Jie Huang

Viral nervous necrosis (VNN), caused by nervous necrosis virus (NNV), is a devastating viral disease affecting marine fish aquaculture worldwide, particularly during larval and juvenile stages. Although NNV has a broad and expanding host range, the susceptibility of greenfin horse-faced filefish (Thamnaconus septentrionalis) has not been established. To determine whether T. septentrionalis is susceptible to NNV, we investigated an outbreak of abnormal mortality in juveniles from a marine aquaculture farm in Rizhao, Shandong Province, China. A comprehensive etiological investigation combined bacterial isolation and identification, real-time quantitative RT-PCR (RT-qPCR), tissue-distribution analysis, histopathology, transmission electron microscopy (TEM), and RNA2-based phylogenetic analysis. Diseased fish exhibited characteristic VNN-like neurological signs, including spiral swimming, loss of equilibrium, anorexia, and body darkening. Several bacterial taxa, including Vibrio- and Tenacibaculum-related isolates, were recovered from diseased fish, indicating the possibility of bacterial involvement. However, the concurrent detection of abundant NNV RNA in neural tissues, together with characteristic neurological signs, compatible histopathological lesions, and NNV-like particles, provided convergent evidence supporting natural NNV infection. NNV RNA was detected in all ten diseased fish, whereas the other screened viral pathogens were not detected. Pooled brain–eye samples contained 4.62 × 10⁸ to 1.33 × 10⁹ NNV RNA copies/μg total RNA, and abundant NNV RNA was also detected in brain, spleen, kidney, and trunk tissues. Histological examination showed vacuolar and degenerative lesions in the brain and retina, and TEM revealed numerous non-enveloped, spherical virus-like particles approximately 25 nm in diameter in neural tissues, consistent with NNV morphology. Phylogenetic analysis of the RNA2 capsid protein gene placed the detected virus within the RGNNV RNA2 lineage. Together, the neurological signs, NNV RNA detection, compatible histopathological lesions, NNV-like particles in neural tissues, and RNA2 phylogenetic evidence provide evidence of natural NNV infection in T. septentrionalis. These findings document evidence of natural NNV infection in T. septentrionalis under aquaculture conditions, highlight the need for further investigations into pathogenicity and transmission, and provide a basis for VNN surveillance and biosecurity management in filefish aquaculture.

Koch’s postulates remain unachievable for most crustacean viruses due to uncultivable pathogens and chronic infection dynamics. We propose viromic Koch’s postulates, a culture-independent framework, to address this limitation. The modified postulates uncover comprehensive information about viruses throughout the chain from clinical samples to viral preparations to challenge trials by combining the virome from metatranscriptomic, proteomic, or molecular analyses with diagnoses from clinical or pathological observations. Applying this approach to iron prawn syndrome (IPS), a chronic disease that causes stunted growth in Macrobrachium rosenbergii , we identified infectious precocity virus (IPV) as the exclusive viral correlate through metatranscriptomic analysis. IPV showed 100% prevalence in symptomatic prawns, but was absent in controls. Viral preparations from diseased tissue reproduced IPS pathology in healthy prawns upon inoculation, with IPV replication kinetics correlating to growth retardation. Histopathology and in situ hybridization localized IPV to neuroendocrine tissues, confirming tropism and replication sites. Our findings definitively establish IPV as the causative agent of IPS. Furthermore, this study demonstrates how systematizing existing culture-independent verification steps—referred to here as viromic Koch’s postulates—can serve as a practical, standardized operational roadmap for pathogen verification in uncultivable systems, facilitating the investigation of viral diseases in aquaculture. IMPORTANCE: Koch’s postulates remain difficult to apply to most crustacean and other uncultivable viruses because pathogen isolation and cell culture are often not feasible. To address this limitation, we propose “viromic Koch’s postulates,” a culture-independent framework that integrates whole-virome background exclusion, comprehensive inoculum characterization, and in vivo spatiotemporal validation of viral replication. Using this approach, we establish IPV as the etiological agent of IPS in Macrobrachium rosenbergii . By linking sequence-based discovery to biological causation without relying on virus culture, this work establishes an operational roadmap for rigorous pathogen identification in crustaceans and other host systems where traditional isolation-based criteria are not feasible.

