Limei Xu

Crayfish hematopoietic tissue cells but not hemocytes are permissive for white spot syndrome virus replication

Hemocytes are the major immune cells of crustaceans which are believed to be essential for the pathogenesis of white spot syndrome virus (WSSV) infection. Crayfish hemocytes and hematopoietic tissue (HPT) cells have been found to be susceptible to WSSV infection, but the procedure of WSSV infection to both cell types has not yet been carefully investigated. In this study, we analyzed the infection and proliferation of WSSV in crayfish hemocytes as well as HPT cells in detail through transmission electronic microscopy (TEM). The results showed that WSSV could enter both hemocytes and HPT cells through endocytosis, but the production of progeny virus was only achieved in HPT cells. Further investigation demonstrated that although WSSV could transcribe its genes in both cell types, viral genome replication and structural protein expression were unsuccessful in hemocytes, which may be responsible for the failure of progeny production. Therefore, we propose that both hemocytes and HPT cells are susceptible to WSSV infection but only HPT cells are permissive to WSSV replication. These findings will extend our knowledge of the interaction between WSSV and the host immune system.

Identification of three immediate-early genes of white spot syndrome virus.

Viral immediate-early (IE) genes generally encode regulatory proteins that are critical for viral replication. Their transcription, which is independent of de novo viral protein synthesis, is driven directly by host transcription factors. In this study, we examined promoter activities of 12 predicted regulatory genes of white spot syndrome virus (WSSV) belonging to the zinc finger protein family by EGFP-reporter assays in High Five cells. The results showed that the promoters of three genes (wsv056, wsv403 and wsv465) could drive reporter gene expression, and RT-PCR analysis revealed that their expression in WSSV-infected primary crayfish hemocytes was insensitive to the protein synthesis inhibitor cycloheximide (CHX). Therefore, they are IE genes of WSSV.

Analysis of white spot syndrome virus envelope protein complexome by two-dimensional blue native/SDS PAGE combined with mass spectrometry

White spot syndrome virus (WSSV) is a large enveloped virus, but the organization of its envelope proteins remains largely unknown. In the present study, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and SDS-PAGE in combination with mass spectrometry to analyze the envelope protein complexome of WSSV. Our results show that the viral envelope consists of multi-protein complexes (MPCs). Within them, the envelope protein VP19 exists as a homotrimer, while another major envelope protein, VP28, mainly exists as a homotetramer. The most notable feature is that the majority of MPCs include VP26 and VP24, suggesting that these two proteins might serve as hub proteins to recruit low-abundance proteins to MPCs and play crucial roles in the process of protein complex formation. Furthermore, we found significant evidence for interactions between several low-abundance proteins, such as VP52B/VP38/VP33 and VP12/VP150. The result of this study may promote the further research on WSSV envelope assembly.

VP24 is a chitin-binding protein involved in white spot syndrome virus infection

ABSTRACT Oral ingestion is the major route of infection for the white spot syndrome virus (WSSV). However, the mechanism by which virus particles in the digestive tract invade host cells is unknown. In the present study, we demonstrate that WSSV virions can bind to chitin through one of the major envelope proteins (VP24). Mutagenesis analysis indicated that amino acids (aa) 186 to 200 in the C terminus of VP24 were required for chitin binding. Moreover, the P-VP24186–200 peptide derived from the VP24 chitin binding region significantly inhibited the VP24-chitin interaction and the WSSV-chitin interaction, implying that VP24 participates in WSSV binding to chitin. Oral inoculation experiments showed that P-VP24186–200 treatment reduced the number of virus particles remaining in the digestive tract during the early stage of infection and greatly hindered WSSV proliferation in shrimp. These data indicate that binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSV per os infection and provide new ideas for preventing WSSV infection in shrimp farms. IMPORTANCE In this study, we show that WSSV can bind to chitin through the envelope protein VP24. The chitin-binding domain of VP24 maps to amino acids 186 to 200 in the C terminus. Binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSV per os infection. These findings not only extend our knowledge of WSSV infection but also provide new insights into strategies to prevent WSSV infection in shrimp farms.

Isolation and preliminary characterization of a new pathogenic iridovirus from redclaw crayfish Cherax quadricarinatus.

