Coinfection with Yellow Head Virus Genotype 8 (YHV-8) and Oriental Wenrivirus 1 (OWV1) in Wild Penaeus chinensis from the Yellow Sea

At present, there are few studies on the epidemiology of diseases in wild Chinese white shrimp Penaeus chinensis. In order to enrich the epidemiological information of the World Organisation for Animal Health (WOAH)-listed and emerging diseases in wild P. chinensis, we collected a total of 37 wild P. chinensis from the Yellow Sea in the past three years and carried out molecular detection tests for eleven shrimp pathogens. The results showed that infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), yellow head virus genotype 8 (YHV-8), and oriental wenrivirus 1 (OWV1) could be detected in collected wild P. chinensis. Among them, the coexistence of IHHNV and DIV1 was confirmed using qPCR, PCR, and sequence analysis with pooled samples. The infection with YHV-8 and OWV1 in shrimp was studied using molecular diagnosis, phylogenetic analysis, and transmission electron microscopy. It is worth highlighting that this study revealed the high prevalence of coinfection with YHV-8 and OWV1 in wild P. chinensis populations and the transmission risk of these viruses between the wild and farmed P. chinensis populations. This study enriches the epidemiological information of WOAH-listed and emerging diseases in wild P. chinensis in the Yellow Sea and raises concerns about biosecurity issues related to wild shrimp resources.


Introduction
The seed industry is a fundamental core supporting modern aquaculture. Viral diseases have been considered a vast hazard to the shrimp farming industry worldwide [1]. Wild shrimp stocks were often used for hatching and breeding to maintain the genetic diversity of farmed populations [2]. Meanwhile, the national stock enhancement program for natural fisheries resources requires artificial hatching of postlarvae from captured broodstock of local shrimp species [3], in which the interaction between the wild aquatic animal populations and aquaculture systems was highly involved. However, the epidemiological status of diseases in wild shrimp stocks is often overlooked. Pathogens, such as infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV), and yellow head virus (YHV), which have caused substantial economic losses

Transmission Electron Microscopy
For transmission electron microscopy (TEM), samples were prepared in two ways: purifying viruses from gills for negative staining and embedding gills and lymphoid organs for ultrathin sections.
Small pieces (~1 mm 3 ) of the lymphoid organ and gills were sampled and fixed in the TEM fixative (2% paraformaldehyde, 2.5% glutaraldehyde, 160 mM NaCl, and 4 mM CaCl 2 in 200 mM PBS, pH 7.2). Ultrathin sections were cut, mounted on collodion-coated grids, and stained with aqueous uranyl acetate/lead citrate using standard procedures as we previously published [24].

Virus Purification
Fifteen grams of P. chinensis gill filaments from individuals collected in 2022 were chopped and added with 100 mL of SM buffer (50 mM Tris-HCl, 10 mM MgSO 4 , 100 mM NaCl, pH 7.5) with 0.5 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) (Solarbio, Beijing, China). The tissue was homogenized by a homogenizer in an ice bath at a speed of 10,000 rpm for 5 s. These steps were repeated several times (> 3) until a uniform tissue homogenate was obtained. The homogenate was centrifuged at 1400 g for 15 min at 4 • C (CR21GIII, Hitachi, Tokyo, Japan). A 20 mL of SM buffer was added to the pellet and homogenized in an ice bath at 10,000 rpm for 10 s, followed by centrifugation at 6000 g for 15 min at 4 • C. These two supernatants obtained in the above steps were combined, filtered through a 38 µm mesh, and the filtrate was centrifuged at 10,000 g for 25 min at 4 • C. The final supernatant was again filtered through a 38 µm mesh, mixed with an equal volume of SM buffer, and centrifuged at 40,000 g for 2 h at 4 • C (CP100WX; Hitachi, Tokyo, Japan) to pellet viral particles [49]. Finally, 500 µL of SM buffer was added to the pellet to suspend it.

Phylogenetic Analyses
For the genus Okavirus, the approach proposed by Mohr was employed using primers YH30 m/31 m to get a partial sequence of the open reading frame 1b (ORF1b) region to clarify the genotype [19]. For the genus Wenrivirus, we constructed four pairs of primers ( Table 2) with overlapping regions for the virus and obtained the full length of the RNAdirected RNA polymerase (RdRp) of the virus via Geneious version 2022.1.1 (Biomatters Ltd., Auckland, New Zealand) [50] splicing to obtain the sequence of the RdRp region of OWV1 in the sample. Then we used Geneious version 2022.1.1 to translate the obtained nucleotide sequences into amino acid sequences. For determining the evolutionary relationship of Wenrivirus in the samples collected in this paper, the entire RdRp region of Wenrivirus and some Phenuiviridae viruses were selected (Refer to another published article for virus names, etc. [24]). Multiple sequence alignments of Okavirus-related nucleic acid sequences were performed using the MUSCLE program in the MEGA version 11.0.10 software [51], and "Find Best DNA Models" were used to determine the most suitable models for Okavirus. Then based on the lowest Bayesian information criterion score, the Tamura-Nei model with discrete gamma distribution (TN93+G) was determined as the best model for Okavirus. Then based on the lowest Bayesian information criterion score, the LG with frequencies model gamma distributed with invariant sites (LG+G+I+F) was determined as the best model for Wenrivirus. The maximum likelihood phylogenetic tree was constructed using the best model for Okavirus and Wenrivirus. Phylogenetic testing was performed using the bootstrap method with 1000 replicates. All relevant sequences have been submitted to NCBI.

