Isolation and the pathogenicity characterization of Decapod iridescent virus 1 (DIV1) from diseased Macrobrachium nipponense and its activation on host immune response.

The high morbidity and mortality of Macrobrachium nipponense occurred in several farms in China, with cardinal symptoms of slow swimming, loss of appetite, empty of intestine, reddening of the hepatopancreas and gills. The pathogen has been confirmed as Decapod Iridescent Virus 1 (DIV1), namely DIV1-mn, by molecular epidemiology, histopathological examination, TEM observation, challenge experiment, and viral load detection. Histopathological analysis showed severe damage in hepatopancreas and gills of diseased prawns, exhibited few eosinophilic inclusions and pyknosis, and TEM of diseased prawns revealed that icosahedral virus particles existed in hepatopancreas and gill, which confirmed the disease of the farmed prawns caused by the DIV1 infection. Besides, challenge tests showed LD50 of DIV1 to M. nipponense was determined to be 2.14 × 104 copies/mL, and real-time PCR revealed that M. nipponense had a very high DIV1 load in the hemocytes, gills and hepatopancreas after infection. Furthermore, qRT-PCR was undertaken to investigated the expression of six immune-related genes in DIV1-infected M. nipponense after different time points, and the results revealed UCHL3, Relish, Gly-Cru2, CTL, MyD88 and Hemocyanin were significantly up-regulated in hemocytes, gills and hepatopancreas, which revealed various expression patterns in response to DIV1 infection. This study revealed that DIV1 infection is responsible for the mass mortality of M. nipponense, one of the important crustacean species, indicating its high susceptibility to DIV1. Moreover, this study will contribute to exploring the interaction between the host and DIV1 infection, specifically in terms of understanding how M. nipponense recognizes and eliminates the invading of DIV1.

Immune responses and histopathological analyses of giant river prawn (Macrobrachium rosenbergii, De Man 1879) challenged with a sub-lethal dose of decapod iridescent virus 1 (DIV1) and chemical control investigation.

Decapod iridescent virus 1 (DIV1) is a lethal virus that has a significant influence on the shrimp and prawn culture industries. The mechanism through which infected prawns respond to the DIV1 virus is currently unknown. Here, we examined in detail the clinical signs, histopathology, and humoral, cellular, and immune-related gene responses after a sub-lethal dose of DIV1 during the acute infection period of 0-120 h post infection (hpi). Interestingly, at the end of the experiment, DIV1-infected prawns had black lesions on several external regions. The DIV1-infected prawns also exhibited few karyopyknotic nuclei in the gills and intestine tissues and exhibited increasing immunological responses, as revealed by significant increases in all examined parameters, including total hemocytes, phagocytosis, lysozyme, and overall bactericidal activity, from 6 to 48 hpi. In addition, between 72 and 120 hpi, all immune response activities of DIV1-infected prawn were impaired compared with those of normal prawns, indicating negative impacts on immunological parameters. A viral load analysis of various tissues by qPCR indicated that hemocytes were the dominant initial viral target tissues, followed by the gills and hepatopancreas. An expression analysis of crucial immune-related genes by qRT‒PCR revealed various expression patterns in response to DIV1 infection; in particular, fold changes in the relative expression of anti-lipopolysaccharide factors (ALFs), prophenoloxidase (proPO), lipopolysaccharide and ß-1,3-glucan binding protein (LGBP) were observed. Additionally, five common chemicals, calcium hypochlorite [Ca(OCl)2] at 16.25-130 ppm, hydrogen peroxide (H2O2) at 8.75-70 ppm, povidone iodine (PVP-I) at 3-24 ppm, benzalkonium chloride (BKC) at 20-160 ppm, and formalin at 25-200 ppm, had a significant effect on the killing of DIV1 particles in vitro within 24 h after exposure. These data will be helpful for determining the health status and immune defense mechanisms of giant river prawns during DIV1 infection periods. The study performed the first application of very common disinfectants, and the obtained information will be useful for implementing effective strategies to prevent and control DIV1 infection in both hatchery and grow-out ponds.

Rapid detection of Decapod iridescent virus 1 (DIV1) by recombinase polymerase amplification.

A recombinase polymerase amplification (RPA) assay was established for the rapid detection of Decapod iridescent virus 1 using primers targeted to the virus's ATPase gene (ORF114R). Optimization experiments showed that the optimal amplification temperature of the RPA assay was 37 °C and that the reaction could be completed within only 15 min. The target band of 15 min. is bright enough. In order to shorten the operational reaction time, consequently, 15 min was the optimal amplification time for our new RPA assay for DIV1. Specificity tests showed that the RPA assay did not exhibit any cross-reactivity with other shrimp pathogens(TSV, MrNV, YHV-1, WSSV, EHP, AHPND, EHNV, RSIV, RGV and IHHNV). Sensitivity tests further showed that the detection limit of the new RPA assay was 200 copies/50 µL, indicating that this assay was more sensitive than a nested polymerase chain reaction (PCR) method. A total of 509 clinical samples were assayed using the RPA and the PCR assays; analysis showed that the RPA method could detect weak-positive samples more effectively than the PCR method. Collectively, these findings indicated that the RPA assay was fast, simple, specific, sensitive and has significant potentials for clinical and on-site testing.

Establishment of a real-time Recombinase Polymerase Amplification (RPA) for the detection of decapod iridescent virus 1 (DIV1).

A rapid and simple real-time recombinase polymerase amplification (RPA) assay was developed to detect decapod iridescent virus 1 (DIV1). The assay was developed using optimized primers and probes designed from the conserved sequence of the DIV1 major capsid protein (MCP) gene. Using the optimized RPA assay, the DIV1 test was completed within 20 min at 39 ℃. The RPA assay was specific to DIV1 with a detection limit of 2.3 × 101 copies/reaction and there was no cross-reactivity with the other aquatic pathogens (WSSV, IHHNV, NHPB, VpAHPND, EHP, IMNV, YHV-1 and GAV) tested. Four out of 45 field-collected shrimp samples tested positive for DIV1 by real-time RPA. The same assay results were obtained by both methods. Thus, the real-time RPA assay developed could be a simple, rapid, sensitive, reliable and affordable method for the on-site diagnosis of DIV1 infection and has significant potential in helping to control DIV1 infections and reduce economic losses to the shrimp industry.