Use of reverse transcription-real time polymerase chain reaction (real time RT-PCR) assays with Universal Probe Library (UPL) probes for the detection and genotyping of infectious pancreatic necrosis virus strains isolated in Chile

Calleja, Felipe; Godoy, Marcos; Cárcamo, Juan G.; Bandı́n, Isabel; Yáñez, Alejandro J.; Dopazo, Carlos P.; Kibenge, Fred; Avendaño‐Herrera, Rubén

doi:10.1016/j.jviromet.2012.03.022

Cited by 25 publications

(29 citation statements)

References 31 publications

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“…In Chile, the types of IPNV that are present and have been genetically characterized to date belong only to genogroups 1 and 5 (Mutoloki & Evensen 2011, Eissler et al 2011, Calleja et al 2012. However, the geographic zones examined have been limited to very few locations, and the great majority of samples have been from freshwater outbreaks.…”

Section: Introductionmentioning

confidence: 99%

Detection and phylogenetic analysis of infectious pancreatic necrosis virus in Chile

Tapia¹,

Eissler²,

Torres³

et al. 2015

Dis. Aquat. Org.

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Infectious pancreatic necrosis virus (IPNV) is the etiological agent of a highly contagious disease that is endemic to salmon farming in Chile and causes great economic losses to the industry. Here we compared different diagnostic methods to detect IPNV in field samples, including 3 real-time reverse transcription PCR (qRT-PCR) assays, cell culture isolation, and indirect fluorescent antibody test (IFAT). Additionally, we performed a phylogenetic analysis to investigate the genogroups prevailing in Chile, as well as their geographic distribution and virulence. The 3 qRT-PCR assays used primers that targeted regions of the VP2 and VP1 genes of the virus and were tested in 46 samples, presenting a fair agreement within their results. All samples were positive for at least 2 of the qRT-PCR assays, 29 were positive for cell culture, and 23 for IFAT, showing less sensitivity for these latter 2 methods. For the phylogenetic analysis, portions of 1180 and 523 bp of the VP2 region of segment A were amplified by RT-PCR, sequenced and compared with sequences from reference strains and from isolates reported by previous studies carried out in Chile. Most of the sequenced isolates belonged to genogroup 5 (European origin), and 5 were classified within genogroup 1 (American origin). Chilean isolates formed clusters within each of the genogroups found, evidencing a clear differentiation from the reference strains. To our knowledge, this is the most extensive study completed for IPNV in Chile, covering isolates from sea-and freshwater salmon farms and showing a high prevalence of this virus in the country.

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Section: Introductionmentioning

confidence: 99%

Detection and phylogenetic analysis of infectious pancreatic necrosis virus in Chile

Tapia¹,

Eissler²,

Torres³

et al. 2015

Dis. Aquat. Org.

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“…Posteriormente, Nishizawa et al (2005) revelaron la existencia de un séptimo genogrupo integrado únicamente por cepas japonesas, y recientemente en Australia se aisló un nuevo Aquabirnavirus desde trucha arcoíris, que fue clasificado en un octavo genogrupo (McCowan et al, 2015;Mohr et al, 2015). En Chile, los virus IPN que han sido caracterizados genéticamente pertenecen a los Genogrupos 1 y 5, i.e., norteamericanos y europeos, respectivamente (Eissler et al, 2011;Mutoloki & Evensen, 2011;Calleja et al, 2012;Tapia et al, 2015;Jorquera et al, 2016), dentro de los cuales forman sus propios subtipos o subgrupos (Tapia et al, 2015). Sin embargo, existe muy poca información acerca de la predominancia de cada uno de estos genogrupos y su interacción con las distintas especies hospederas de salmónidos cultivadas en el país.…”

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“…Las secuencias obtenidas se compararon y clasificaron en base a cepas de referencia de birnavirus acuáticos chilenos y extranjeros disponibles en Genbank y reportadas en publicaciones científicas (i.e., Blake et al, 2001;Eissler et al, 2011;Mutoloki & Evensen, 2011;Calleja et al, 2012;Ruane et al, 2015;Jorquera et al, 2016). Además se incluyen algunas muestras utilizadas también en el trabajo realizado por Tapia et al (2015) (ver Tabla 1).…”

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“…1) muestra que los virus de este estudio se clasifican dentro de los dos genogrupos previamente reportados en el país, el Genogrupo 1 y 5 (Eissler et al, 2011;Mutoloki & Evensen, 2011;Calleja et al, 2012;Tapia et al, 2015;Jorquera et al, 2016). En la Figura 1 se observan dos subgrupos dentro del Genogrupo 5, el 5A que incluyó los virus reportados para Chile, en este y otros trabajos anteriores y el 5B que agrupó cepas de origen australiano.…”

