Large icosahedral viruses belonging to the Iridoviridae family have diameters between 120 and 350 nm, and some of them can reach 350 nm (e.g. genus Lymphocystivirus) (King et al., 2011). This family of viruses is distinguished by the broad range of vertebrate and invertebrate hosts it can infect, including fish, amphibians, and reptiles as well as invertebrates (such as insects) (King et al., 2011). Based on the particle size, host range, and the presence of methyltransferase, International Committee on Taxonomy of Viruses (ICTV) classifies the Iridoviridae family as Iridovirus, Chloriridovirus, Daphniairidovirus, Decapodiridovirus, Ranavirus, Lymphocystivirus, and Megalocytivirus, a total of 7 genera (ICTV, 2021). In farmed shrimps and crabs, a newly discovered virus called Decapod iridescent virus 1 (DIV1) has resulted in significant economic losses (Xu et al., 2016; Qiu et al., 2017; Qiu et al., 2019a; Qiu et al., 2021; Qiu et al., 2022) and was designated as the only species of the genus Decapodiridovirus within the Iridoviridae family by the ICTV (ICTV, 2019). DIV1 is a double-stranded DNA (dsDNA) virus with a 166 kbp-long genome (Li et al., 2017; Qiu et al., 2018b). It has two original isolations, Cherax quadricarinatus iridovirus (isolate CQIV CN01) and Shrimp hemocyte iridescent virus (isolate SHIV 20141215) (ICTV, 2019). According to the current database, only these two original isolates have completed whole-genome sequencing with very high sequence similarity. Previous studies have confirmed that DIV1 susceptible hosts meeting the World Organization for Animal Health (WOAH) criteria (WOAH, 2022) include P. vannamei, P. monodon, Macrobrachium rosenbergii, M. nipponense, Exopalaemon carinicauda, Procambarus clarkii, Cherax quadricarinatus, Portunus trituberculatus (Xu et al., 2016; Qiu et al., 2017; Qiu et al., 2019a; Chen et al., 2019; Srisala et al., 2021; Qiu et al., 2022). Eriocheir sinensis and Pachygrapsus crassipes, two species of crab, were intramuscularly injected with DIV1 in an invasive experimental challenge (Pan et al., 2017), rendering them unable to be designated as susceptible species just yet. P. chinensis, M. superbum, Nereis succinea, Pomacea canaliculata, Plexippus paykulli, and a few other cladocera can all have DIV1 detected by PCR (Qiu et al., 2017; Qiu et al., 2018c; Qiu et al., 2019a; Qiu et al., 2019b; Qiu et al., 2021), however the infection status has not been determined at the histology level. Therefore, more research is required to determine whether these species are susceptible hosts, vectors, or carriers.

Crustaceans provide nutritious and high-quality protein, unsaturated fatty acids, vitamin D, and other micronutrients for human beings. With the increase in market demand, the scale of shrimp and crab farming is expanding year by year. However, due to the lack of biosecurity awareness in the rapid rise of intensive aquaculture, disease has become one of the main factors restricting the increase of shrimp and crab production. The kuruma shrimp, Penaeus japonicus, is one of the most essential cultured and captured shrimps in the world and is farmed in Southeast Asian nations such as China, Japan, Korea, Australia, and others (Hewitt and Duncan, 2001; Ren et al., 2020; Cao et al., 2022). Due to its excellent desiccation resistance, tasty flavor, and abundant nutritional value, it is regarded as one of the most commercially important species of the Penaeidae family (Zhang et al., 2022). In 2021, China’s annual P. japonicus aquaculture production totaled 44,548 tons (Bureau of Fisheries et al., 2022). DIV1 was detected in samples of P. japonicus raised in China, according to target surveillance, in 2017 and 2018 (Qiu et al., 2018c; Qiu et al., 2019b). He et al. (2021) infected healthy P. japonicus with DIV1 by intramuscular injection as an invasive challenge. DIV1 had an inhibitory effect on the immune enzyme activity of shrimp, according to the enzyme activity study and transcriptomics analysis, and HSP70, C-type lectins, and caspase may be crucial defenses against DIV1 infection (He et al., 2021). The intramuscular injection, however, does not satisfy the WOAH requirements for listing species susceptible to a pathogen because it is not a technique that mimics natural transmission channels. Therefore, it is necessary to confirm further that P. japonicus is susceptible to DIV1.

