In 2016, we collected 200 P. chinensis individuals (12–14 cm in body length) from a hatchery in Shandong Province, China, and transported them alive (about two and a half hours) to the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences for use in bioassays. Within 2 weeks of being stocked across several aquaria, the shrimp displayed evidence of morbidity without typical clinical signs but suffered from ∼20% mortality from unknown causes. Cephalothorax tissues were sampled from the moribund shrimp to screen pathogens using transmission electron microscopy (TEM) and various PCR/RT-PCR methods. Total RNA and DNA were separately extracted from three pooled samples, and each pooled sample included five cephalothorax tissues. The molecular diagnosis of shrimp pathogens, including white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV), infectious myonecrosis virus (IMNV), Enterocytozoon hepatopenaei (EHP), and acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus (Vp_AHPND), was performed using RT-PCR or PCR as previously described (Dangtip et al., 2015; OIE, 2015; Jaroenlak et al., 2016; Chen et al., 2018).

Small pieces (∼1 mm³) of the lymphoid organ, hepatopancreas, and gills were sampled and fixed in the TEM fixative (2% paraformaldehyde, 2.5% glutaraldehyde, 160 mM NaCl, and 4 mM CaCl₂ in 200 mM PBS, pH 7.2) to screen any pathogens. Ultrathin sections were cut, mounted on collodion-coated grids, stained with aqueous uranyl acetate/lead citrate using standard procedures, and examined using TEM at the Equipment Center of the Medical College, Qingdao University.

Approximately 3 g soft cephalothorax tissues excluding hepatopancreas were sampled from the moribund shrimp and homogenized in 10 mL TNEP (100 mM Tris-HCl, 400 mM NaCl, 10 mM EDTA, pH 7.4) containing 200 μL protease inhibitor (0.675 g PMSF in 40 mL isopropyl alcohol) using a TissueLyser (Qiagen). The homogenate was clarified by low-speed centrifugation, and the supernatant was then centrifuged at 10,000 × g for 30 min at 4°C. The pellet was homogenized again in 10 mL TNEP and centrifuged at 8,000 × g for 20 min at 4°C. The supernatants were then combined and filtered sequentially through 0.45 μm and 0.22 μm membrane syringe filters to remove bacteria before being ultra-centrifuged in a P90AT rotor at 130,000 × g for 170 min at 4°C (CP100WX Ultracentrifuge, Hitachi, Japan). The pellets were resuspended in the TN buffer (100 mM Tris-HCl, 400 mM NaCl, pH 7.4) and loaded onto a sucrose density gradient prepared by layering equal volumes of 20.0, 31.5, 43.0, 54.5, and 66.0% (w/v) sucrose and ultra-centrifuged in a P40ST rotor at 130,000 × g for 170 min at 4°C. The density gradients were fractionated. Aliquots of the band were diluted with TN buffer and ultra-centrifuged again in a P90AT rotor at 130,000 × g for 120 min at 4°C. The pellets obtained were then resuspended in a total volume of 1 mL TN buffer. Drops of the resuspended materials were placed unto grids, negatively stained with 2% phosphotungstic acid (PTA) (pH 6.5), and examined using TEM (HT7700 electron microscopy, Hitachi, Japan) operating at 80 kV.

Total RNA was extracted from the viral extracts using TRIzol Reagent (Invitrogen), and ribosomal RNA (rRNA) was depleted using a Ribo-zero™ rRNA Removal Kit (Epicenter, United States). RNA sequencing libraries were generated from the rRNA-depleted RNA using NEBNext^® Ultra™ Directional RNA Library Prep Kit for Illumina^® (NEB, United States). Nest generation sequencing (NGS) was performed on the Illumina Hiseq platform, employing a 150 bp paired-end sequencing strategy. This step and the subsequent bioinformatics analyses were performed as described previously (Dong et al., 2020).

To obtain the full-length genome sequence, we designed PCR primers based on the consensus contigs assembled from the Illumina Hiseq reads (Table 1). RNA was extracted from purified virions using a UNlQ-10 Column Trizol Total RNA Isolation Kit (Sangon Biotech, Shanghai). cDNA was synthesized with random primers using the M-MuLV First Strand cDNA Synthesis Kit (Sangon Biotech, China) and was then amplified by PCR using LA Taq (Takara Bio, Japan). To determine the 5′ and 3′ terminal sequences of RNA segments, we conducted rapid amplification of cDNA ends (RACE) using a 5′/3′ RACE kit (Invitrogen). The open reading frames (ORFs) in the RNA segments were predicted using Geneious (version 11.1.5) and annotated using the Conserved Domain Database (CDD) (Marchler-Bauer et al., 2007).

