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Simple Summary: Decapod iridescent virus 1 (DIV1), a new virus, has posed significant challenges to the Macrobrachium nipponense industry, and little is known about the mechanism of the host response to DIV1 infection. In order to understand the immune response of M. nipponense to DIV1 infection, transcriptome analysis was conducted on the hepatopancreas of M. nipponense to examine the global expression patterns at 48 hpi with DIV1. Our results showed that multiple immune-related genes (e.g., lectin, dorsal, wnt6, hsp70, integrin and caspase) may play a significant role in M. nipponense against DIV1 infection, and the immune-related signaling pathways were significantly activated. This study has the potential to enhance our comprehension of the immune response of M. nipponense to DIV1 infection, which is advantageous for future treatment strategies for disease caused by DIV1. Macrobrachium nipponense is a significant cultivated species in China. However, decapod iridescent virus 1 (DIV1), as a newly discovered crustacean-lethal virus, has resulted in significant financial losses for the M. nipponense industry. In order to examine the immunological response of M. nipponense to DIV1, we conducted transcriptome analysis of the hepatopancreas from M. nipponense infected with DIV1 using RNA-seq. RNA sequencing analysis identified a combined total of 41,712 assembled unigenes, and 7014 genes that showed differential expression were identified in the group infected with DIV1, compared to the control group. Among these DEGs, 3952 were found to be up-regulated, while 3062 were down-regulated; many well-characterized DEGs were involved in innate immune defense, particularly involving the C-type lectin receptor signaling pathway, complement and coagulation cascades, phagosome, lysosome and PPAR signaling pathway. Moreover, the expression levels of well-known immune-related genes (dorsal, wnt6, lectin, caspase, integrin, hsp70) in the hepatopancreas and hemolymph were investigated by Quantitative real-time PCR (qRT-PCR), and the findings demonstrated a significant increase in gene expression in the hepatopancreas and hemolymph at various time points after infection. The results acquired in this study offered further comprehensive understanding of the immunological response of M. nipponense to DIV1 infection. [ABSTRACT FROM AUTHOR]

As a newly discovered virus, Decapoda iridovirus 1 (DIV1) can cause a mortality rate of up to 100% in crustaceans, leading to huge economic losses. At present, there is no effective prevention and control measures for this disease. In the present study, the specific primers targeting highly conserved regions of MCP gene were designed, and then a quantitative real-time PCR method was established. The results indicate that DIV1 quantitative real-time PCR established has good specificity and does not cross react with other pathogens including white spot syndrome virus (WSSV), infectious subcutaneous and hematopoietic necrosis virus (IHHNV) and Vibrio parahaemolyticus induced acute hepatopancreatic necrosis disease (VpAHPND). The real-time PCR was capable of detecting DIV1 DNA at a minimum concentration of 10 copies/µL within 34 cycles. The method has good repeatability, with intra group and inter group coefficients of variation both less than 2%. Thirty-two clinical samples were assessed using both the real-time PCR and conventional PCR. The results shown real-time PCR we established are more sensitive than conventional PCR. In conclusion, this method has strong specificity, stable repeatability, and high sensitivity, providing technical support for clinical diagnosis, epidemiology investigation and monitoring of DIV1. [ABSTRACT FROM AUTHOR]

Macrobrachium nipponense is a significant cultivated species in China. However, decapod iridescent virus 1 (DIV1), as a newly discovered crustacean-lethal virus, has resulted in significant financial losses for the M. nipponense industry. In order to examine the immunological response of M. nipponense to DIV1, we conducted transcriptome analysis of the hepatopancreas from M. nipponense infected with DIV1 using RNA-seq. RNA sequencing analysis identified a combined total of 41,712 assembled unigenes, and 7014 genes that showed differential expression were identified in the group infected with DIV1, compared to the control group. Among these DEGs, 3952 were found to be up-regulated, while 3062 were down-regulated; many well-characterized DEGs were involved in innate immune defense, particularly involving the C-type lectin receptor signaling pathway, complement and coagulation cascades, phagosome, lysosome and PPAR signaling pathway. Moreover, the expression levels of well-known immune-related genes (dorsal, wnt6, lectin, caspase, integrin, hsp70) in the hepatopancreas and hemolymph were investigated by Quantitative real-time PCR (qRT-PCR), and the findings demonstrated a significant increase in gene expression in the hepatopancreas and hemolymph at various time points after infection. The results acquired in this study offered further comprehensive understanding of the immunological response of M. nipponense to DIV1 infection.

