Deborah Kukielka

Metagenomic Detection of Viral Pathogens in Spanish Honeybees: Co- Infection by Aphid Lethal Paralysis, Israel Acute Paralysis and Lake Sinai Viruses

The situation in Europe concerning honeybees has in recent years become increasingly aggravated with steady decline in populations and/or catastrophic winter losses. This has largely been attributed to the occurrence of a variety of known and “unknown”, emerging novel diseases. Previous studies have demonstrated that colonies often can harbour more than one pathogen, making identification of etiological agents with classical methods difficult. By employing an unbiased metagenomic approach, which allows the detection of both unexpected and previously unknown infectious agents, the detection of three viruses, Aphid Lethal Paralysis Virus (ALPV), Israel Acute Paralysis Virus (IAPV), and Lake Sinai Virus (LSV), in honeybees from Spain is reported in this article. The existence of a subgroup of ALPV with the ability to infect bees was only recently reported and this is the first identification of such a strain in Europe. Similarly, LSV appear to be a still unclassified group of viruses with unclear impact on colony health and these viruses have not previously been identified outside of the United States. Furthermore, our study also reveals that these bees carried a plant virus, Turnip Ringspot Virus (TuRSV), potentially serving as important vector organisms. Taken together, these results demonstrate the new possibilities opened up by high-throughput sequencing and metagenomic analysis to study emerging new diseases in domestic and wild animal populations, including honeybees.

One-step real-time quantitative PCR assays for the detection and field study of Sacbrood honeybee and Acute bee paralysis viruses

Two one-step real-time RT-PCR assays, based on SYBR Green (SG) chemistry, were developed or adapted respectively, for the detection, differentiation, and quantitation of two important honeybee viruses: Sacbrood virus (SBV) and Acute bee paralysis virus (ABPV). Both reactions were optimized to yield the highest sensitivity and specificity. The genome equivalent copies (GEC) detection limit per reaction was 389.3 for the ABPV RT-PCR. The GEC detection limit per reaction was 298.9 for the SBV RT-PCR. Viral detection and identification were confirmed by melting curve analysis and sequencing of the PCR products. Both techniques were used to evaluate Spanish field samples and establish the distribution of these viruses. Acute bee paralysis virus was not detected, and Sacbrood virus was present at low frequencies. The one-step real-time SG RT-PCR methods are fast, accurate, and useful for detecting and quantifying these honeybee viruses, which cause inapparent infections and contribute to the increasing depopulation of honeybee colonies.

Utility of a Panviral Microarray for Detection of Swine Respiratory Viruses in Clinical Samples

ABSTRACT Several factors have recently converged, elevating the need for highly parallel diagnostic platforms that have the ability to detect many known, novel, and emerging pathogenic agents simultaneously. Panviral DNA microarrays represent the most robust approach for massively parallel viral surveillance and detection. The Virochip is a panviral DNA microarray that is capable of detecting all known viruses, as well as novel viruses related to known viral families, in a single assay and has been used to successfully identify known and novel viral agents in clinical human specimens. However, the usefulness and the sensitivity of the Virochip platform have not been tested on a set of clinical veterinary specimens with the high degree of genetic variance that is frequently observed with swine virus field isolates. In this report, we investigate the utility and sensitivity of the Virochip to positively detect swine viruses in both cell culture-derived samples and clinical swine samples. The Virochip successfully detected porcine reproductive and respiratory syndrome virus (PRRSV) in serum containing 6.10 × 102 viral copies per microliter and influenza A virus in lung lavage fluid containing 2.08 × 106 viral copies per microliter. The Virochip also successfully detected porcine circovirus type 2 (PCV2) in serum containing 2.50 × 108 viral copies per microliter and porcine respiratory coronavirus (PRCV) in turbinate tissue homogenate. Collectively, the data in this report demonstrate that the Virochip can successfully detect pathogenic viruses frequently found in swine in a variety of solid and liquid specimens, such as turbinate tissue homogenate and lung lavage fluid, as well as antemortem samples, such as serum.

Development and evaluation of a SYBR Green real-time RT-PCR assay for evaluation of cytokine gene expression in horse

Cytokine secretion is one of the main mechanisms by which the immune system is regulated in response to pathogens. Therefore, the measurement of cytokine expression is fundamental to characterizing the immune response to infections. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) is widely used to measure cytokine mRNA levels, but assay conditions should be properly evaluated before analyzing important equine infections through relative quantification of gene expression. The aim of this study was to develop and evaluate a set of RT-qPCR assays for a panel of the most common cytokines in horses involved in innate and adaptive immune responses. Eight cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-10, IL-12, TNFα, IFNβ and IFNγ) and a housekeeping gene (β-actin) were detected and amplified with the same annealing temperature in a SYBR Green RT-qPCR assay of samples of mitogen-stimulated peripheral blood mononuclear cells from a healthy horse and whole blood from a horse infected with African horse sickness virus. The method gave good efficiency for all genes tested, allowing quantification of relative expression levels. These SYBR Green RT-qPCR assays may be useful for examining cytokine gene expression in horses in response to exposure to economically important pathogens.