It is necessary to identify a pathogen’s susceptible hosts for international trade and disease control (Qiu et al., 2022). Therefore, healthy kuruma shrimp were experimentally infected using invasive and non-invasive experimental techniques to test P. japonicus’ susceptibility to DIV1. In this investigation, clinical observation, pathogen detection, and histology were used to examine the infection status of the individuals. Additionally, we conducted the DIV1 analysis on the cultivated P. japonicus in a few Chinese coastal provinces.

International Committee on Taxonomy of Viruses (ICTV) now classifies the Iridoviridae family comprising 7 genera, including Chloriridovirus, Daphniairidovirus, Decapodiridovirus and Iridovirus, belonging to Betairidovirinae, which mainly infecting insects and crustaceans, while Lymphocystivirus, Megalocytivirus and Ranavirus, belong to the Alphairidovirinae, which mainly infecting amphibians, reptiles and fish (ICTV, 2021). The infection with Decapod iridescent virus 1 (iDIV1) is an important emerging disease, often causing mortalities higher than 80%. It has been included in the Quarterly Aquatic Animal Disease Report (QAAD) by the Network of Aquaculture Centres in Asia-Pacific (NACA) in 2016 and listed by the World Organization for Animal Health (WOAH) in 2021. China has classified iDIV1 as a Class II animal pandemic disease in 2022.

According to the Criteria for Listing Species as Susceptible to Infection with a Specific Pathogen of WOAH, species of aquatic animals are considered susceptible to infection with a pathogenic agent when the presence of a multiplying or developing pathogenic agent has been demonstrated by the occurrence of natural cases or by experimental exposure that mimics natural transmission pathways (WOAH, 2022). Therefore, Pr. clarkii, M. rosenbergii, P. vannamei, P. monodon, M. nipponense and C. quadricarinatus have been shown to be susceptible hosts of DIV1 by the occurrence of natural cases (Xu et al., 2016; Qiu et al., 2017; Qiu et al., 2019a; Srisala et al., 2021), and E. carinicauda and Po. trituberculatus has been confirmed by experimental exposure that mimics natural transmission pathways (Chen et al., 2019; Qiu et al., 2022).

China’s targeted surveillance detected DIV1 in farmed P. japonicus samples in 2017 and 2018 (Qiu et al., 2018c; Qiu et al., 2019b). Ren et al. (2022) also detected DIV1 in cultured P. japonicus samples collected from Dalian, Liaoning Province of China, in 2020 through epidemiological investigation. He et al. (2021) challenged P. japonicus with DIV1 by intramuscular injection, which can lead to infection in healthy individuals. Although the above reports add to the evidence that P. japonicus may be susceptible to DIV1, there is no complete confirmation that P. japonicus is a susceptible host of DIV1. In this study, healthy P. japonicus were artificially infected with SHIV (an original isolate of DIV1) by per os (the pathway that mimics natural transmission) and intramuscular injection (invasive pathway). A variety of methods, nucleic acid detection, histopathological analyses, TEM, and ISDL, were then used to confirm whether P. japonicus was successfully infected with DIV1 and whether DIV1 is multiplying or developing in the cells. Through the present research, P. japonicus could be verified as a susceptible host of DIV1 because it fully complied with the Criteria for Listing Species as Susceptible to Infection with a Specific Pathogen of WOAH (WOAH, 2022).