BLASTx of the deduced RdRp, glycoprotein precursor (GPC), and nucleocapsid (N) protein sequences against the GenBank database was performed, and the top BLASTx hits to OWV1 were downloaded. Multiple sequence alignment was performed using MAFFT (Nakamura et al., 2018), and the conserved domains were extracted using TrimAl (Capella-Gutierrez et al., 2009) with a heuristic selection of the automatic method based on similarity statistics (-automated1). Phylogenetic analyses were performed using PhyML3.1 (Guindon et al., 2010), with 1,000 bootstrap replicates and the LG amino acid substitution model.

We developed a reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) targeting the S1 fragment. The product fragment (71 bp) was amplified using the forward primer OWV1-S1-F (5′-CCG ACA TGG ATG CGT TCA-3′), the reverse primer OWV1-S1-R (5′-CAA GGC TGC AAC AAT GAC CTT-3′), and the TaqMan probe (5′-6FAM-CGC AGA CAT CCA GTT CCA GGG CTT T-TAMRA-3′). The TaqMan probe-based RT-qPCR for OWV1 (TaqMan-RTqPCR-OWV1) in a 20 μL mixture, containing 10 μL Probe 1-step RT-qPCR 5G Premix (with 0.08 mM dNTP, 0.01 mM Mg²⁺, mutated MMLV reverse transcriptase, TOROIVD 5G DNA polymerase, RNase inhibitor, reaction buffer, and stabilizer, etc.) (TOROIVD^®, China), 0.5 μM OWV1-S1-F and OWV1-S1-R primers each, 0.2 μM probe, and 1 μL RNA template. The amplification reaction was performed using a CFX96 Quantitative Fluorescence Instrument (BIO-Rad, California, United States), and the reaction procedure was initiated at 52°C for 5 min and 95°C for 10 s, followed by 40 cycles of 95°C for 10 s and 60°C for 20 s.

DNA and RNA extracted from soft tissues of cephalothoraxes of the moribund P. chinensis individuals were pooled into three samples, with each including five cephalothorax tissues. Commonly known shrimp pathogens were screened using various PCR or RT-PCR tests, including white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV), infectious myonecrosis virus (IMNV), Enterocytozoon hepatopenaei (EHP), and acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus (Vp_AHPND). Among the three pooled samples, one sample tested positive for WSSV.

TEM examination of the ultrathin lymphoid organ and gill tissue sections from moribund P. chinensis identified spherical- to ovoid-shaped virions (80 nm∼115 nm in diameter) in the cytoplasm of the lymphoid cells (Figures 1A,B) and branchial cells (Figures 1C,D). In addition, electron microscopy of the negatively stained OWV1 particles purified from soft cephalothorax tissues identified virions morphologically similar to those detected in the infected cells (Figure 1E). Consistent with the PCR detection data, TEM also revealed the presence of WSSV particles (Figure 1F).

A total of 81,931,726 raw reads were obtained from the NGS data of the viral extracts, including 76,860,336 clean reads, which were de novo assembled using Trinity. BLASTx searches of the ORFs predicted in the assembled contigs identified three contigs showing homologies to the L, M, and S proteins of bunyaviruses.

In detail, 71,968 reads were mapped back to the L RNA segment (coverage: 1,482 ± 639), 48,574 mapped to the M RNA segment (coverage: 2,139 ± 1,051), and 8,038 mapped to the S RNA segment (coverage: 924 ± 477). To confirm the 3′ and 5′ terminal sequences of each RNA segment, we designed primers for Sanger sequencing to generate 5′- and 3′-RACE clones (Table 1). Sanger sequencing of the clones verified the contigs obtained from NGS. The conserved reverse complementary decamer sequences on the two ends were: 5′-ACACAAAGAC…and …GUCUUUGUGU-3′. These inverted terminal repeat sequences were identical to those of MoV and WzSV-1 (Li et al., 2015; Cowley, 2021) and similar to those of the closely related terrestrial uukuviruses and phleboviruses (Abudurexiti et al., 2019; Kuhn et al., 2020). Like other bunyaviruses, the inverted terminal repeats of OWV1 likely form short panhandles found in bunyaviruses that are important for the N protein binding and nucleocapsid assembly (Mir and Panganiban, 2005).