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), classified as pattern recognition receptors (PRRs), play crucial roles in innate immune responses. However, research on this topic in crustaceans remains limited. In the present study, 27 NLR family members were identified and characterized based on the redclaw crayfish genome. Gene structure, conserved domain, and motif analyses revealed that these NLR genes were classified into four subfamilies with three major domains, and phylogenetic and structural analyses further confirmed their evolutionary conservation. qRT-PCR analysis showed that the relative expression levels of several NLR genes changed significantly under the DIV1 challenge. More importantly, in vivo silencing of NAWD1 gene in Cherax quadricarinatus promoted DIV1 replication . These findings, together with those of previous studies, suggest that NLR genes in crustaceans may play a key role in immune responses to viral infections.

Abstract Objectives This study was to identify and characterize Decapod iridescent virus 1 (DIV1) in the outbreaks reported in two whiteleg shrimp farms and one black tiger shrimp farm located in northern Taiwan in 2020. Methods The histopathology, electron microscopy and polymerase chain reaction (PCR) specific for the DIV1 were used to identify the virus, and the phylogenetic analysis was performed by comparing the major capsid protein gene fragment of DIV1s from Taiwan with reference sequences of the family Iridoviridae. Results DIV1 was identified by diagnostic PCR and caused mild mortality (20%) in cultured Penaeus monodon and high mortality (100%) in cultured whiteleg shrimp. Cultured P. monodon was first found to be infected with DIV1 through natural route of infection. Histopathological examination showed dark‐eosinophilic cytoplasmic inclusions in the degenerative cells of targeted hematopoietic tissues. For electron microscopy, a non‐enveloped virus particle was observed from homogenates of mixed target organs through negative staining with a diameter of 112±2 nm. Nucleotide sequences of DIV1 isolates from the Taiwanese outbreak are 100% identical to those from the PRC. Conclusions Based on the clinical evidence, mortality rates, histopathology, electron microscopy examinations and phylogenetic analysis, it is believed that DIV1 is the causative agent of the outbreak. This is the first report of DIV1 in cultured shrimp in Taiwan. The emergence of DIV1 signals a warning to shrimp aquaculture farmers worldwide.