Due to its advantages of a short production cycle, wide market demand, and profitability, decapod crustacean aquaculture has become one of the fastest‐growing food production systems. However, disease outbreaks have become one of the most important factors limiting the sustainability of decapod crustaceans. This review synthesizes current advancements in the understanding of emerging viral pathogens in decapod crustacean aquaculture, with emphasis on their genomic‐based taxonomical classification, susceptible hosts, global distribution, clinical manifestations, pathogenic mechanisms, diagnostic developments, and management strategies. This paper summarizes details of emerging viruses with confirmed pathogenicity (such as decapod iridescent virus 1), as well as viruses with uncertain or yet‐unconfirmed pathogenic roles. In addition, we highlight the role of next‐generation sequencing in virus discovery and examine the challenges posed by coinfection with other microorganisms. This review provides information to help the decapod crustacean aquaculture industry in setting up early warning systems to prevent the introduction of emerging pathogens into the production systems.

Decapod iridescent virus 1 (DIV1) is a newly recognized pathogen that causes severe disease in farmed crustaceans, leading to substantial economic losses in the shrimp and crab aquaculture industries. This study investigates the thermal persistence and heat inactivation conditions of DIV1, aiming to establish effective heat treatment protocols for seafood safety in international trade and aquaculture waste management. DIV1 was treated at different temperatures (56°C, 60°C, and 70°C) for varying durations, and infectivity was assessed through experimental infection of Penaeus vannamei . Results indicated that treatment at 56°C for 30 min, 60°C for 15 min, or 70°C for 1 min effectively inactivated DIV1, with these parameters being less stringent than the current WOAH recommendation of 80°C for 30 min. Additionally, this study revealed that DIV1 could remain infectious at room temperature for more than 168 h and at ultralow temperatures (−80°C) for more than 8 years. These findings provide critical data for optimizing heat treatment strategies to mitigate pathogen transmission risks in aquaculture and seafood products, ensuring biosecurity and safety in international markets.

The World Organisation for Animal Health (WOAH) has assessed crustacean diseases, such as infections with white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), decapod iridescent virus 1 (DIV1), and acute hepatopancreatic necrosis disease (AHPND), as listed diseases, and infection with Ecytonucleospora hepatopenaei (EHP) as an emerging disease, all of which significantly threaten the shrimp industry. This study developed a quintuplex EvaGreen-based melting curve real-time PCR method for the simultaneous detection of WSSV, IHHNV, DIV1, AHPND-causing Vibrio ( V _AHPND ), and EHP. In the specific assay, only the target pathogen demonstrated efficient and detectable amplification, thereby indicating that the method exhibits high specificity. Regarding sensitivity testing, the five pathogens were detected at a concentration of 1.0 × 10 ¹ copies/μL. Each concentration gradient was evaluated in triplicate, and the coefficient of variation for each gradient remained below 3.38%, thereby affirming that the method demonstrates highly repeatability. We tested the diagnostic sensitivity (DSe) and the diagnostic specificity (DSp) of our method using a total of 800 clinical samples which were gathered from the shrimp farming regions in China. The newly established method in this study demonstrated a DSe above 89.74% for the five pathogens and a DSp of 100%. The quintuplex EvaGreen real-time PCR method developed here offers an accurate and efficient approach for EHP, WSSV, V _AHPND , IHHNV, and DIV1. IMPORTANCE: Crustacean diseases, such as infections with WSSV, IHHNV, DIV1, V _AHPND , and EHP, pose a significant threat to the global shrimp industry, leading to substantial economic losses. Rapid, accurate, and simultaneous detection of these pathogens is crucial for effective disease management and biosecurity in shrimp farming. In this study, we developed a quintuplex EvaGreen-based melting curve real-time PCR method that enables the simultaneous detection of these five major shrimp pathogens with exceptional specificity, sensitivity, and repeatability. By evaluating 800 clinical samples, our method demonstrated high diagnostic sensitivity and specificity, making it a valuable tool for early pathogen detection and disease control. This novel approach can help mitigate disease outbreaks, improve shrimp farm productivity, and support the sustainable development of the aquaculture industry.

