Natural Infection of Covert Mortality Nodavirus in Small Yellow Croaker in Coastal Water

Covert mortality nodavirus (CMNV) is an alphanodavirus mainly infecting marine shrimp and co-inhabiting organisms in aquaculture ponds. To evaluate the possibility of CMNV prevalence in the wild fish species, epidemiological survey of CMNV infection in Larimichthys polyactis, the dominant species in the Yellow Sea and East China Sea, were conducted in the present study. We performed CMNV RT-LAMP assay in samples of L. polyactis collected in August 2018 and 2019 and found that CMNV prevalence was 18% and 7%, respectively. The nucleotide sequences of both RdRp and capsid protein genes of CMNV from L. polyactis were 99% similar to those of CMNV isolated from shrimp. CMNV-positive L. polyactis exhibited necrosis of cardiac muscle, oocytes loosely arranging, severe cytoplasmic vacuolation of hepatocytes, moderate pyknosis of brain pyramidal cells, degenerate renal tubular cells with ill-defined margins, and declined spleen cells in the histological examination. Moreover, CMNV-positive signals were further observed in pyramidal cells of the brain, the cortical area of the kidney, oocyte growth rings, and in necrotic tissues of cardiac muscle, liver, and spleen in the in situ hybridization assay. The results revealed that CMNV had colonized in the wild populations of L. polyactis and the ecological risk of CMNV spread and epidemic in wild fish in the coastal water was non-negligible.


INTRODUCTION
Viral covert mortality disease (VCMD), also being named as running mortality syndrome (RMS), impacted greatly on the shrimp farming industry in the Southern East Asia and China in the past decade (Pooljun et al., 2016;Thitamadee et al., 2016;Zhang et al., 2017;Varela, 2018). Covert mortality nodavirus (CMNV), an alphanodavirus, was determined to be the causative agent for VCMD Zhang, 2019). CMNV could infect the most economic shrimp species including Penaeus vannamei, P. monodon, P. chinensis, P. japonicus, as well as a variety of co-inhabiting crustaceans in the shrimp ponds Liu et al., 2019). Recently, CMNV has been reported to switch its hosts to fish by cross-species transmission and could infect teleosteans, such as the gold fish Carassius auratus, olive flounder Paralichthys olivaceus, and Abe's mangrove goby Mugilogobius abei Wang et al., 2019). However, the CMNV prevalence in the wild teleost species has not been well investigated and evaluated yet.
The small yellow croaker (Larimichthys polyactis), an important commodity species in China, Japan, and South Korea, is widely distributed in the Bohai sea, Yellow sea, and East China Sea (Guo et al., 2006;Li et al., 2006;Tian, 2007;Shan et al., 2017). L. polyactis was known as one of the "four major fishery products" in the East China Sea. However, the volumes of L. polyactis landings continuously declined since the 1970s, due to human activities and environmental degradation (Jin, 1996). Although the resource of L. polyactis had recovered significantly since the 1980s under the protection measures of the Chinese government and the scientific community, the landings were still decreasing (Cheng et al., 2004;Lin, 2004;Guo et al., 2006). Recently, its population showed a tendency to shrink, and adult individuals tended to show characters of reduced size and early maturation (Jin, 1996;Qi et al., 2020). In view of the key role of its playing in the marine ecology, the L. polyactis has been the focus of research on fish species in the Yellow Sea and northern East China Sea. Biological characteristics, ecological habits, and stock assessment of L. polyactis had been well reported (Liu, 1990;Jin, 1996;Xue et al., 2004;Guo et al., 2006;Wang and Sun, 2006;Zhang et al., 2008;Shan et al., 2017). A few studies have focused on its diseases, let alone how the disease affects the wild L. polyactis population.
In the present study, we first conducted a systematic investigation of CMNV presence and infection in L. polyactis from the Yellow Sea and northern East China Sea through molecular detection, histopathology, and in situ hybridization (ISH). The results revealed that CMNV, an aquaculture animal virus, had colonized in the wild populations of L. polyactis in the investigated seas.

Sample Collection
In August 2018 and August 2019, bottom trawl samplings were conducted at 120 designated sites (south of 38 • 20 N, west of 120 • 30 E, Figure 1) in the Yellow Sea and northern East China Sea. Three to five individuals of L. polyactis (Figure 2A) were sampled at every designated sampling site. From each individual, the tissues of liver, kidney, spleen, brain, heart, and ovary (if available) were dissected, and half of the tissues were preserved in 4% paraformaldehyde (4% PFA) solution (Sinopharm, Beijing, China) for histopathological and in situ hybridization analysis. Another half was further divided into two parts, one part was preserved in RNAstore solution (Tiangen, Beijing, China) for RT-PCR analyses, the other part was smeared on Whatman FTA R Elute card (GE Healthcare Life Sciences, Marlborough, MA, United States) for RT-LAMP analyses. The tissue-smeared FTA R cards were air dried for 30 min at room temperature, and then stored at -20 • C.

