Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus

Abstract

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5’-Taq nuclease-3’ minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses ( Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak ( n = 11), as well as samples collected from partridges during surveillance programs ( n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

Influence of flavivirus co‐circulation in serological diagnostics and surveillance: A model of study using West Nile, Usutu and Bagaza viruses

“…Serum samples from a susceptible avian host for the three flaviviruses, the red‐legged partridge ( Alectoris rufa ), were obtained after experimental inoculation of different strains of WNV (lineages 1 and 2), USUV and BAGV (Table ) following the methodology previously described (Llorente et al, ; Pérez‐Ramírez, Llorente, Del Amo, Nowotny, & Jiménez‐Clavero, ; Sotelo, Gutierrez‐Guzmán, et al, ). Prior to the experiments, all individuals were tested serologically using a competitive ELISA (INgezim West Nile Compac 10.WNV.k3, Ingenasa, Madrid, Spain) and micro‐VNT (as described below) and virologically by real‐time RT‐PCR in feather pulps (Del Amo et al, ; Buitrago et al, ) to ensure that previous exposure to WNV, USUV or BAGV had not occurred. Animal care, handling and experimental procedures were authorized by the Ethics and Animal Welfare Commitees of the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) according to Council Directive 2010/63/EU.…”

A Duplex Quantitative Real-Time Reverse Transcription-PCR for Simultaneous Detection and Differentiation of Flaviviruses of the Japanese Encephalitis and Ntaya Serocomplexes in Birds

“…It is well-known that the design of generic molecular tools may be challenging to cover the range of target pathogens and may limit their sensitivity (43), especially when clinical material is analyzed. However, the new dRRT-PCR has demonstrated to be a specific and highly sensitive tool, capable of detecting the wide range of JE and Ntaya flaviviral species analyzed, and posing a performance similar to the RRT-PCR methods used as reference (34,35). On the other hand, most PCR methods described for flaviviruses detection have been verified with human or mosquito samples, when available (28,(31)(32)(33).…”

“…Two previously validated techniques, namely a triplex RRT-PCR for WNV (lineages 1 and 2) and USUV simultaneous detection (34) and a single RRT-PCR for BAGV detection (35), were employed as reference methods in comparative assays. For detection of other flaviviruses, a widely used conventional RT-PCR (27) was performed with minor modifications, and amplification products were further sequenced to confirm the virus identity.…”

“…Initially, the analytical sensitivity of the dRRT-PCR was studied by analyzing duplicates of 10-fold serial dilutions of viral suspensions of WNV-L1 (Spain/2010/H-1b), WNV-L2 (B956), USUV (SAAR 1776/1958), and BAGV (Spain H/2010). All experiments were run in parallel with the equivalent reference RRT-PCR method (34,35), obtaining a similar (BAGV) or at least 10 times higher (WNV-L1, WNV-L2, USUV) sensitivity with the dRRT-PCR (Supplementary Table 1).…”

Estudio de la patogenia el Maedi-Visna ovino y su aplicación en el diagnóstico y control de la enfermedad = Study of ovine Maedi-Visna pathogenia and its application in diagnostic and illness control

“…El virus Visna/maedi (VMV) forma parte de la familia Retroviridae, subfamilia Orthoretrovirinae, género Lentivirus, y subgénero Lentivirus de los pequeños rumiantes (LVPR) (Buitrago et al, 2012) que engloba las distintas cepas de MV y de la Artritis Encefalitis Caprina (CAEV). Dentro del género Lentivirus, se encuentran otros virus como el HIV o el SIV con los que comparte características patológicas similares (Pétursson et al, 1991;Leroux et al, 2010), resultando su estudio de gran interés.…”