Testing Results of Pooled Samples
To understand the pathogen information in wild P. chinensis populations, we detected the pooled samples for seven known pathogens, including DIV1, IHHNV, CMNV, EHP, WSSV, IMNV, and TSV. The PCR and qPCR results ( Table 2) show that all shrimp samples collected in three years were negative for five pathogens, including CMNV, EHP, WSSV, IMNV, and TSV. The samples collected in 2020 showed DIV1 and IHHNV positive, and the samples collected in 2022 had IHHNV positive as well.

Testing Results of Individual Samples for YHVs and OWV1
All individual samples collected in three years were positive for two pathogens, including OWV1 and YHVs ( Figure S1, Table 3). The Ct value of the individual OWV1 detection in three years changed from 9.2 ± 0.1 to 35.0 ± 1.7 cycles, in which the batch average Ct value of OWV1 was 19.2 ± 7.6, 16.3 ± 6.6, and 25.0 ± 6.7 in 2020, 2021, and 2022 (with significant differences to 2020 and 2021), respectively. After sequencing and BLAST in the NCBI database, we confirm that all YHVs positives are YHV-8.

TEM Examination
TEM performed with ultrathin sections of lymphoid organs and gills of P. chinensis showed spherical to oval virus particles (80 nm-115 nm) of OWV1 in the cytoplasm of the gill tissue ( Figure 1A,B). However, no YHVs particle was observed in the gill and lymphoid organ tissues. Alternatively, the supernatant and pellet suspension from virus purification were observed with TEM. The results showed a few rod-like structures in the supernatant, with multiple linear structures but no envelope similar to our previous observation [49] ( Figure 1C,D).

Homology and Phylogenetic Analyses
To unambiguously identify the genotype of YHVs, we performed a phylogenetic analysis of the samples collected in this study. The products of the YHV ORF1b region obtained were sent for sequencing. After NCBI Nucleotide-BLAST (Table 4), it shows that the ORF1b part of the YHV isolates collected in this study had a 98.32%-99.24% similarity to the corresponding segment of YHV-8 20120706 (NC_048215.1). Furthermore, the results of the phylogenetic analysis show that YHVs sequenced in this study cluster with YHV-8 previously isolated from farmed P. chinensis populations [22] and are separated from the other seven genotypes (Figure 2). Therefore, YHV-8 was identified from the wild P. chinensis collected in this study. The obtained product of OWV1 RdRp region was sent for sequencing and translated into the amino acid sequence using Geneious version 2022.1.1. After NCBI Protein-BLAST (Table 5), it can be seen that the RdRp of the OWV1 isolates collected in this study had a 99.14-99.57% similarity to the corresponding segment of OWV1 (QHW05228.1). Moreover, the phylogenetic analysis results show that OWV1 isolates of wild P. chinensis cluster with the OWV1 reference sequence previously isolated from farmed P. chinensis [24] and then gather with Wenzhou shrimp virus 1 (WzSV-1) and Mourilyan virus (MoV) into the genus Wenrivirus (Figure 3).