Detection of Covert Mortality Nodavirus by RT-LAMP
From each of the tissue-smeared FTA R card, an area (about 2 mm 2 ) was cut off and immersed into TE buffer to elute RNA. The eluted RNA was denatured at 70 • C for 5 min, immediately placed on ice, and used as the template for CMNV RT-LAMP assay with the protocol described by Zhang et al. (2017).

RNA Extraction
The RNA was extracted from the CMNV RT-LAMP-positive RNA samples by using the Trizol RNA Extraction kit (Tiangen, Beijing, China), and then the amount and purity of the purified RNA was measured using the Nanodrop 2000 (Thermo Scientific, Waltham, MA, United States).

Primer Design, cDNA Synthesis, and PCR Amplification
To determine the targeted gene sequence of CMNV from the RT-LAMP-positive L. polyactis, six pairs of primers (Table 1) were designed based on the CMNV RNA1 and RNA2 genome segments (GenBank accession numbers MT270123 and MT270124) using the software Primer Premier 5 (Premier Biosoft, San Francisco, CA, United States).
cDNA was synthesized from the extracted RNA with SMART R MLV-reverse transcriptase (TaKaRa) according to the procedure described by Xu et al. (2020).
PCR was carried out in a 25-µl reaction mixture containing 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 4 mM MgCl 2 , 1.5 mM dNTP, 0.2 µM primers, 2 µl of cDNA, and 2.5 U of TaKaRa EX Taq DNA polymerase (TaKaRa). The PCR was conducted according to a previous report (Xu et al., 2020) with modifications of annealing temperatures and extension time described in Table 1. DNA amplifications were performed for 35 cycles in all the reactions.

Sequence Analysis
A total of 20 viral genomic sequences (Supplementary Table 1) from the National Center for Biotechnology Information 1 were downloaded and aligned with that of CMNV from L. polyactis by ClustalW as implemented (Tamura et al., 2011) using default settings. The phylogenetic tree was generated by the neighbor-joining method with bootstrap analysis (1,000 replicates) using MEGA 5.0.

Histopathological Section
The fish tissues were first preserved in 4% PFA for 12-24 h, and then transferred and stored into 70% ethanol. Paraffin sections were prepared and stained with hematoxylin and eosin-phloxine (H&E) according to the standard procedures (Lightner, 1996).

In situ RNA Hybridization
After examinng the H&E stained sections and determining the location of the lesions, the corresponding unstained sections were subjected to ISH with digoxigenin (DIG)-labeled CMNV RNA probe. In situ RNA hybridization was conducted according to the procedures reported (Piette et al., 2008;Chen et al., 2014). Final detection was performed with anti-digoxigenin antibody conjugated to alkaline phosphatase that was visualized using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NTB). Nuclear Fast Red solution (Sigma-Aldrich, St. Louis, United States) was dropwise added onto the surface of the sections and stained the nucleus for 2 min. The sections were then visualized and photographed with a Nikon Eclipse E80i microscope (Nikon Co., Tokyo, Japan).

Prevalence of Covert Mortality Nodavirus in Larimichthys polyactis
In August 2018, a total of 76 L. polyactis samples were collected from 30 sites out of the 120 designated sampling sites. The results of RT-LAMP analysis showed that 14 samples (18%) were detected to be CMNV positive ( Figure 2B). The CMNV-positive  samples were collected from 13 different sites, and the positive rate of sampling sites was determined to be 40% (Figures 2B,C).
In August 2019, a total of 102 L. polyactis samples were collected from 29 out of the 120 designated sampling sites. These 29 sites were different from those of 2018 ( Figure 2C). RT-LAMP analysis results showed that seven samples (7%) from five sites were positive for CMNV (Figure 2B), and the positive rate of sampling sites was determined as 17% (Figures 2B,C).
In general, whether in 2018 or 2019, L. polyactis with positive CMNV were smaller than those with negative CMNV.

Histopathological Observation of Tissues From Covert Mortality Nodavirus (CMNV)-Positive Larimichthys polyactis
The tissues of L. polyactis that were determined to be positive for CMNV in RT-LAMP and RT-PCR assay were processed for histological evaluation. The histopathological observation revealed obvious histopathological changes and lesions in the tissues of the brain, kidney ovary cardiac muscle, liver, and spleen. Cerebral cortical tissue was severely vacuolated, and moderate karyopyknosis was noticed in pyramidal cells (Figure 5A). In the kidney, tubular degeneration and unclear boundaries, and vacuolar degeneration of the epithelial cells of the tubular wall were found. Meanwhile, the renal parenchyma showed diffuse hemorrhage, erythrocytes increased, and inflammatory cells exuded (Figure 5B). The oocytes in the ovary were loosely arranged ( Figure 5C). Mild muscle fragmentation tending toward muscular lysis was noticed in cardiac muscle (Figure 5D). In the liver, the shape of the hepatocytes was deformed, and severe cytoplasmic vacuolation occurred in almost all hepatocytes ( Figure 5E). Cells in the spleen were degenerated, and hemosiderin was obviously precipitated, forming "nodules" (Figure 5F). However, no typical lesions were observed in the above tissues of CMNV-free L. polyactis ( Supplementary  Figures 2A-F).