Objectives: This study was to identify and characterize Decapod iridescent virus 1 (DIV1) in the outbreaks reported in two whiteleg shrimp farms and one black tiger shrimp farm located in northern Taiwan in 2020. Methods: The histopathology, electron microscopy and polymerase chain reaction (PCR) specific for the DIV1 were used to identify the virus, and the phylogenetic analysis was performed by comparing the major capsid protein gene fragment of DIV1s from Taiwan with reference sequences of the family Iridoviridae. Results: DIV1 was identified by diagnostic PCR and caused mild mortality (20%) in cultured Penaeus monodon and high mortality (100%) in cultured whiteleg shrimp. Cultured P. monodon was first found to be infected with DIV1 through natural route of infection. Histopathological examination showed dark‐eosinophilic cytoplasmic inclusions in the degenerative cells of targeted hematopoietic tissues. For electron microscopy, a non‐enveloped virus particle was observed from homogenates of mixed target organs through negative staining with a diameter of 112±2 nm. Nucleotide sequences of DIV1 isolates from the Taiwanese outbreak are 100% identical to those from the PRC. Conclusions: Based on the clinical evidence, mortality rates, histopathology, electron microscopy examinations and phylogenetic analysis, it is believed that DIV1 is the causative agent of the outbreak. This is the first report of DIV1 in cultured shrimp in Taiwan. The emergence of DIV1 signals a warning to shrimp aquaculture farmers worldwide. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Penaeid shrimp aquaculture is impacted by various diseases. However, most published studies on physiological responses to pathogens have focused on the changes in one or two tissues of shrimp infected by a single pathogen, or the effects of two pathogens infecting the shrimp in a single tissue. There has been limited systematic examination on the similarities and differences of immune responses in multiple tissues under various pathogen infection. Here, the transcriptomic changes of three immune tissues (gill, hepatopancreas and hemocytes) under the infection of white spot syndrome virus (WSSV), Vibrio parahaemolyticus acute hepatopancreatic necrosis disease (VPAHPND), and decapod iridovirus 1 (DIV1) were examined to provide new insights regarding the immune responses of the most important cultured shrimp, Penaeus vannamei. The results showed tissue-specific differences in the immune responses of shrimp tissues. The significant differentially expressed genes (DEGs) in gill are mainly related to environmental information processing and cellular processes. The DEGs in hemocytes are mostly involved in cellular processes, while those in hepatopancreas are primarily associated with metabolism. In addition, cytoskeleton-related proteins, MAPK signaling pathway, complement and coagulation level pathway, and thermogenesis may play key roles in the shrimp–pathogen interactions across tissues. These findings shed light on the typical immune responses of Penaeus vannamei under the infection of pathogens and contribute to the sustainable development of penaeid shrimp farming. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two shrimp proteins, i.e. cuticle protein 8-like (CP8) and glutamine-fructose-6-phosphate aminotransferase [isomerizing] 2-like isoform X7, were identified to interact with DIV1-168L. • The expression of CP8 was mainly distributed in gill and epidermis. • The expression level of CP8 was significantly inhibited during DIV1 infection. Decapod iridescent virus 1 (DIV1) stands as a significant pathogen affecting crustaceans, posing a grave threat to the shrimp industries in aquaculture dependent nations. Within the Iridoviridae family, the conserved envelope protein DIV1-168L plays a pivotal role in virion entry. Nonetheless, the host factors that interact with 168L remain unidentified. To address this gap, we established a cDNA library derived from Litopenaeus vannamei gill tissue and conducted yeast two-hybrid screening to identify host factors that interact with 168L. Additionally, we performed co-immunoprecipitation assays to verify the interaction between cuticle protein 8 (CP8) and 168L. Expression pattern analysis revealed the presence of CP8 transcripts in the gill and epidermis. Furthermore, immunohistochemistry results demonstrated the expression of CP8 in gill cells and its localization in the gill filament epithelium. Fluorescence analysis indicated that full-length CP8 colocalized with 168L in the cytoplasm of Sf9 cells. Removal of the signal peptide from the N -terminal of CP8 eliminated its concentration in the cytoplasm. Additionally, CP8 expression was significantly inhibited during DIV1 infection. Therefore, our research contributes to a better understanding of the entry mechanism of iridovirids. The GenBank accession number for the DIV1 sequence is MF197913.1. [ABSTRACT FROM AUTHOR]

Decapod iridescent virus 1 (DIV1) is an emerging viral pathogen that seriously threatens crustacean farming and has caused enormous economic losses in recent years. This virus was originally isolated and identified from diseased crayfish Cherax quadricarinatus and shrimp Litopenaeus vannamei in Fujian and Zhejiang, China, in 2014 by two research groups. In 2019, the Executive Committee of the International Committee on Taxonomy of Viruses approved DIV1 as a member of the new genus Decapodiridovirus in the family Iridoviridae. DIV1 is icosahedral in shape and contains a single linear double‐stranded DNA genome of approximately 165 kb. DIV1 can infect many host species, including freshwater and seawater crustaceans, but infected hosts do not show unique pre‐clinical signs, which increases the difficulties in the diagnosis and treatment of this disease. Increasing progress has been made towards understanding many aspects of this virus, including its classification, genomic features, transmission mode, host range, pathogenicity, clinical symptoms, histopathology, detection method and host immunity in response to infection. In this review, we provide an overview of recent progress towards understanding the epidemiology and pathobiology of disease caused by DIV1 and highlight the future research prospects in control and prevention strategies. [ABSTRACT FROM AUTHOR]