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“…Sin embargo, el genoma de la cepa VUV/84, no ha sido sometido a la misma presión selectiva ni ha competido con la actual población de virus chilenos en los procesos de mutación, por lo que no formaría parte de este clúster. Al analizar en conjunto los datos publicados de todos los virus IPN secuenciados en Chile durante los últimos años (Eissler et al, 2011;Mutoloki & Evensen, 2011;Calleja et al, 2012;Tapia et al, 2015;Jorquera et al, 2016), se observa que están distribuidos en todas las regiones del país donde hay producción masiva de salmónidos (i.e., Los Ríos, Los Lagos, Aysén y Magallanes), y que existe una predominancia de las cepas clasificadas en el Genogrupo 5 (78,8%) sobre el Genogrupo 1 (21,2%) (Tabla 2). Además, el virus infecta a todas las especies de salmónidos cultivadas en el país, con una mayor incidencia en S. salar, como también lo demuestran los informes sanitarios de SERNAPESCA, donde esta especie es la que muestra mayor porcentaje de mortalidad causada por IPN y el mayor número de diagnósticos de la enfermedad (SERNAPESCA, 2014a).…”

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Genotipificacion y relacion hospedador especifica del virus de la necrosis pancreatica infecciosa en Chile

Torres

Eissler

Tapia

et al. 2016

lajar

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RESUMEN. Se analizaron muestras de órganos (riñón y bazo) de las principales especies de salmónidos cultivados en Chile a través de la reacción en cadena de la polimerasa anidada (PCR anidado) para detectar la presencia del virus de la necrosis pancreática infecciosa (IPNV). La técnica resultó ser eficiente y sensible, permitiendo amplificar material genético del virus para su secuenciación directamente desde los tejidos, incluso desde muestras que no pudieron ser aisladas en cultivo celular. A través del análisis filogenético se detectaron los dos genogrupos descritos previamente en el país, i.e., europeo (Genogrupo 5) y americano (Genogrupo 1), siendo cuantitati-vamente predominantes los IPNV del Genogrupo 5 (78,8%). Dentro del Genogrupo 1, se observó claramente la formación de un sub-grupo de virus chilenos separados de las cepas de referencia (e.g., WB, VR-299), por lo que se propone su denominación como genotipo chileno. Adicionalmente, se observó una relación estadística-mente significativa hospedador-específica entre los genogrupos identificados: los virus del Genogrupo 5 provienen de Salmo salar mientras que los del Genogrupo 1 provienen de Oncorhynchus mykiss u O. kisutch (P < 0,001). La asociación de esta relación hospedador-específica con la virulencia puede proveer información importante para el manejo y control de IPNV en Chile. Palabras clave: virus, IPNV, PCR anidado, genotipificación, filogenia, relación hospedador-específica, gen VP2. Genotyping and host-specific relationship of infectious pancreatic necrosis virus in ChileABSTRACT. Samples of fish organs (kidney and spleen) from the main salmonid species farmed in Chile were analyzed through the nested polymerase chain reaction (nested PCR) to detect the presence of infectious pancreatic necrosis virus (IPNV). The technique proved to be efficient and sensitive, allowing amplification of viral genetic material for sequencing directly from tissue, even from samples that could not be isolated in cell culture. The phylogenetic analysis showed the two genogroups previously described in the country, i.e., European (Genogroup 5) and American (Genogroup 1), being the IPNV that belong to Genogroup 5 the dominant one (78.8%). It is clear that the Chilean IPN viruses are clustered within the Genogroup 1 forming a sub-group that is separated from the reference strains (e.g., WB, VR-299), hence we propose its denomination as a Chilean genotype. Additionally, a statistically significant host-specific relationship was observed between the genogroups identified: viruses from Genogroup 5 were detected in Salmo salar, while the ones from Genogroup 1 were detected in Oncorhynchus mykiss or O. kisutch (P < 0.001). The association of this hostspecific relationship with the virulence can provide important information for the management and control of IPNV in Chile.

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Phylogenetic analysis of infectious pancreatic necrosis virus in Ireland reveals the spread of a virulent genogroup 5 subtype previously associated with imports

et al. 2014

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Infectious pancreatic necrosis is a significant disease of farmed salmonids resulting in direct economic losses due to high mortality and disease-management costs. Significant outbreaks of the disease occurred in farmed Atlantic salmon in Ireland between 2003 and 2007, associated with imported ova and smolts. As the virus was known to occur in the country since the development of aquaculture in the 1980s, this study examined archived samples to determine whether these older isolates were associated with virulent forms. The study showed that two genotypes of IPNV were present in the 1990s, genotype 3 and genotype 5. A more virulent subtype of the virus first appeared in 2003 associated with clinical outbreaks of IPN, and this subtype is now the most prevalent form of IPNV found in the country. The data also indicated that IPNV in Ireland is more closely related to Scottish and continental European isolates than to Norwegian, Chilean and Australasian genogroup 5 isolates.

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Use of reverse transcription-real time polymerase chain reaction (real time RT-PCR) assays with Universal Probe Library (UPL) probes for the detection and genotyping of infectious pancreatic necrosis virus strains isolated in Chile

Cited by 25 publications

References 31 publications

Detection and phylogenetic analysis of infectious pancreatic necrosis virus in Chile

Detection and phylogenetic analysis of infectious pancreatic necrosis virus in Chile

Genotipificacion y relacion hospedador especifica del virus de la necrosis pancreatica infecciosa en Chile

Phylogenetic analysis of infectious pancreatic necrosis virus in Ireland reveals the spread of a virulent genogroup 5 subtype previously associated with imports

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