Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

Area of Science:

• Aquatic Animal Health
• Molecular Diagnostics
• Virology

Background:

• Infectious hematopoietic necrosis virus (IHNV) is a significant pathogen affecting salmonid fish globally.
• Existing diagnostic methods for IHNV include conventional PCR and virus isolation, which have limitations.
• Phylogenetic analysis has identified five major genogroups of IHNV worldwide.

Purpose of the Study:

• To develop and validate a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the nucleocapsid (N) gene.
• To evaluate the diagnostic sensitivity (DSe) and specificity (DSp) of the new RT-rPCR assay.
• To compare the performance of the RT-rPCR assay against OIE-accepted conventional PCR and virus isolation methods.

Main Methods:

• Development of a universal IHNV RT-rPCR assay targeting the N gene.
• Validation of DSe and DSp using laboratory-challenged steelhead trout (Oncorhynchus mykiss).
• Comparative analysis with conventional PCR and virus isolation in cell culture, including field testing on hatchery samples.

Main Results:

• The IHNV N gene RT-rPCR assay demonstrated 100% DSp and DSe.
• The RT-rPCR assay exhibited a higher diagnostic odds ratio (DOR) compared to virus culture and conventional PCR.
• The assay proved highly repeatable within and reproducible between laboratories, detecting more positive samples in field testing than cell culture.

Conclusions:

• The developed universal IHNV RT-rPCR assay is highly sensitive, specific, repeatable, and reproducible.
• This RT-rPCR assay is suitable for use in diagnostic settings for IHNV detection in salmonid fish.
• The new assay offers an improved diagnostic tool for managing IHNV outbreaks.