As a newly discovered virus, Decapoda iridovirus 1 (DIV1) can cause a mortality rate of up to 100% in crustaceans, leading to huge economic losses. At present, there is no effective prevention and control measures for this disease. In the present study, the specific primers targeting highly conserved regions of MCP gene were designed, and then a quantitative real-time PCR method was established. The results indicate that DIV1 quantitative real-time PCR established has good specificity and does not cross react with other pathogens including white spot syndrome virus (WSSV), infectious subcutaneous and hematopoietic necrosis virus (IHHNV) and Vibrio parahaemolyticus induced acute hepatopancreatic necrosis disease (VpAHPND). The real-time PCR was capable of detecting DIV1 DNA at a minimum concentration of 10 copies/μL within 34 cycles. The method has good repeatability, with intra group and inter group coefficients of variation both less than 2%. Thirty-two clinical samples were assessed using both the real-time PCR and conventional PCR. The results shown real-time PCR we established are more sensitive than conventional PCR. In conclusion, this method has strong specificity, stable repeatability, and high sensitivity, providing technical support for clinical diagnosis, epidemiology investigation and monitoring of DIV1., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhao, Liu, Zheng, Zhang, Han, Zhou, Ma, Wang and Chen.)

In the past few years, outbreaks of infections with infectious precocious virus (IPV) and Decapod iridescent virus 1 (DIV1) have significantly hindered the development of the aquaculture industry of giant freshwater prawn (Macrobrachium rosenbergii) and caused severe economic losses in China. To understand the epidemiological information of farmed M. rosenbergii , we collected 79 samples from 23 farming ponds in Jiangsu Province from 2022 to 2023 and detected 8 common pathogens. The results showed that IPV and DIV1 could be detected in collected samples. Infections with IPV or DIV1 and the resulting diseases were confirmed by clinical signs, histopathologic examination, and qPCR. At the same time, we found coinfection with IPV and DIV1 in the same individuals by clinical diagnosis, histopathologic examination, and molecular testing. However, iron prawn syndrome (IPS) or IPS equivale and clinical infection with DIV1 (iDIV1) cases showed mutual exclusion in individuals and ponds. The results revealed significant differences in loads of the two viruses in the same individuals and ponds, with an overall negative correlation. This is the first report of coinfection with IPV and DIV1 in farmed M. rosenbergii. • We found the coinfection of DIV1 and IPV in cultured Macrobrachium rosenbergii individuals and ponds. • There was an extremely significant negative correlation between the loads of the two pathogens in individual samples. • IPS or IPS equivocal and clinical iDIV1 cases showed mutual exclusion in individuals and ponds. • There was no significant difference in IPV load between IPS symptomatic and IPS equivocal samples. [ABSTRACT FROM AUTHOR]

Penaeid shrimp aquaculture is impacted by various diseases. However, most published studies on physiological responses to pathogens have focused on the changes in one or two tissues of shrimp infected by a single pathogen, or the effects of two pathogens infecting the shrimp in a single tissue. There has been limited systematic examination on the similarities and differences of immune responses in multiple tissues under various pathogen infection. Here, the transcriptomic changes of three immune tissues (gill, hepatopancreas and hemocytes) under the infection of white spot syndrome virus (WSSV), Vibrio parahaemolyticus acute hepatopancreatic necrosis disease (VPAHPND), and decapod iridovirus 1 (DIV1) were examined to provide new insights regarding the immune responses of the most important cultured shrimp, Penaeus vannamei. The results showed tissue-specific differences in the immune responses of shrimp tissues. The significant differentially expressed genes (DEGs) in gill are mainly related to environmental information processing and cellular processes. The DEGs in hemocytes are mostly involved in cellular processes, while those in hepatopancreas are primarily associated with metabolism. In addition, cytoskeleton-related proteins, MAPK signaling pathway, complement and coagulation level pathway, and thermogenesis may play key roles in the shrimp–pathogen interactions across tissues. These findings shed light on the typical immune responses of Penaeus vannamei under the infection of pathogens and contribute to the sustainable development of penaeid shrimp farming.

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.