Compared with the typical ‘white head’ of M. rosenbergii infected with DIV1, the penaeid shrimp is often thought to present with some atypical clinical signs, including an empty guts and stomach, softshell, expanded and deeper pigmentation spots on the epithelium, and reddish body (Qiu et al., 2017; Qiu et al., 2019a; Qiu et al., 2021). In this study, in addition to anorexia and retardation, the most obvious appearance symptom of DIV1-infected P. japonicus was that almost all of the challenged individuals showed the evident reddish body, consistent with the clinical signs published by He et al. (2021). Similarly, previous reports have shown that many individuals of P. vannamei infected with DIV1 exhibit an evident reddish body (Qiu et al., 2017; Qiu et al., 2021; Guo et al., 2022). The clinical sign of reddish body have been reported in many cases of shrimp disease in China and other Asian countries, but the earlier etiology is thought to be mainly due to infection with V. parahaemolyticus or WSSV (Huang et al., 1995; Alapide-Tendencia and Dureza, 1997; Liu et al., 1999; Fan et al., 2006; Jayasree et al., 2006; Zheng et al., 2011; Felix et al., 2011; Cao et al., 2014). The clinical sign of reddish body is a general sign indicating an imbalance in shrimp, but it may be caused by several pathogens and other uninfectious factors. In addition, the hepatopancreas and digestive tract of the infected P. japonicus did not show significant abnormalities compared with the healthy individuals in appearance and dissection. It is worth noting that the lymphoid organ of infected P. japonicus showed a swollen and whitish appearance, which could be clearly observed in front of the hepatopancreas in the central longitudinal dissection of the shrimp. Therefore, like the ‘white head’ of M. rosenbergii, the swollen and whitish lymphoid organs of P. japonicus may also be regarded as the typical symptom of iDIV1, which can be used to assist the clinical diagnosis. Unlike prawns, species such as crabs or crayfish infected with DIV1 cannot easily observe signs in their internal organs from the outside due to their thick and opaque carapace (Xu et al., 2017; Qiu et al., 2019a).

At the histopathological level, unique characteristics of karyopyknosis and eosinophilic inclusions and minute basophilic stains were combined in lymphoid organs, hematopoietic tissues, and gills of diseased P. japonicus, which was additionally confirmed by the ISDL blue signals at the same site in these tissues. In contrast to hematopoietic tissues and gills, lymphoid organs indicate disorder of the lymphoid tubule matrix followed by aberrant morphology of the nucleus plus the presence of karyorrhectic and pyknotic nuclei, and such lesions are more easily observed at low magnification. These lesions in lymphoid organs have also been reported previously and can be used as a preliminary marker to speed up screening for DIV1 infections (Sanguanrut et al., 2021). The eosinophilic inclusions were also present in cytoplasm in hemocytes, the cells of hematopoietic tissues and lymphoid organs of DIV1-infected shrimp, crayfish and crabs (Qiu et al., 2019a; Qiu et al., 2021; Qiu et al., 2022), which is very similar to some reported cases caused by the putative iridovirus in penaeid shrimp Protrachypene precipua (Lightner and Redman, 1993). The karyopyknosis is similar to some fish cases caused by grouper sleepy disease iridovirus (GSDIV) (Mahardika et al., 2004) and iridovirus in African lampeye and dwarf gourami (Sudthongkong et al., 2002). For ISDL results, in addition to lymphoid organs, hemopoietic tissues, and gills, DIV1 positive signals were also detected in epithelial tissues, hepatopancreas tubules, and muscle, but the signals in these three tissues were more concentrated in the nucleus. However, upon closer inspection, no obvious abnormalities were found in any of the three tissues. Therefore, for penaeid shrimp samples, lymphoid organs and hematopoietic tissues are the most suitable tissues for the pathological diagnosis of iDIV1. Sanguanrut et al. (2021) reported that a preliminary diagnosis could be made through lymphoid organs, and a further comprehensive diagnosis could be made combined with hematopoietic tissues. From TEM, the agminate or dispersive DIV1 virions were observed in lymphoid organs, the nervous tissue, the stomach, and hemocytes of gills and the heart of diseased P. japonicus. In addition, the virogenic stroma and the process of nucleocapsid formation could be observed in the cytoplasm. Xu et al. (2016), also observed clearly icosahedral DIV1 particles in the cytoplasm of hematopoietic tissue cells of diseased C. quadricarinatus by TEM (Xu et al., 2016; Qiu et al., 2017; Qiu et al., 2019a; Qiu et al., 2021; Qiu et al., 2022). In this study, hematopoietic tissues of prawn individuals were difficult to be identified by naked eye, so hematopoietic tissues could not be obtained during TEM sampling. In TEM observation of target tissue of P. vannamei, M. rosenbergii and Po. tritomatus, virion formation took place in the cytoplasmic morphologically distinct regions, termed virogenic stromata and the assembly of nucleocapsid can be observed, which was consistent with the results in this paper (Qiu et al., 2017; Qiu et al., 2019a; Qiu et al., 2022). Notably, some dispersive DIV1 particles were also observed in neural tissues, but virus replication and assembly were not observed. In terms of lymphoid organs, histopathological observation showed disorganization of the lymphoid tubule matrix accompanied by abnormal morphology of the nuclei plus the presence of karyorrhectic and pyknotic nuclei after DIV1 infection, while TEM also showed a large number of viruses in and out of lymphoid organ cells and a large number of abnormal cells, which also explained the swollen and whitish appearance of lymphoid organs from the histopathological and cytopathological levels.

