Insights on Minimizing False Positives in IHHNV Detection: Experiences from Ecuador's Penaeus vannamei Aquaculture

Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp aquaculture is complicated by endogenous viral elements (EVEs) causing false positives in conventional PCR assays. This study analyzed 277 Penaeus vannamei samples from Ecuador using World Organization for Animal Health (WOAH)-recommended short-fragment primers (IHHNV-309, -389, -392, -77012; ~1.5 kb amplicons) and long-amplicon PCR (LA-PCR; ~3.7 kb, 90% of the genome), complemented by histopathology. Short-fragment primers showed high positivity rates (72.9-83.0% individually; 69.3% combined), while LA-PCR reduced positives to 29.6%, with 95.1% overlap indicating true infections as a subset of conventional results. Approximately 55.6% of samples likely contained EVEs mimicking IHHNV, and 14.8% were true negatives. Histopathology confirmed classic IHHNV lesions (Cowdry A-type inclusions) in only one sample (0.36%), which also showed co-infections (hepatopancreatic atrophy, gregarines, and unidentified viral inclusions), suggesting multifactorial pathology. These findings highlight inflated IHHNV prevalence due to single-primer PCR, particularly in Ecuador, where reliance on WOAH-suggested primers (e.g., 389F/R) fails to distinguish infectious IHHNV from EVEs or confirm subclinical status, risking misattribution of losses to IHHNV while overlooking pathogens like Vibrio spp. We advocate LA-PCR and histopathology to enhance diagnostics and support sustainable shrimp fisheries.