In situ Hybridization Analysis of Tissues From Covert Mortality Nodavirus-Positive Larimichthys polyactis
The ISH assay results showed that purple hybridization signals of the CMNV probe could be observed in the pyramidal cells of the brain (Figure 5A), cortical tissue in the kidneys (Figure 5B), growth ring of the oocytes (Figure 5C), necrotic cardiac muscle cells (Figure 5D), necrotic hepatocytes (Figure 5E), and cells in the spleen (Figure 5F). However, no positive hybridization signals were detected on the sections without the addition of a CMNV DIG-labeled RNA probe (Supplementary Figures 3A-F).

DISCUSSION
Up to now, CMNV had been prevalent in farming shrimp for 10 years and heavily attacked the production of farming shrimp. High incidence of CMNV infection in coastal ponds was causing increasing concerns of its spread to wild populations in the near seas. To evaluate the possibility of CMNV infection in the wild fish species, the infection and prevalence of CMNV in populations of wild L. polyactis, the dominant species in the Yellow Sea and the East China Sea, were investigated in the present study.
The investigation found that 18% (2018) and 7% (2019) of the L. polyactis samples collected from the Yellow sea and East China sea were CMNV positive. The rates of CMNVpositive sampling sites were determined to be 43.33 and 13.33% in 2018 and 2019, respectively. The results indicated that CMNV had colonized wildly in the Yellow Sea and East China Sea, but its prevalence in L. polyactis has declined in 2019 compared with that in 2018. Also, its large-scale spread might be related to the annual migration of L. polyactis populations in the Yellow Sea and East China Sea. The multiple sequence alignment and phylogenetic tree analysis showed that CMNV isolated from L. polyactis was highly homologous to the CMNV isolate from cultured Penaeus vannamei. It could be deduced that L. polyactis-CMNV isolate might originate from the aquacultured shrimp. Wang et al. (2019) reported that CMNV infection in goldfish Carassius auratus could cause nervous tissue vacuolation and cardiac muscle necrosis. Zhang et al. (2018) found that CMNV infection in M. abei could induce extensive skeletal muscle necrosis, nervous tissue vacuolation in the retina of the eyes and the cerebellum of the brain. In this study, CMNV infection was revealed in multiple tissues of the L. polyactis individuals, including the brain, cardic muscle, liver, kidneys, spleen, and ovaries, by ISH analysis. The liver, the largest digestive gland of fish, has the functions of secretion, detoxification, storage, and excretion. Kidney and spleen are the main immune organs of fish. Hence, CMNV infection could affect the digestion and immune function of L. polyactis. Vertical transmission of CMNV has been reported in redgetail prawn Exopalaemon carinicauda . A high percentage of oocytes were found to be infected by CMNV, which had raised our concerns about the possibility of vertical transmission of CMNV in L. polyactis populations. However, L. polyactis is a benthic fish species, which will die due to the rupture of the swim bladder when caught during fishing (Liao, 1995), making it very difficult to obtain a live fish for the challenge test. Moreover, the technology of artificial cultivation and breeding of L. polyactis has not yet been achieved. Therefore, it is hard to perform challenge experiments to prove the vertical transmission ability of CMNV in L. polyactis at present.
The results of the present study raise concerns about the potential effects of CNNV on fish populations in coastal waters. A similar beta-nodavirus-like agent was suspected to be responsible for the mortality of mullets Liza saliens in the Caspian Sea (Mohammad et al., 2014;Maryam et al., 2015). Whether the decline in landings of L. polyactis was associated with CMNV infection remains to be determined. Lately, it was found that L. polyactis had gradually replaced anchovy (Engraulis japonicus) in the Bohai sea ecosystem and became an important food source for many larger predatory fish (Zhang, 2018). Although there was no direct evidence to prove the horizontal transmission ability of the CMNV from L. polyactis to other co-inhabiting fish or shrimp, the risk of CMNV propagation and spread in the wild fish species in the coastal waters could not be negligible considering that L. polyactis is a key prey in the Bohai sea ecosystem, especially preyed on by predators, and it might enhance the possibility of CMNV propagation and spread in the wild fish species in the coastal waters.
In conclusion, the molecular, histopathological, and epidemiological evidences in the present study indicated that CMNV had colonized in the populations of L. polyactis, one of the dominant species in the Yellow Sea and East China Sea. In addition, the ecological risk of CMNV spread and epidemic in wild fish in the coastal waters is non-negligible.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by the Ethics Committee of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences.

AUTHOR CONTRIBUTIONS
QZ, CY, and XS designed the experiments. TX, YL, and JH executed the experiments. TX and QZ analyzed the data. QW contributed to the sampling. TX wrote the manuscript. QZ, KT, and CY revised the manuscript. All authors interpreted the data, critically revised the manuscript for important intellectual contents, and approved the final version.