Acipenserid Herpesvirus 2 Genome and Partial Validation of a qPCR for Detection in White Sturgeon (Acipenser transmontanus)

Authors: QUIJANO CARDE, EVA MARIE - University Of California, Davis; ANENSON, KELSEY - University Of California, Davis; Waldbieser, Geoffrey - Geoff; BROWN, C.TITUS - University Of California, Davis; GRIFFIN, MATT - Mississippi State University; HERDERSON, EILEEN - University Of California, Davis; YUN, SUSAN - University Of California, Davis; SOTO, ESTEBAN - University Of California, Davis

Submitted to: Diseases of Aquatic Organisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/25/2023
Publication Date: 2/1/2024
Citation: Quijano Carde, E., Anenson, K., Waldbieser, G.C., Brown, C., Griffin, M., Herderson, E., Yun, S., Soto, E. 2024. Acipenserid Herpesvirus 2 Genome and Partial Validation of a qPCR for Detection in White Sturgeon (Acipenser transmontanus). Diseases of Aquatic Organisms. 157:45-59. https://doi.org/10.3354/dao03768.
DOI: https://doi.org/10.3354/dao03768

Interpretive Summary: The Acipenserid herpesvirus 2 (AciHV-2) causes up to 10% adult mortality and 80% juvenile mortality in white sturgeon, the primary species used for caviar production in the United States. In order to produce a diagnostic tool for this virus, researchers at the University of California, Davis, Mississippi State University, and the USDA, ARS, Warmwater Aquaculture Research Unit in Stoneville, MS, produced genome sequence assemblies of four isolates of the virus, then developed a quantitative molecular test to detect the virus in sturgeon tissues. The test was specific and sensitive in sturgeon cell lines and tissues of infected fish. This new test gives diagnosticians and researchers an improved tool to detect AciHV-2 outbreaks and better understand the dynamics of AcviHV-2 infections.

Technical Abstract: White sturgeon (Acipenser transmontanus) is the primary species used for high-quality caviar and sturgeon meat production in the United States. An important pathogen of white sturgeon is Acipenserid herpesvirus 2 (AciHV-2), which causes up to 10% mortality in adult and 80% mortality in juvenile white sturgeon. A current limiting factor in the research efforts is that a complete genome is not currently available for the white sturgeon or for AciHV-2. In addition, only a few methods are available for the diagnosis of AciHV-2. Thus, the purpose of this study was to assemble the complete genome of AciHV-2 and design and validate a quantitative PCR (qPCR) assay for the diagnosis of AciHV-2 in white sturgeon tissues. Four isolates from previous natural outbreaks throughout the past 30 years were sequenced via Illumina and Nanopore technologies. After filtering out contaminants, the obtained annotated assemblies of approximately 134 Kb cluster together in a similarity matrix with a published partial AciHV-2 sequence and separate from other herpesviruses. Initial characterization of open reading frames (ORFs) revealed various potential targets for further host-pathogen interactionsinteraction studies. Candidate ORFs for qPCR were selected based on sequence conservation among all four isolates of AciHV-2 and low sequence homology with AciHV-1, another important viral pathogen of sturgeons. NucleotideA nucleotide comparison of the top candidate ORF suggests this is the ATPase subunit of the terminase. The efficiency of the optimized qPCR was of 96% with an R2 of 0.9885. Spiking with 250 ng of white sturgeon fin DNA revealed a decrease in efficiency to 88% but no significant difference in the Ct values obtained per dilution. The limit of detection for this assay was 104 copies/reaction with a protocol of 35 cycles. Analytical specificity assessment revealed no cross-reaction with other known viruses that affect white sturgeon or other closely related sturgeon species. Relative accuracy was assessed from fin clip samples obtained from white sturgeon juveniles in an AciHV-2 immersion challenge. Viral culture was used as the gold standard diagnostic test for comparison. Both the True Negative and True Positive Rates were 100%, giving this assay a 100% relative accuracy when compared to viral culture during an active outbreak. Survivors were tested by qPCR, but only 6% tested positive via this assay 82 days post-exposure. The availability of a whole genome sequence for AciHV-2 and a highly specific and sensitive qPCR assay for the diagnosis of AciHV-2 in white sturgeon not only opens the door to further studies on the effects of AciHV-2 on the immune system of the host, but also directly impacts the ability to provide specific and rapid diagnosis of this agent in captive and wild populations