Shrimp genome sequence contains independent clusters of ancient and current Endogenous Viral Elements (EVE) of the parvovirus IHHNV

BACKGROUND: Shrimp have the ability to accommodate viruses in long term, persistent infections without signs of disease. Endogenous viral elements (EVE) play a role in this process probably via production of negative-sense Piwi-interacting RNA (piRNA)-like fragments. These bind with Piwi proteins to dampen viral replication via the RNA interference (RNAi) pathway. We searched a genome sequence (GenBank record JABERT000000000) of the giant tiger shrimp (Penaeus monodon for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). RESULTS: The shrimp genome sequence contained three piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in pseudochromosome 35 (PC35). Both PC35 clusters contained multiple sequences with high homology (99%) to GenBank records DQ228358 and EU675312 that were both called “non-infectious IHHNV Type A” (IHHNV-A) when originally discovered. However, our results and those from a recent Australian P. monodon genome assembly indicate that the relevant GenBank records for IHHNV-A are sequence-assembly artifacts derived from scrambled and fragmental IHHNV-EVE. Although the EVE in the two PC35 clusters showed high homology only to IHHNV-A, the clusters were separate and distinct with respect to the arrangement (i.e., order and reading direction) and proportional content of the IHHNV-A GenBank records. We conjecture that these 2 clusters may constitute independent allele-like clusters on a pair of homologous chromosomes. The third EVE cluster was found in pseudochromosome 7 (PC7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against current types of infectious IHHNV. One disadvantage was that some EVE in PC7 can give false positive PCR test results for infectious IHHNV. CONCLUSIONS: Our results suggested the possibility of viral-type specificity in EVE clusters. Specificity is important because whole EVE clusters for one viral type would be transmitted to offspring as collective hereditary units. This would be advantageous if one or more of the EVE within the cluster were protective against the disease caused by the cognate virus. It would also facilitate gene editing for removal of non-protective EVE clusters or for transfer of protective EVE clusters to genetically improve existing shrimp breeding stocks that might lack them. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-022-08802-3

AP4 method for two-tube nested PCR detection of AHPND isolates of Vibrio parahaemolyticus

Our previous work on the mechanism of virulence for the unique isolates of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (VPAHPND) revealed that it was mediated by a binary Pir-like toxin pair ToxA and ToxB. These toxins are located on the pVA plasmid, a plasmid carried by AHPND-causing strain of V. parahaemolyticus with a size of approximately 69 kbp. Using the coding sequences of ToxA, a one-step PCR detection method for VPAHPND was introduced in June 2014 but had the limitation that attempts to adapt it into a nested PCR protocol were unsuccessful. As a result, low levels of VPAHPND in shrimp or other samples could not be detected without first preparing an enrichment broth culture to allow bacterial growth before extraction of template DNA. Here, we describe the AP4 (abbreviation of AHPND detection version 4) method, a two-tube nested PCR method that targets the tandem genes ToxA and ToxB, including the 12 bp spacer that separates them on pVA plasmid. Testing of the method revealed that it gave 100% positive and negative predictive values for VPAHPND using a panel of 104 bacterial isolates including 51 VPAHPND isolates and 53 non-AHPND isolates, the latter including 34 isolates of V. parahaemolyticus and 19 isolates of other bacteria found in shrimp ponds, including other Vibrio species. The AP4 nested PCR method was 100 times more sensitive (100 fg total DNA template) than the one-step AP3 (10 pg total DNA template) method, and it could detect VPAHPND in experimentally challenged shrimp by 6 h post immersion (n = 2/3), while AP3 could not detect is until 12 h post immersion (n = 1/3). Thus, the AP4 method may be useful in detecting VPAHPND isolates in samples where target material is limited (e.g., small tissue quantity or archived DNA) and enrichment cannot be employed (i.e., frozen samples or samples preserved in alcohol)

Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.

Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VPAHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VPAHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24-48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. JALL01000066.1) and similarity to known binary insecticidal toxins called 'Photorhabdus insect related' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set

Examples of HP histology of moribund shrimp administered single or combined doses of ToxA and ToxB by reverse gavage.