Discussion
Shrimp diseases have become an important factor hindering the shrimp industry's green development of the shrimp farming industry [52]. This study revealed that multiple pathogens, including YHV-8, OWV1, DIV1, and IHHNV, can be detected in wild P. chinensis populations in the Yellow Sea. Furthermore, we observed coinfection with YHV-8 and OWV1 in all P. chinensis individuals by molecular diagnosis, phylogenetic analysis, and TEM. As the P. chinensis farming industry still largely relays on the wild P. chinensis broodstock [3], the consistently high prevalence of YHV-8 and the existence of DIV1 in wild P. chinensis reveal significant risks in the shrimp farming industry and the stock enhancement program for fishery resources of P. chinensis in East Asia.
Unlike human health, surveillance for aquatic animal health usually emphasizes the population-based approach. Detection of a lower prevalence in a larger population requires a larger sample size [53], which requires more tests. Therefore, pooling individual samples before a diagnostic test is a commonly used strategy. WOAH standard recommends a pooling rate of less than 5:1 [15], which may still result in a large number of tests for a large population. Our previous study using the TaqMan-qPCR method to evaluate the pooling rate revealed that a 50:1 pooling rate could have a similar diagnostic sensitivity to the pooling rate of 5:1 [54]. All the pooling modes from 5:1 to 150:1 have good diagnostic specificity. The tests of our present study using a pooling rate of no more than 20:1 should not significantly impact the diagnostic specificity and sensitivity.
Compared with the farmed samples, the accessibility of captured wild P. chinensis is much more difficult due to the declined fishery resources and uncertainty of seasonal migration caused by climate change [55,56]. Meanwhile, weak and diseased shrimp individuals may much easier be predated by carnivorous fishes or sink to the deep bottom. In facing these challenges, we used two different PCR methods targeting two sequence locations for each of the seven pathogens except YHVs and OWV1. This strategy can verify false results due to low specificity, low sensitivity, and contamination in sample preparation or PCR methods. Nevertheless, the PCR and qPCR results for each pathogen were highly coincident, which indicated that the molecular results were reliable.
Unlike the research on the viral diseases of farmed crustaceans, the reports on the spread of wild crustacean viruses and coinfection with multi-pathogen are still very limited [7]. Spann et al. [57] reported that coinfection with MoV and GAV is very common in diseased P. monodon. Notably, YHV-8 and GAV belong to Okavirus, and MoV and OWV1 belong to Wenrivirus. The similarity of the virus taxonomy and the coinfection in Spann et al.'s and our results raise the interesting issue of whether the Okavirus and Wenrivirus have a synergistic mechanism in the concomitance of coinfection. Teixeira-Lopes et al. [58] revealed that viral coinfection could regulate the viral load in the host, and there may be a negative correlation between the two viruses when they coexist. On the other hand, a number of studies reported that shrimp previously infected with IHHNV gained resistance to infection with WSSV and significantly reduced the mortality of shrimp. However, the survival rate of shrimp infected with TSV and YHV was significantly higher than that of shrimp infected only with YHV [10,59]. Further study on interactions between concomitant YHV-8 and OWV1 in P. chinensis populations may help to discover the deep infection mechanism of shrimp viruses.
The confirmatory diagnosis of a suspected case of infection with a specific pathogen is generally based on meeting the criterium of two or more independent tests in the WOAH Manual [15]. The diagnosis of infection with DIV1 also follows the same principle [60]. With qPCR and nested PCR, this study confirmed that wild P. chinensis in the Yellow Sea has been infected with IHHNV and DIV1. IHHNV natively originated from the Western Hemisphere. It was first detected from farmed P. vannamei in 2001 in China [61]. P. chinensis has not been listed as a susceptible species of IHHNV [15]. However, IHHNV positives have been detected from farmed P. chinensis in the National Target Surveillance Program of China in 2019 [62]. IHHNV detected in wild populations of P. chinensis implies the transmission risk of viruses between farmed and wild shrimp populations. DIV1 was detected in the wild P. monodon captured from the northeastern Indian Ocean [6]. DIV1 detected from wild P. chinensis indicated a broader virus spread in the wild. We did not find viral particles like DIV1 or IHHNV with TEM, which may be related to the selected tissues, centrifugal force, and low viral load. After subsequent virus purification, a large number of suspected nucleocapsid complex structures, likely YHV-8 virions, were observed in the supernatant. However, no complete complex structure was found, which might be caused by a large centrifugal force [49].
Okavirus isolates from captured wild P. chinensis cluster with YHV-8 previously identified from farmed P. chinensis [22] and separated from the other seven genotypes in the phylogenetic analysis. Similarly, the phylogenetic analysis of OWV1 showed that OWV1 isolates from wild and farmed shrimp [24] cluster together. These phylogenetic analyses provide information for tracing the source of YHV-8 and OWV1 infection in wild and farmed P. chinensis. Conclusively, the sequence similarity of the isolates from farmed and wild P. chinensis for both viruses implies the transmission risk of viruses between farmed and wild shrimp populations.
This study has detected multiple pathogens in wild P. chinensis, which reminds pathogenic risks in wild shrimp populations are nonnegligible. Wild shrimp used for broodstock must be strictly inspected and quarantined to select specific pathogen-free stocks [4,29,63]. Diseased and virus-carrying wild shrimp should be strictly prevented from entering the aquaculture systems. The national surveillance plan targeting specific pathogens may need to be extended to wild shrimp [64]. On the other hand, hatcheries for stock enhancement should implement strictly high-quality biosecurity measures to ensure pathogens from aquaculture systems will not be released into wild populations [3]. Implementation of biosecurity measures is critical not only for preventing disease risks in shrimp farms but also for securing the ecosystem of wild shrimp populations.

Conclusions
This study is the first to report the coexistence of multiple viruses, including DIV1, IHHNV, YHV-8, and OWV1. Notably, the results reveal a consistently high prevalence of coinfection with YHV-8 and OWV1 in wild P. chinensis populations and the transmission risk of viruses between wild and farmed populations. The findings imply that we should pay attention to the epidemiology of diseases in wild P. chinensis, implement disease surveillance on wild shrimp, and introduce biosecurity policies and measures to prevent disease risks both in shrimp farms and hatcheries for stock enhancement.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/v15020361/s1, Figure S1: Agarose gel electrophoresis of positive pathogens by PCR detection. Figure S2: Agarose gel electrophoresis of negative pathogens by PCR detection.