China’s targeted surveillance showed that DIV1 was detected in farmed P. japonicus samples in 2017 and 2018, and the positive rate was 7.7% (1/13 in samples) in both years (Qiu et al., 2018b; Qiu et al., 2019b). Ren et al. (2022) showed that the positive rate of DIV1 in cultured P. japonicus collected from Liaoning Province of China in 2020 was 5.3% (9/157 in samples). In this study, DIV1 test results of epidemiological P. japonicus samples from five provinces in 2022 showed that a total of 4 samples were DIV1 (MCP & ATPase) positive, with a total positive rate of 2.6% (4/153 in samples). According to the above test results of collected samples, the positive rate of DIV1 in the farmed P. japonicus samples was relatively low. However, considering that the above surveys are active surveillance and the samples with disease onset are not investigated, the prevalence of DIV1 in the farmed P. japonicus cannot be ignored. In the present study, considering 17 batches of cultured samples investigated were all healthy asymptomatic shrimp, to improve the accuracy of detection, two published real-time PCR assays (Qiu et al., 2018a; Qiu et al., 2020) targeting two different genes (MCP and ATPase) were selected to detect DIV1. Samples were judged to be DIV1 (MCP & ATPase) positive only if both assays were positive. The results of the two methods were significantly different, which showed that the positive rate of the real-time PCR method for the MCP gene was significantly higher than that of the method for ATPase. Since the P. japonicus samples collected in this paper were all healthy and asymptomatic prawns, the DIV1 in the samples may be in a state of low load carrying rather than infection. Therefore, the difference in sensitivity and the instability in the detection of low-load samples may be the main reason for the different detection results of the two real-time PCR methods. Besides, the above results suggest that at least two gene fragments of DIV1 genome should be detected in the monitoring investigation of healthy samples, and only when two DIV1 gene fragments are detected positive can a positive result be given. In the Aquatic Manual of WOAH, the definition of confirmed case of pathogens such as WSSV and IHHNV is based on the detection of two gene fragments of pathogens at the same time (WOAH, 2021). Since the WOAH manual of iDIV1 has not yet been formulated, such double gene detection criteria should also be used in the identification of confirmed cases.

Similar to the results of surveillance surveys in previous reports (Qiu et al., 2018b; Qiu et al., 2019b; Ren et al., 2022), the detection rate of DIV1 in cultivated P. japonicus samples investigated in this paper was also relatively low. However, since these surveys did not fully cover the breeding ponds with clinical signs, and P. japonicus has also been confirmed to be susceptible hosts of DIV1, which can be infected through natural ingestion, therefore, the potential threat of DIV1 to P. japonicus aquaculture should raise alarm.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

LQ and JH designed the experiments. X-MG, Y-HF, and J-YX carried out the experiments. X-YR contributed to the shrimp collection and sampling. LQ contributed to the funding of research. LQ, X-MG, and JH wrote the manuscript. All authors contributed to the article and approved the submitted version.