Decapod iridescent virus 1 (DIV1) results in severe economic losses in shrimp aquaculture. However, little is known about the physiological effect of DIV1 infection on the host. In this study, we found that the lethal dose 50 of DIV1-infected Litopenaeus vannamei after 48, 72, 96, and 156 h were 4.86 × 106, 5.07 × 105, 2.13 × 105, and 2.38 × 104 copies/μg DNA, respectively. In order to investigate the mechanisms of DIV1 infection, a comparative transcriptome analysis of hemocytes from L. vannamei, infected or not with DIV1, was conducted. The BUSCO analysis showed that the transcriptome was with high completeness (complete single-copy BUSCOs: 57.3%, complete duplicated BUSCOs: 41.1%, fragmentation: 0.8%, missing: 0.8%). A total of 168,854 unigenes were assembled, with an average length of 601 bp. Based on homology searches, Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and cluster of orthologous groups of proteins (KOG) analysis, 62,270 (36.88%) unigenes were annotated. Among them, 1,112 differentially expressed genes (DEGs) were identified, of which 889 genes were up-regulated and 223 genes were down-regulated after DIV1 infection. These genes were mainly annotated to the major metabolic processes such as fructose and mannose metabolism, carbon metabolism, and inositol phosphate metabolism. Among these metabolic pathways, the triosephosphate isomerase (TPI) family was the most eye-catching DEG as it participates in several metabolic processes. Three types of TPI, LvTPI-like, LvTPI-Blike, and LvTPI-Blike1, were obtained for gene silencing by RNA interference. The results showed that LvTPI-like and LvTPI-Blike1 silencing caused a high mortality rate among L. vannamei. However, LvTPI-like and LvTPI-Blike silencing reduced DIV1 replication in DIV1-infected L. vannamei. All the results indicated that TPI-like genes play an important role during DIV1 infection, which provides valuable insight into the infection mechanism of DIV1 in shrimp and may aid in preventing viral diseases in shrimp culture.

Decapod iridescent virus 1 (DIV1) results in severe economic losses in shrimp aquaculture. However, little is known about the physiological effect of DIV1 infection on the host. In this study, we found that the lethal dose 50 of DIV1-infected Litopenaeus vannamei after 48, 72, 96, and 156 h were 4.86 × 106, 5.07 × 105, 2.13 × 105, and 2.38 × 104 copies/μg DNA, respectively. In order to investigate the mechanisms of DIV1 infection, a comparative transcriptome analysis of hemocytes from L. vannamei , infected or not with DIV1, was conducted. The BUSCO analysis showed that the transcriptome was with high completeness (complete single-copy BUSCOs: 57.3%, complete duplicated BUSCOs: 41.1%, fragmentation: 0.8%, missing: 0.8%). A total of 168,854 unigenes were assembled, with an average length of 601 bp. Based on homology searches, Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and cluster of orthologous groups of proteins (KOG) analysis, 62,270 (36.88%) unigenes were annotated. Among them, 1,112 differentially expressed genes (DEGs) were identified, of which 889 genes were up-regulated and 223 genes were down-regulated after DIV1 infection. These genes were mainly annotated to the major metabolic processes such as fructose and mannose metabolism, carbon metabolism, and inositol phosphate metabolism. Among these metabolic pathways, the triosephosphate isomerase (TPI) family was the most eye-catching DEG as it participates in several metabolic processes. Three types of TPI, LvTPI-like, LvTPI-Blike , and LvTPI-Blike1 , were obtained for gene silencing by RNA interference. The results showed that LvTPI-like and LvTPI-Blike1 silencing caused a high mortality rate among L. vannamei. However, LvTPI-like and LvTPI-Blike silencing reduced DIV1 replication in DIV1-infected L. vannamei. All the results indicated that TPI-like genes play an important role during DIV1 infection, which provides valuable insight into the infection mechanism of DIV1 in shrimp and may aid in preventing viral diseases in shrimp culture. [ABSTRACT FROM AUTHOR]

