Qingli Zhang

Covert mortality nodavirus (CMNV) is the pathogen causing viral covert mortality disease (VCMD) in cultured shrimp, and cross-species transmission is an important characteristic of this virus. The lack of stable, continuously passable shrimp cell lines has long been a bottleneck for in-depth studies on CMNV pathogenesis, virus-host interactions, and antiviral strategies. In this study, we systematically evaluated the infectivity, replication kinetics, and serial passage stability of CMNV in eight cell lines of different origins, including three insect cell lines (Sf21, C6/36, and S2) and five fish cell lines (EMRB, EPC, ZF4, TiB, and GiCB). Based on CPE observations and viral RNA replication profiles across three blind passages, cell lines exhibiting both obvious CPE and viral RNA replication in at least two passages were classified as susceptible, including EPC (P1 and P3), TiB (P1 and P3), and S2 (P1-P3). ZF4 (P1-P3) and C6/36 (P1 and P3), despite lacking obvious CPE, supported viral RNA replication across multiple passages and were therefore tentatively designated as atypically susceptible. In contrast, Sf21 and EMRB showed no detectable viral RNA replication in any of the three passages, while GiCB supported replication only at P1; these three cell lines were accordingly classified as non-susceptible. These findings identify multiple non-shrimp cell lines that support stable CMNV replication at the RNA level and serial passage, providing useful experimental systems for studies of viral pathogenesis, host interactions, diagnostic development, and antiviral product screening.

Climate change and human activities have increased the risk of virus spillover from wildlife, posing a threat to human health. A human ocular disease called persistent ocular hypertensive viral anterior uveitis (POH-VAU) has recently emerged; however, the cause is unclear. Here we report that POH-VAU is associated with covert mortality nodavirus (CMNV) of aquatic origin. CMNV is prevalent in farmed and wild aquatic animals worldwide. We confirmed CMNV infection in ocular tissues and seroconversion in 70 patients with POH-VAU. An exposure survey and analysis of logistic regression models revealed that CMNV exposure frequency, number of severe exposures and exposure severity were associated with an increased risk of POH-VAU. Epidemiological data indicate that frequent unprotected processing of aquatic animals and consumption of raw aquatic animals were commonly reported exposure events, collectively accounting for 71.4% of investigated cases. Challenge tests revealed that CMNV can cause elevated intraocular pressure and pathological damage to ocular tissues in mice and can infect mammalian cells in vitro. This study reveals that an aquatic animal virus is associated with an emerging human disease.

Antarctic krill Euphausia superba, one of the most abundant species on the planet, is a keystone species of the Southern Ocean ecosystem. In the present study, we analyzed the RNA virome of Antarctic krill via metatranscription methods. The results showed that only 0.39% (49/12, 558) of the resultant unigenes could be assigned to known viral taxa, which were most similar to 17 known viruses, including nine invertebrate viruses, two vertebrate viruses, three protozoan viruses and three mycoviruses. However, most of the detected viruses possessed low amino acid similarity with counterparts in the viral databases. Penaeus vannamei picornavirus (PvPV; Family Picornaviridae) and covert mortality nodavirus (CMNV; Family Nodaviridae) were the two most abundant viruses in the Antarctic krill RNA virome. Notably, PvPV and CMNV are known pathogens to multiple aquatic animals according to epidemiological survey and exposure experiments, whereby PvPV positive krill caused clinical symptoms and histopathological lesions to P. vannamei and similarly, CMNV infection altered the swimming and feeding behavior of parent marine medaka Oryzias melastigma and caused tissue damage and even spinal curvature of the offspring. Results herein reveal, for the first time, the high abundance and taxonomic diversity of viruses in Antarctic krill while simultaneously highlighting the risk of an important virus reservoir to global aquaculture, and the potential impact on animals in the Antarctic ecosystem.

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (β-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.

Efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, shrimp viral vector of pUC19-IHHNV-PH-GUS and baculoviral vector of Bacmid or Bacmid-VP28 encoding shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which had shortened inverted terminal repeats, into pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and gene transfer efficiency of this system had been evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but GUS expression could be detected only in the cases when the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.

Translucent post-larvae disease (TPD), caused by Vibrio parahaemolyticus ( Vp _TPD ), has become an emerging shrimp disease, affecting more than 70%–80% of coastal shrimp nurseries in China in spring 2020. Here, we investigated the key virulence factors of Vp _TPD by analyzing protein fragments, related genomic information, as well as experimental challenge tests. After investigating the toxic effects of purified protein fragments with different molecular weights (MWs) from Vp _TPD, we found that only the protein fragments with MW >100 kDa showed similar lethality to live Vp _TPD in the experimental challenge test using post-larvae shrimp. Meanwhile, similar histopathological changes exhibiting in the hepatopancreas and midgut of the diseased individuals were observed in the post-larvae shrimp challenged with either bacterial protein fragments (MW >100 kDa) or live Vp _TPD . Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses, two novel proteins, Vibrio high virulent protein (VHVP)-1 and VHVP-2, were identified as the candidates of key virulence factors to cause TPD. Moreover, VHVP-1 and VHVP-2 were found to be encoded by two genes ( vhvp-1 and vhvp-2 ) tandemly located on a 187,791-bp plasmid and were predicted to depend on the same promoter following a comparative genomic analysis. Further epidemiological investigation and challenge test indicated that the V. parahaemolyticus isolate carrying only the vhvp-1 gene and lacking vhvp-2 gene could not cause mortality of experimental Penaeus vannamei post-larvae. The mutant (Δvhvp-2) by deleting vhvp-2 gene could only cause 4.92% of accumulative mortality of post-larvae that is similar to the non- Vp _TPD Vibrio strain. Additionally, the complemented strains, Δvhvp-2/pBT3-vhvp-2 and Δvhvp-2/pwtCas9-vhvp-2, showed similar virulence to the wild-type Vp _TPD . The results demonstrated that V. parahaemolyticus becomes lethal to post-larval shrimp by acquiring the VHVP-2 virulence factor. This study sheds light on further investigations of the pathogenic mechanism of Vp _TPD and the development of strategies for early diagnosis of TPD in shrimp hatcheries. IMPORTANCE: As a severe emerging shrimp disease, TPD has heavily impacted the shrimp aquaculture industry and resulted in serious economic losses in China since spring 2020. This study aimed to identify the key virulent factors and related genes of the Vp _TPD , for a better understanding of its pathogenicity of the novel highly lethal infectious pathogen, as well as its molecular epidemiological characteristics in China. The present study revealed that a novel protein, Vibrio high virulent protein-2 (MW >100 kDa), is responsible to the lethal virulence of V. parahaemolyticus to shrimp post-larvae. The results are essential for effectively diagnosing and monitoring novel pathogenic bacteria, like Vp _TPD , in aquaculture shrimps and would be beneficial to the fisheries department in early warning of Vp _TPD emergence and developing prevention strategies to reduce economic losses due to severe outbreaks of TPD. Elucidation of the key virulence genes and genomics of Vp _TPD could also provide valuable information on the evolution and ecology of this emerging pathogen in aquaculture environments.