We report the preliminary characterization of a new iridovirus detected in diseased Cherax quadricarinatus collected from a farm in Fujian, China. Transmission electron microscopy identified numerous icosahedral particles (~150 nm in diameter) in the cytoplasm and budding from the plasma membrane of hematopoietic tissue cells. SDS-PAGE of virions semi-purified from the hemolymph of moribund C. quadricarinatus identified 24 proteins including a 50 kDa major capsid protein (MCP). By summing the sizes of DNA restriction endonuclease fragments, the viral genome was estimated to be ~150 kb in length. A 34 amino acid sequence deduced from a 103 bp MCP gene region amplified by PCR using degenerate primers targeted to MCP gene regions conserved among iridoviruses and chloriridoviruses was most similar (55% identity) to Sergestid iridovirus. Based on virion morphology, protein composition, DNA genome length, and MCP sequence relatedness, the virus identified has tentatively been named Cherax quadricarinatus iridovirus (CQIV). In addition, experimental infection of healthy C. quadricarinatus, Procambarus clarkii, and Litopenaeus vannamei with CQIV caused the same disease and high mortality, suggesting that CQIV poses a potential threat to cultured and wild crayfish and shrimp.

Identification of the interaction domains of white spot syndrome virus envelope proteins VP28 and VP24.

VP28 and VP24 are two major envelope proteins of white spot syndrome virus (WSSV). The direct interaction between VP28 and VP24 has been described in previous studies. In this study, we confirmed this interaction and mapped the interaction domains of VP28 and VP24 by constructing a series of deletion mutants. By co-immunoprecipitation, two VP28-binding domains of VP24 were located at amino acid residues 46-61 and 148-160, while VP24-binding domain of VP28 was located at amino acid residues 31-45. These binding domains were further corroborated by peptide blocking assay, in which synthetic peptides spanning the binding domains were able to inhibit VP28-VP24 interaction, whereas same-size control peptides from non-binging regions did not.

Genomic characterization of a novel iridovirus from redclaw crayfish Cherax quadricarinatus: evidence for a new genus within the family Iridoviridae

A novel iridovirus, Cherax quadricarinatus iridovirus (CQIV), was identified from diseased C. quadricarinatus in 2014. This virus is considered as a new threat to crustacean aquaculture because it is lethal to both peneaid shrimp and crayfish. Here, we determined the complete genome sequence of CQIV. The double-stranded DNA genome is 165 695 bp in length with a G+C content of 34.6 %. A total of 178 open reading frames (ORFs) have been predicted, encoding hypothetical proteins ranging from 50 to 1327 amino acids. Forty-seven of these exhibit similarities to proteins of known functions. Phylogenetic analysis based on multiple alignments of conserved proteins shows that CQIV clusters with the members of the family Iridoviridae, but is placed in a distinct clade from all the five known genera. It indicates that CQIV may represent a new genus in the family Iridoviridae, for which we propose the name Cheraxvirus based on the host organism.

Low-abundance envelope protein VP12 of white spot syndrome virus interacts with envelope protein VP150 and capsid protein VP51

VP12 and VP150 are two minor envelope proteins of white spot syndrome virus (WSSV). In our previous studies, VP12 was found to co-migrate with 53-kDa form of VP150 on two-dimensional Blue Native/SDS-PAGE, suggesting that there is an interaction between them. In this study, we confirmed the interaction by co-immunoprecipitation assay and demonstrated that the binding region with VP12 is located between residues 207 and 803 of VP150. Further studies found that VP12 can be attached to WSSV capsids by interacting with capsid protein VP51. These findings suggest that VP12 may function as a linker protein participating in the linkage between VP12/VP150 complex and viral nucleocapsid.

Different roles of crayfish hemocytes in the uptake of foreign particles

ABSTRACT Crustacean hemocytes are known to remove invading pathogens by phagocytosis. In this study, we investigated how the semigranular cells (SGCs) and granular cells (GCs) of crayfish Cherax quadricarinatus participated in this process. By injecting the animals with excessive amounts of fluorescent microspheres (FMs), we showed that only a small portion of the circulating hemocytes were phagocytic cells, and they took up FMs in a size‐dependent manner. The 0.2 &mgr;m FMs were internalized almost entirely by SGCs, while GCs and SGCs both contributed to the uptake of 2 &mgr;m FMs. Further analysis of the hemocytes from the animals injected with a mixture of FMs suggested that there were a subpopulation of SGCs specifically ingesting 0.2 &mgr;m FMs. The size‐dependent manner was also applied to biological particles. Escherichia coli was internalized by both SGCs and GCs, whereas white spot syndrome virus (WSSV) was mostly ingested by SGCs. However, the bacterial cells were rapidly taken and cleared from the circulation by the hemocytes, while the WSSV virions were gradually internalized and remained in the cells for a relatively longer period of time. These findings provide basic information of the phagocytic hemocytes of crayfish and how they respond to different foreign particles. HighlightsOnly a small portion of crayfish hemocytes are phagocytic cells, including semigranular and granular cells.The phagocytic hemocytes internalize biological and abiological particles in a size‐dependent manner.The internalization kinetics of hemocytes to bacterial cells is different from that to fluorescent microspheres and virus.