Although positive- and negative-sense single-stranded RNA viruses are ubiquitous in nature, there is currently no evidence of recombination or reassortment between viruses with these two major forms of genome organization. Here, we describe the discovery of brine shrimp virga-like virus 1 (BSVV1), a novel positive-sense single-stranded RNA virus with a recombinant genome structure derived from two viral phyla with differing genome organizations. The genome of BSVV1 comprises three open reading frames (ORFs). ORF1 resembles the RNA-dependent RNA polymerase of Ips virga-like virus 1 (a positive-sense RNA virus), while ORF2, transcribed in the positive orientation, is related to the glycoprotein of Hubei bunya-like virus 10 and other negative-sense RNA viruses. The predicted ORF3 was unique to BSVV1 without known homologs identified. The presence of the three protein products was verified by mass spectrometry. Notably, our analysis also revealed that BSVV1 is geographically widespread and found in brine shrimp from at least eight countries on four continents. In addition, BSVV1 was successfully cultured and proliferated to high viral loads during brine shrimp development. In sum, we provide compelling evidence of an ancient recombination event between negative- and positive-sense single-stranded RNA viruses, enriching our understanding of the evolution of genome structures in RNA viruses.

Infectious precocity virus (IPV) poses a significant economic threat to the aquaculture industry by causing sexual precocity and slow growth in Macrobrachium rosenbergii. In this study, we conducted an in-depth investigation into the genetic evolution of IPV from 2011 to 2024 by collecting 31 IPV variants through epidemiological surveys and public databases, including 29 variants with complete genomic sequences. The phylogenetic analysis revealed that these complete genomic sequences clustered into two distinct phylogenetic clades as follows: the Southeast Asian clade and the Chinese clade. Nucleotide and protein variation analyses demonstrated a high degree of similarity, with nucleotide identity ranging from 98.5% to 100% and protein identity from 99.4% to 100%. Further analysis of protein variations within the putative coding region identified two distinct variation patterns. The average dN/dS ratio of 0.12 highlights the strong purifying selection acting on IPV, particularly on structural proteins. In conclusion, this study significantly expands the genomic database of IPV and provides valuable insights into its genetic evolution. These findings offer critical scientific evidence to enhance detection protocols and support sustainable M. rosenbergii aquaculture practices.

Crustaceans are important food sources worldwide and possess significant ecological status in the marine ecosystem. However, our understanding of the diversity and evolution of RNA viruses in crustaceans, especially in economic crustaceans, is still limited. Here, 106 batches of economic crustaceans including 13 species were collected from 24 locations in China during 2016–2021. We identified 90 RNA viruses, 69 of which were divergent from the known viruses. Viral transcripts were assigned to 18 different viral families/clades and three unclassified groups. Among the identified viruses, five were double-stranded RNA viruses, 74 were positive-sense single-stranded RNA (+ssRNA) viruses, nine were negative-sense single-stranded RNA (−ssRNA) viruses, and two belonged to an unclassified RNA virus group. Phylogenetic analyses showed that crustacean viruses were often clustered with viruses identified from invertebrates. Remarkably, most crustacean viruses were closely related to those from different host species along the same food chain or ecological aquatic niche. In addition, the genome structures of the newly discovered picornaviruses exhibited remarkable diversity. Our study significantly expands the diversity of viruses in important economic crustaceans and provides essential data for the risk assessment of the pathogens spreading in the global aquaculture industry. IMPORTANCE: The study delves into the largely uncharted territory of RNA viruses in crustaceans, which are not only vital for global food supply but also play a pivotal role in marine ecosystems. Focusing on economic crustaceans, the research uncovers 90 RNA viruses, with 69 being potentially new to science, highlighting the vast unknown viral diversity within these marine organisms. The findings reveal that these viruses are often related to those found in other invertebrates and tend to share close relationships with viruses from species within the same food web or habitat. This suggests that viruses may move between different marine species more frequently than previously thought. The discovery of such a wide variety of viruses, particularly the diverse genome structures of newly identified picornaviruses, is a significant leap forward in understanding the crustacean virology. This knowledge is crucial for managing disease risks in aquaculture and maintaining the balance of marine ecosystems.

Perinereis species are essential benthonic animals in coastal ecosystems and have significant roles as live feed in aquaculture, owing to their high-protein and low-fat nutritional profile. Despite their ecological importance, the viral communities associated with these organisms need to be better understood. In this study, we generated 2.6 × 10⁸ reads using meta-transcriptomic sequencing and de novo assembled 5.3 × 10³ virus-associated contigs. We identified 12 novel RNA viruses from two species, Perinereis aibuhitensis and P. wilsoni, which were classified into four major viral groups: Picobirnaviridae, Marnaviridae, unclassified Picornavirales, and unclassified Bunyavirales. Our findings revealed the hidden diversity of viruses and genome structures in Perinereis, enriching the RNA virosphere and expanding the host range of Picobirnaviridae, Marnaviridae, and Bunyavirales. This study also highlighted the potential biosecurity risk of the novel viruses carried by Perinereis to aquaculture.