Analytical sensitivity and specificity of a RT-PCR for the diagnosis and characterization of the spatial distribution of three Apis mellifera viral diseases in Spain

The occurrence and spatial distribution of deformed wing virus (DWV), black queen cell virus (BQCV), and Kashmir bee virus (KBV) were assessed in 294 honeybee colonies in Spain by employing a SYBR-Green based real time RT-PCR. 60% of them were positive for both DWV and BQCV, and those two viruses were detected in 84% and 68% of the samples, respectively. Conversely, KBV was detected in only 1.7% of the samples. Increments in the number of bee colonies per region, adjusted by the number of samples collected, were associated with increased risk of finding DWV, BQCV, and KBV, as estimated by mixed Bayesian regression models. The residual risk for DWV, BQCV, and KBV decreased northerly and westerly, suggesting that factors or forces that favour the presence of these viruses could be more prevalent in southern and eastern regions of Spain. Results will be useful in the design and implementation of effective honeybee viral disease control and surveillance programs in Spain.ZusammenfassungSpanien gilt als der weltweit drittgrösste Produzent für Honigbienen (Apis mellifera). Demgegenüber stehen von Viren wie dem Verkrüppeltem Flügelvirus (DWV), dem Schwarzen Königzellenvirus (BQCV) und dem Kaschmir Bienenvirus (KBV) hervorgerufene Krankheiten, die als die wichtigsten Ursachen für virale Krankheiten der Honigbiene gelten und schwerwiegende epidemiologische und wirtschaftliche Schäden in befallenen Bienenständen verursachen können. Voraussetzung für die Kontrolle dieser viralen Infektionen ist die Entwicklung hochspezifischer und hochsensitiver Diagnosemethoden und die Kenntnis der landesweiten Verbreitung dieser Krankheiten. Für Spanien gilt es insbesondere auch, vorläufige Daten über die Auswirkungen dieser Krankheiten auf die Honigbienen produzierende Industrie zu erfassen. Hauptziel dieser Arbeit war es, die analytische Sensitivität und Spezifität von RT-PCR-Methoden für die Identifizierung von DWV-, BQCV- und KBV-Infektionen in spanischen Bienenvölkern zu ermitteln. Insgesamt wurden zwischen den Jahren 2004 und 2006 Proben aus 294 Bienenvölkern der 14 von 15 Verwaltungsregionen Spaniens (ohne Inselregionen) gesammelt (Abb. 1). Für die Detektion von DWV und BQCV nutzten wir eine auf SYBR-Green-Inkorporation beruhende Einschritt-Realtime RT-PCR-Methode (Kukielka et al., 2008) und für die KBV-Detektion modifizierten wir eine konventielle RT-PCR-Methode (Stoltz et al., 1995) für Realtime-Bedingungen.Die analytische Spezifität der RT-PCR-Methoden wurde durch Sequenzanalyse der Amplifikationsprodukte überprüft. Die Sensitivität der Realtime RT-PCR-Ansätze lag bei 10−9 Viruspartikel für DWV und BQCV und bei 10]t-5 für KBV. Zur Analyse der Verbindung zwischen Virusvorkommen und Anzahl und räumlicher Verteilung der Bienenvölker nutzten wir ein auf einem Bayes’schen Verfahren beruhendes Modell. Dabei zeigte sich, dass DBV mit 84,0 % das am häufigsten vorkommende Virus in den Stockproben war, gefolgt von BQCV (68,0 %) und KBV (1,7 %). Eine Ko-Infektion durch zwei Viren, meist mit BQCV und DWV, wurde in 60,4 % der Proben gefunden. Die visuelle Begutachtung des krankheitsspezifischen Restrisikos lässt erwarten, dass hier nicht erfasste Faktoren einen Anstieg des Risko für einen DWV-, BQCV- und KBV-Befall in Spanien in östlicher und südlicher Richtung mit sich bringen können (Abb. 2).Der Anteil der in dieser Studie gefundenen positiven Probe für pathogene Bienenviren entspricht dem von Schätzwerten aus anderen Ländern. Der Anteil positiver Proben war auch assoziiert mit der Anzahl an Bienenvölkern in der jeweiligen Region. In Abwesenheit aktiver und formaler Präventions- und Überwachungsprogramme ist deshalb zu erwarten, dass Bienenviren vor allem in Regionen mit der grössten Bienendichte eingeführt werden und sich dort aufgrund der erhöhten Kontaktwahrscheinlichkeiten mit infizierten Bienen verbreiten können. Die Ergebnisse dieser Studie sollten von Nützlichkeit sein für die Entwicklung von Überwachungs- und Kontrollprogrammen für Viruskrankheiten, die die Honigbienenindustrie Spaniens treffen können.