhibitors and despite positive detection, variable banding intensity in electrophoretic analysis of amplified products necessitated the use of reference cultures to validate diagnosis. The developed microarray incorporated 152 pathogen-specific and control probes to facilitate diagnosis and taxonomic classification of detected pathogens. The array was challenged with pure culture extracts of the five target pathogens, selected related and non-target, unrelated pathogens of tomato. Positive detection of each of the pathogens was demonstrated but the production of hybridisation signals was highly variable and extremely sensitive to minor technical differences. Each of the five pathogens were successfully detected in combination proving that different classes of seed-borne pathogens could be detected from a single sample using the developed microarray. This prototype microarray has good potential for phytodiagnostic screening of the five targeted pathogens, and further validation, optimisation and extension for testing tomato seed samples may facilitate incorporation of this array into standard diagnostic protocols