BLASTp analyses revealed that the OWV1 RdRp (2,052 aa) encoded by the L RNA segment (6,317 nt) (Figure 2A) was most similar to the RdRp of WzSV-1/MoV (79.6%/79.4% identity; E-value = 0; query coverage: 100%/100%), followed by Wenling crustacean virus 7 (WCV7, 34.3% identity; E-value = 0, query coverage: 95%) and bunya-like brown spot virus (BBSV; 36.7% identity; E-value = 0, query coverage: 79%) (Li et al., 2015; Grandjean et al., 2019). Moreover, CDD search found an endonuclease domain at the N-terminus of the predicted RdRp of OWV1 (L_protein_N superfamily domain cl20015; aa 62-133; E-value ≤ 1.71e-11) and also a Bunya_RdRp superfamily domain cl20265 (aa 600-1293; E-value ≤ 9.41e-97).

The GPC glycoprotein (912 aa), encoded by the M RNA segment (2,978 nt) of OWV1, possessed two transmembrane domains: the Phlebovirus_G1 superfamily domain cl06326 (aa 91-370; E-value ≤ 4.34e-13) and the Phlebovirus_G2 superfamily domain cl09431 (aa 430-874; E-value ≤ 1.35e-35) (Figure 2B). These domains were one of the specific characterizations of the genus Phlebovirus, responsible for anchoring these glycoproteins to the viral envelope (Halldorsson et al., 2016). BLASTp searches revealed that the OWV1 GPC protein was most similar to those of WzSV-1/MoV (71.1%/70.8% identity, E-value: 0, coverage: 99%/99%).

Similarly, the nucleoprotein (N) protein (243 aa) encoded by the S RNA segment (1,164 nt) was most similar to those of WzSV-1/MoV (65.0%/65.2% identity; E-value ≤ 2e-99, coverage: 90%/92%), followed by BBSV (32.0% identity; E-value ≤ 3e-19, coverage: 80%) (Figure 2).

However, it was reported that MoV contained an additional small (S2) RNA segment (1,364 nt) encoding a putative non-structural protein (NSs2; 394 aa) (Oanh et al., 2011; Cowley, 2016). BLASTx searches were repeated using contigs assembled from the OWV1 library, which successfully identified a near-complete RNA segment encoding a homolog of the MoV/WzSV-1 NSs2 protein. A total of 24,110 reads (coverage: 2,508 ± 1,572) were mapped to this contig. In addition, 5′ and 3′ RACE showed that the fourth RNA segment also possessed the complementary decamer terminal sequence identical to those present in the other three RNA segments. Unlike structural proteins, however, the NSs2 protein (401 aa) encoded by the fourth RNA segment (S2; 1,382 nt) of OWV1 was moderately similar to those of WzSV-1/MoV (57.1%/56.6% identity). Apart from the NSs2 protein of MoV/WzSV-1, neither BLASTp nor CDD searches found more homologs of the OWV1 NSs2 protein, whose biological functions thus remain unknown.

Phylogenetic analyses of the RdRp sequences of OWV1 and other phenuiviruses revealed that OWV1 fell within the genus Wenrivirus together with WzSV-1/MoV (Figure 3). However, other related viruses, e.g., WCV7, BBSV, and two phenuiviruses discovered in different crustacean hosts (Li et al., 2015; Grandjean et al., 2019), did not cluster together with OWV1. Similarly, phylogenetic analyses of the GPC and N protein sequences also revealed that OWV1, WzSV-1, and MoV clustered and formed a well-supported lineage (Supplementary Figures 1, 2).

The TaqMan-RTqPCR-OWV1 method was established using OWV1 standard templates in nine concentrations from 1 × 10¹ to 1 × 10⁹ copies/μL. The method calculated the logarithmic OWV1 copies per ng total RNA (copies/ng-RNA) in inverse proportion to the threshold cycles (CT) with a slope of -4.118 and an intercept of 48.66. The established standard curve had a correlation coefficient of 0.994. Three parallel CT values of each sample are nearly the same, indicating that the intra-assay repeatability was reliable. We collected 32 P. chinensis samples from two markets, which had no obvious gross sign when sampling. After being tested with TaqMan-RTqPCR-OWV1, 23 samples were positive for OWV1 at 1.17 × 10²—1.90 × 10⁷ copies/ng-RNA of OWV1 in gills of P. chinensis.