In the early 1980s, the emergence of Decapod Penstylhamaparvovirus 1, more commonly referred to as the infectious hypodermal and hematopoietic necrosis virus (IHHNV), in blue shrimp (Penaeus stylirostris), led to a significant shift in the way crustaceans were cultivated. These outbreaks triggered a necessary adjustment in the preferred cultured species, from blue shrimp to a more resistant but smaller sized species, the pacific white shrimp (Penaeus vannamei) (Dhar et al., 2019). Currently, P. vannamei represents the most successful and economically important cultured species of crustacean, representing over 70% of farmed shrimp worldwide.

IHHNV is virus is the smallest of the known shrimp viruses belonging to the family Parvoviridae, sub-family Hamaparvoviridae, and was the first shrimp virus for which a genome sequence was published in 2000 (Shike et al., 2000, Tijssen et al., 2012, Pénzes et al., 2020). At the ultrastructure level, the virions are icosahedral, non-enveloped, measure around 22 nm in size, and contain a single-stranded DNA genome approximately 4.1 kb in length (Mari et al., 1993; Shike et al., 2000; recently reviewed in Dhar et al., 2019). The genome of IHHNV contains three major open reading frames (ORFs), the left, middle, and right, which are typical of the family Parvoviridae ( Shike et al., 2000; Cotmore et al., 2019).

Almost four decades have elapsed since the discovery of IHHNV, and yet shrimp disease experts are still striving to develop an efficient diagnostic technique for detection of this virus. A SCOPUS (https://www.scopus.com/search/form.uri?display=basic#basic) search for the keywords “infectious hypodermal and hematopoietic necrosis virus and PCR” yields 135 results with 29 hits on the development of molecular diagnostics for IHHNV. These articles span from 2000 to 2019 (Nunan et al., 2000, Arunrut et al., 2019) underscoring the ongoing efforts to establish adequate diagnostic techniques to prevent the movement of infected stocks around the world and mitigate the negative effects of the virus. The main challenges that are encountered with IHHNV detection are the presence of fragments of the IHHNV genome integrated in the host genome as endogenous viral elements (EVE) and a high mutation rate (Tang and Lightner, 2006, Robles-Sikisaka et al., 2010, Saksmerprome et al., 2011, Cowley et al., 2018). It is possible that the combination of these factors has made PCR-based detection of IHHNV complicated and unreliable. Furthermore, the differentiation of the EVEs from infectious forms of the virus is fundamental since false positives have enormous implications leading to the destruction of valuable breeding stocks and the rejection of shipments of live broodstock and post-larvae.

In 2019, detection of IHHNV in three US facilities and concurrent movement of post-larvae led to the spread of the virus to Canada and the United Kingdom (Dhar et al., 2022). Whole genome sequencing of the virus, phylogenetic analysis, and modeling of the tertiary structure of the viral capsid protein were used to confirm the IHHNV isolate detected in the US was indeed an infectious form of the virus (Dhar et al., 2022).

A genome-integrated form of IHHNV that contained a fragment of the capsid protein gene within a microsatellite locus was initially described in P. monodon originating in East Africa, where samples that tested positive by PCR failed to cause infection in an experimental challenge to specific pathogen free P. vannamei (Tang and Lightner, 2006). Subsequently, Saksmerprome and colleagues detected several other fragments of the viral genome scattered throughout the P. monodon genome as well as in some stocks of P. vannamei originating in Thailand (Saksmerprome et al., 2011). While screening P. monodon and P. vannamei stocks that were presumably free of IHHNV using primer sets spanning the entire IHHNV genome, these authors reported IHHNV non-specific amplification by the 309 F/R conventional PCR primers recommended by the WOAH and with the IQ2000 commercial kit developed using these primer sets (Saksmerprome et al., 2011). Later data indicated that P. monodon and P. vannamei stocks screened by these authors contained an IHHNV EVE, which the primers used couldn't amplify within the host genome. As a result, they recommended the use of multiple primer sets to reduce the risk of false positives.

Routine screening of thousands of shrimp samples by conventional PCR for certification of stocks free of infectious IHHNV is time consuming and less sensitive than real-time PCR based assay. The fast-growing food producing shrimp aquaculture sector urgently needs a fast and efficient assay for large-scale screening of shrimp products and feed ingredients. A real-time PCR assay that can discriminate between EVE and the infectious form of the virus in a reliable and reproducible manner would be very beneficial. Since the two original reports of IHHNV detection by real-time PCR in 2001 (Dhar et al., 2014, Tang and Lightner, 2001), several papers are now published that described IHHNV detection (Fig. 1). Although the TaqMan real-time PCR described by Tang and Lightner (2001) is the WOAH-recommended assay, there are reports of non-specific amplification using this method in P. monodon. Recently, Cowley et al., (2018) reported a TaqMan real-time assay to screen P. monodon samples for IHHNV and developed primer sets that can differentiate EVE from the infectious form of IHHNV. It is worth noting the primers for detecting the infectious form of the virus were based on the same region of the NS1 gene, which is targeted by the primers 309 F/309 R recommended by the WOAH (2023) .

Considering the incomplete knowledge in the published literature and a growing need to ensure fast, reliable, accurate detection of the infectious form of IHHNV in commercially important species of shrimp, calls for a side-by-side comparison of WOAH-recommended and published primer sets in real-time PCR-assay performance using a common set of curated samples. To address this issue, we conducted an exhaustive literature search to find original papers that described IHHNV detection by real-time PCR (Table 1) and compared the performance of the primers described in these papers to distinguish the IHHNV infectious form from EVEs by TaqMan real-time PCR assay using a common set of P. vannamei, P. monodon and P. stylirostris samples. In addition, we designed new sets of primers to ensure that the previously published and newly designed primers cover the entire genome of IHHNV and EVEs reported in the literature. Based on this comparative analysis, we report here two primer sets that can reliably differentiate infectious forms of IHHNV from EVEs; and these primer sets could be valuable in detecting IHHNV in all three species. In addition, we report here a primer set that can amplify a shrimp internal control gene, EF-1α, in all t species.