<p>(A) Tissue sections from a BSA (10/20 μg/g) negative control shrimp (tubule longitudinal sections) and (B) a ToxA (5 μg/g) treated shrimp (tubule cross sections) showing only normal histology with morphology and cell types as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126987#pone.0126987.g001" target="_blank">Fig 1A to 1C</a>. (C) ToxB only (5 μg/g) and (D) ToxA+ToxB (2 μg/g each) showing mostly normal histology, but with some thin HP tubule epithelia (black arrows) when compared to epithelia of normal thickness (grey arrows). (E) ToxA+ToxB (5 g/g each) showing enlarged HP tubules (compared to A-C) with collapsed epithelia (black arrows) but no cell sloughing. (F) ToxA+ToxB (10 μg/g each) showing massive sloughing (black arrows) and dissolution of HP tubule epithelial cells (i.e., severe AHPND histopathology).</p

SDS-PAGE analysis of 60% AS fractions from broth of non-AHPND isolate S02 (Lane 2), and VP_AHPND isolates 5HP (Lane 3) and CN (Lane 4).

<p>Lane 1: 60% AS from culture broth without bacteria. There are 2 major bands (ToxA at ~16kDa and ToxB at ~50 kDa) present in lanes for the VP_<b>AHPND</b> isolates 5HP and CN, but absent or at low level in non-AHPND isolate S02.</p

Bacterial expression of ToxA and ToxB.

<p>(A) ToxA expressed with a 6-His tag and purified by Ni-NTA affinity chromatography. Lane 1: Bacterial cell lysate from a non-induced bacterial culture; Lane 2: Bacterial cell lysate from an IPTG-induced culture; Lane 3: Eluted protein from the Ni-NTA column. The deduced molecular weight for ToxA-His was 12.7 kDa. (B) ToxB was expressed as a GST-fusion protein. Lane 1: Bacterial cell lysate from a non-induced culture; Lane 2: Bacterial cell lysate from an IPTG-induced culture; Lane 3: Eluted fraction from Sepharose 4B beads; Lanes 4&5: Fraction eluted from Sepahrose 4B after thrombin-cut. The estimated molecular weights for GST-ToxB and ToxB were approximately 76 and 50 kDa, respectively.</p

Examples of histopathological sections of hepatopancreatic (HP) tissue from moribund shrimp treated by reverse gavage with 60% AS fractions from <i>V</i>. <i>parahaemolyticus</i> isolates.

<p>(A) A longitudinal section of HP tissue from pre-challenged shrimp showing normal histology with the lumens enclosed by epithelial cell layers comprised of non-vacuolated deeply basophilic (purple stained), embryonic cells (E-cells) at the distal end of the tubule that progress in the proximal direction into a mixture of B-cells with large, single vacuoles, R-cells with multiple vacuoles and F-cells that are non-vacuolated and deeply basophilic. (B) A section of normal HP tissue from the PBS negative control shrimp showing normal tubules mostly in longitudinal section except for a few tubules at the outer (distal) portion of the HP where they are cut in cross-section. The tubule lumens are surrounded by epithelial cells similar to those in (A). (C) Tangential section of HP tissue from shrimp treated with non-AHPND S02 preparation and showing normal HP and showing the same cell types as in (A) and (B). (D) Section HP tubules (mostly in cross-section) from shrimp treated with 5HP preparation and showing AHPND pathology characterized by absence of normal epithelia containing B-cells, R-cells and F-cells as seen in (A) to (C) and instead by massive sloughing of epithelial cells into tubule lumens in the absence of bacteria. The inset shows a magnification of the sloughed epithelial cells in a tubule lumen. (E) Section of HP tubules (cross-section) from shrimp treated with CN preparation and showing AHPND pathology similar to that in (D) but more severe in that all of the tubule lumens are completely filled with sloughed cells except for two tubules cut in cross-section through the E-cell region.</p