Cherax quadricarinatus is a large-sized, highly fecund, and fast-growing species of freshwater crayfish, and has become one of the world's most intensely studied crustaceans. Decapod iridescent virus 1 (DIV1), a newly described species in the family Iridoviridae , is known to infect various crustaceans, including C. quadricarinatus , and may pose a new threat in the shrimp-farming industry. The present study performed de novo transcriptome sequencing of C. quadricarinatus hepatopancreas during DIV1 infection. A total of 114,784 transcripts and 56,418 genes were obtained; 1070 genes were upregulated and 775 genes were downregulated when compared with the uninfected samples (controls). Three pattern recognition receptor genes (fibrinogen-related protein, C-type lectin, and beta-1,3-glucan-binding protein) were upregulated during DIV1 infection. Among the top-30 upregulated unigenes, 9 unigenes were identified as vitellogenin (Vg) genes, and the top-3 upregulated unigenes were identified as involved in Vg lipid transport, lipid localization, and lipid transporter activity, which were all significantly over-representative GO terms in the GO enrichment analysis of total and upregulated differentially expressed genes (DEGs). Many genes associated with Jak-STAT signaling pathway, Endocytosis, Phagosome, MAPK signaling pathway, Apoptosis and Lysosome were positively modified after DIV1 infection. The predicted protein–protein interaction (PPI) analysis showed NF1 and TUBA, CRM1 and TUBB were involved in protein interactions. This research showed that DIV1 infection has a significant impact on the transcriptome profile of C. quadricarinatus hepatopancreas, and the results enhance our understanding of virus–host interactions. Furthermore, the high number of transcripts generated in the present study will provide information for identifying novel genes in the absence of a full C. quadricarinatus genome sequence. • First Cherax quadricarinatus transcriptome aimed to characterize immune response during DIV1 infection. • 1845 DEGs (1070 upregulated and 775 downregulated) were identified. 1070 genes were upregulated and 775. • The expression level of vitellogenin was significantly upregulated during DIV1 infection. • Phagosome and MAPK signaling pathway were positively modified during DIV1 infection. [ABSTRACT FROM AUTHOR]

Viral diseases have become a significant impediment to the sustainable development of the global shrimp aquaculture industry. Decapod iridescent virus 1 (DIV1) is an emerging shrimp virus that has affected shrimp in China recent years. Rapid detection of DIV1 could improve enhance the effectiveness of prevention, control and treatment in the absence of good prevention and control measures. This study established loop-mediated isothermal amplification (LAMP) along with two visual interpretation methods, LAMP-dye and LAMP-LFD, to detect DIV1. The newly developed method would not cause cross-reactions with other shrimp pathogens such as white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus acute hepatopancreatic necrosis disease (Vp AHPND). The detection limit of DIV1 LAMP was as low as 103 copies of DIV1 per reaction, with a reaction time of less than 40 min. The diagnostic sensitivity and diagnostic specificity of this method were determined to be 88% and 100%, respectively, when compared with the conventional PCR. Both of the LAMP-dye and LAMP-LFD methods are cost-effective and do not require expensive amplification equipment. They can be combined with LAMP and other temperature amplification methods for rapid on-site detection, effectively prevent aerosol contamination, and which are convenient and suitable for field testing or preliminary infection rish prediction experiments to predict the risk of infection. • This two visual interpretation methods LAMP-dye and LAMP-LFD for detection of DIV1, with a reaction time of less than 40 min. • Both of LAMP-dye and LAMP-LFD methods do not require expensive equipment, and effectively prevent the aerosol contamination. • The visual interpretation methods were 100% detection for the positive versus negative samples, could meet the requirement for early warning detection of virus in field. [ABSTRACT FROM AUTHOR]

Redclaw crayfish (Cherax quadricarinatus) is a large, tropical freshwater crustacean species with considerable potential of commercial production. In recent years, infection with DIV1 in redclaw crayfish is being reported in aquaculture industries, causing high mortality and huge economic losses. However, many characteristics of this virus, including pathogenesis, transmission mechanism, and host immunity, remain largely unknown.MicroRNAs are known to play important roles in numerous biological processes, and many microRNAs are reported to be involved in the regulation of immune responses. In this study, nine-small RNA libraries were constructed using hemocytes of redclaw crayfish to characterize the differentially expressed miRNAs (DE-miRNAs) at 24 and 48 h postinfection (hpi). A total of 14 and 22 DE-miRNAs were identified in response to DIV1 infection at 24 and 48 hpi, respectively. Further, functional annotation of the predicted host target genes using GO and KEGG pathway enrichment analyses indicated that relevant biological processes and signal pathways underwent miRNA-mediated regulation after DIV1 infection. Our results enhanced the understanding of the mechanisms of miRNA-mediated regulation of immune responses under DIV1 infection in crustaceans. • This was the first report to characterize DE-miRNAs that involved in immune response to DIV1 infection in crustaceans. • Total of 14 and 22 DE-miRNAs were identified in response to DIV1 challenge at 24 and 48 hpi, respectively. • Function annotation revealed that the endocytosis was key pathway in the immune response to DIV1 infection. [ABSTRACT FROM AUTHOR]

In this study, ridgetail white prawns—Exopalaemon carinicauda—were infected per os (PO) with debris of Penaeus vannamei infected with shrimp hemocyte iridescent virus (SHIV 20141215), a strain of decapod iridescent virus 1 (DIV1), and via intramuscular injection (IM with raw extracts of SHIV 20141215. The infected E. carinicauda showed obvious clinical symptoms, including weakness, empty gut and stomach, pale hepatopancreas, and partial death with mean cumulative mortalities of 42.5% and 70.8% by nonlinear regression, respectively. Results of TaqMan probe-based real-time quantitative PCR showed that the moribund and surviving individuals with clinical signs of infected E. carinicauda were DIV1-positive. Histological examination showed that there were darkly eosinophilic and cytoplasmic inclusions, of which some were surrounded with or contained tiny basophilic staining, and pyknosis in hemocytes in hepatopancreatic sinus, hematopoietic cells, cuticular epithelium, etc. On the slides of in situ DIG-labeling-loop-mediated DNA amplification (ISDL), positive signals were observed in hematopoietic tissue, stomach, cuticular epithelium, and hepatopancreatic sinus of infected prawns from both PO and IM groups. Transmission electron microscopy (TEM) of ultrathin sections showed that icosahedral DIV1 particles existed in hepatopancreatic sinus and gills of the infected E. carinicauda from the PO group. The viral particles were also observed in hepatopancreatic sinus, gills, pereiopods, muscles, and uropods of the infected E. carinicauda from the IM group. The assembled virions, which mostly distributed along the edge of the cytoplasmic virogenic stromata near cellular membrane of infected cells, were enveloped and approximately 150 nm in diameter. The results of molecular tests, histopathological examination, ISDL, and TEM confirmed that E. carinicauda is a susceptible host of DIV1. This study also indicated that E. carinicauda showed some degree of tolerance to the infection with DIV1 per os challenge mimicking natural pathway.

Macrobrachium rosenbergii is a valuable freshwater prawn in Asian aquaculture. In recent years, a new symptom that was generally called “white head„ has caused high mortality in M. rosenbergii farms in China. Samples of M. rosenbergii, M. nipponense, Procambarus clarkii, M. superbum, Penaeus vannamei, and Cladocera from a farm suffering from white head in Jiangsu Province were collected and analyzed in this study. Pathogen detection showed that all samples were positive for Decapod iridescent virus 1 (DIV1). Histopathological examination revealed dark eosinophilic inclusions and pyknosis in hematopoietic tissue, hepatopancreas, and gills of M. rosenbergii and M. nipponense. Blue signals of in situ digoxigenin-labeled loop-mediated isothermal amplification appeared in hematopoietic tissue, hemocytes, hepatopancreatic sinus, and antennal gland. Transmission electron microscopy of ultrathin sections showed a large number of DIV1 particles with a mean diameter about 157.9 nm. The virogenic stromata and budding virions were observed in hematopoietic cells. Quantitative detection with TaqMan probe based real-time PCR of different tissues in naturally infected M. rosenbergii showed that hematopoietic tissue contained the highest DIV1 load with a relative abundance of 25.4 ± 16.9%. Hepatopancreas and muscle contained the lowest DIV1 loads with relative abundances of 2.44 ± 1.24% and 2.44 ± 2.16%, respectively. The above results verified that DIV1 is the pathogen causing white head in M. rosenbergii. M. nipponense and Pr. clarkii are also species susceptible to DIV1.

As a new emerging viral pathogen, Decapod iridescent virus 1 (DIV1) seriously threatens crustacean farming in recent years. However, limited research progresses have been made on the immune mechanism between host and viral factors in response to DIV1 infection. In the current study, a natural occurrence of DIV1 infection with obvious clinical signs was found in farmed redclaw crayfish Cherax quadricarinatus , and confirmed by nested PCR detection and histopathological examination. Besides, gene expression profiles were analyzed after being challenged with DIV1, and results showed that 27 immune related genes were upregulated compared with the control group. Moreover, the gut microbiota from healthy and DIV1-infected crayfish were investigated by 16S rDNA high-throughput sequencing. Results showed that significant differences in the microbial composition and function were observed after DIV1 challenge. Furthermore, we discovered that changes in gene expression profiles were correlated with microbiota alterations under DIV1 challenge. Taken together, our findings will provide new insights into the immune response mechanism of DIV1 infection in crustaceans. • DIV1 infection was found in farmed redclaw crayfish by nested PCR detection and histopathological examination. • Significant difference was observed within the composition and function of gut microbiota after being challenged with DIV1. • A high correlation between bacterial genera and immune genes was observed between healthy and DIV1-infected crayfish. [ABSTRACT FROM AUTHOR]

In this study, ridgetail white prawns—Exopalaemon carinicauda—were infected per os (PO) with debris of Penaeus vannamei infected with shrimp hemocyte iridescent virus (SHIV 20141215), a strain of decapod iridescent virus 1 (DIV1), and via intramuscular injection (IM with raw extracts of SHIV 20141215. The infected E. carinicauda showed obvious clinical symptoms, including weakness, empty gut and stomach, pale hepatopancreas, and partial death with mean cumulative mortalities of 42.5% and 70.8% by nonlinear regression, respectively. Results of TaqMan probe-based real-time quantitative PCR showed that the moribund and surviving individuals with clinical signs of infected E. carinicauda were DIV1-positive. Histological examination showed that there were darkly eosinophilic and cytoplasmic inclusions, of which some were surrounded with or contained tiny basophilic staining, and pyknosis in hemocytes in hepatopancreatic sinus, hematopoietic cells, cuticular epithelium, etc. On the slides of in situ DIG-labeling-loop-mediated DNA amplification (ISDL), positive signals were observed in hematopoietic tissue, stomach, cuticular epithelium, and hepatopancreatic sinus of infected prawns from both PO and IM groups. Transmission electron microscopy (TEM) of ultrathin sections showed that icosahedral DIV1 particles existed in hepatopancreatic sinus and gills of the infected E. carinicauda from the PO group. The viral particles were also observed in hepatopancreatic sinus, gills, pereiopods, muscles, and uropods of the infected E. carinicauda from the IM group. The assembled virions, which mostly distributed along the edge of the cytoplasmic virogenic stromata near cellular membrane of infected cells, were enveloped and approximately 150 nm in diameter. The results of molecular tests, histopathological examination, ISDL, and TEM confirmed that E. carinicauda is a susceptible host of DIV1. This study also indicated that E. carinicauda showed some degree of tolerance to the infection with DIV1 per os challenge mimicking natural pathway. [ABSTRACT FROM AUTHOR]

Macrobrachium rosenbergii is a valuable freshwater prawn in Asian aquaculture. In recent years, a new symptom that was generally called "white head" has caused high mortality in M. rosenbergii farms in China. Samples of M. rosenbergii, M. nipponense, Procambarus clarkii, M. superbum, Penaeus vannamei, and Cladocera from a farm suffering from white head in Jiangsu Province were collected and analyzed in this study. Pathogen detection showed that all samples were positive for Decapod iridescent virus 1 (DIV1). Histopathological examination revealed dark eosinophilic inclusions and pyknosis in hematopoietic tissue, hepatopancreas, and gills of M. rosenbergii and M. nipponense. Blue signals of in situ digoxigenin-labeled loop-mediated isothermal amplification appeared in hematopoietic tissue, hemocytes, hepatopancreatic sinus, and antennal gland. Transmission electron microscopy of ultrathin sections showed a large number of DIV1 particles with a mean diameter about 157.9 nm. The virogenic stromata and budding virions were observed in hematopoietic cells. Quantitative detection with TaqMan probe based real-time PCR of different tissues in naturally infected M. rosenbergii showed that hematopoietic tissue contained the highest DIV1 load with a relative abundance of 25.4 ± 16.9%. Hepatopancreas and muscle contained the lowest DIV1 loads with relative abundances of 2.44 ± 1.24% and 2.44 ± 2.16%, respectively. The above results verified that DIV1 is the pathogen causing white head in M. rosenbergii. M. nipponense and Pr. clarkii are also species susceptible to DIV1. [ABSTRACT FROM AUTHOR]