The Complete Genome of an Endogenous Nimavirus (Nimav-1_LVa) From the Pacific Whiteleg Shrimp Penaeus (Litopenaeus) Vannamei.

White spot syndrome virus (WSSV), the lone virus of the genus Whispovirus under the family Nimaviridae, is one of the most devastating viruses affecting the shrimp farming industry. Knowledge about this virus, in particular, its evolution history, has been limited, partly due to its large genome and the lack of other closely related free-living viruses for comparative studies. In this study, we reconstructed a full-length endogenous nimavirus consensus genome, Nimav-1_LVa (279,905 bp), in the genome sequence of Penaeus (Litopenaeus) vannamei breed Kehai No. 1 (ASM378908v1). This endogenous virus seemed to insert exclusively into the telomeric pentanucleotide microsatellite (TAACC/GGTTA)n. It encoded 117 putative genes, with some containing introns, such as g012 (inhibitor of apoptosis, IAP), g046 (crustacean hyperglycemic hormone, CHH), g155 (innexin), g158 (Bax inhibitor 1 like). More than a dozen Nimav-1_LVa genes are involved in the pathogen-host interactions. We hypothesized that g046, g155, g158, and g227 (semaphorin 1A like) were recruited host genes for their roles in immune regulation. Sequence analysis indicated that a total of 43 WSSV genes belonged to the ancestral/core nimavirus gene set, including four genes reported in this study: wsv112 (dUTPase), wsv206, wsv226, and wsv308 (nucleocapsid protein). The availability of the Nimav-1_LVa sequence would help understand the genetic diversity, epidemiology, evolution, and virulence of WSSV.

ICTV Virus Taxonomy Profile: Nimaviridae.

The family Nimaviridae includes the single species White spot syndrome virus, isolates of which infect a wide range of aquatic crustaceans and cause substantial economic losses. Virions are ellipsoid to bacilliform with a terminal thread-like extension. The circular dsDNA genome is 280-307 kbp with several homologous repeat regions. More than 80 structural and functional proteins have been characterized from 531 ORFs. White spot syndrome is a highly lethal, contagious disease associated with white spot syndrome virus infection of shrimps. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Nimaviridae, which is available at www.ictv.global/report/nimaviridae.

Characterization of fossilized relatives of the White Spot Syndrome Virus in genomes of decapod crustaceans.

BACKGROUND: The White Spot Syndrome Virus (WSSV) is an important pathogen that infects a variety of decapod species and causes a highly contagious disease in penaeid shrimps. Mass mortalities caused by WSSV have pronounced commercial impact on shrimp aquaculture. Until now WSSV is the only known member of the virus family Nimaviridae, a group with obscure phylogenetic affinities. Its isolated position makes WSSV studies challenging due to large number of genes without homology in other viruses or cellular organisms. RESULTS: Here we report the discovery of an unusually large amount of sequences with high similarity to WSSV in a genomic library from the Jamaican bromeliad crab Metopaulias depressus. De novo assembly of these sequences allowed for the partial reconstruction of the genome of this endogenized virus with total length of 200 kbp encompassed in three scaffolds. The genome includes at least 68 putative open reading frames with homology in WSSV, most of which are intact. Among these, twelve orthologs of WSSV genes coding for non-structural proteins and nine genes known to code for the major components of the WSSV virion were discovered. Together with reanalysis of two similar cases of WSSV-like sequences in penaeid shrimp genomic libraries, our data allowed comparison of gene composition and gene order between different lineages related to WSSV. Furthermore, screening of published sequence databases revealed sequences with highest similarity to WSSV and the newly described virus in genomic libraries of at least three further decapod species. Analysis of the viral sequences detected in decapods suggests that they are less a result of contemporary WSSV infection, but rather originate from ancestral infection events. Phylogenetic analyses suggest that genes were acquired repeatedly by divergent viruses or viral strains of the Nimaviridae. CONCLUSIONS: Our results shed new light on the evolution of the Nimaviridae and point to a long association of this viral group with decapod crustaceans.

Insect cell surface expression of hemagglutinin (HA) of Egyptian H5N1 avian influenza virus under transcriptional control of whispovirus immediate early-1 promoter.

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

Molecular cloning and characterization of a cathepsin B gene from the Chinese shrimp Fenneropenaeus chinensis.

Cathepsin B is a unique member of the cathepsin superfamily, which acts as both an endopeptidase and peptidyl-dipeptidase. To obtain a better understanding of this enzyme, we cloned a cDNA encoding cathepsin B from the muscle of Fenneropenaeus chinensis (FcCB). FcCB contained a 996-bp open reading frame (ORF) encoding a protein of 331 amino acid residues with a putative signal peptide and a propeptide_C1 at the N-terminal, a glutamine oxyanion hole and active site cysteine, histidine and asparagine residues. A region from residue 79 to 327 conferred the peptidase activity of FcCB. Pair-wise and multiple sequence alignment with 17 other organisms, including ten different vertebrate species, five different invertebrate species and two different plant species, indicated that the signal peptide and the propeptide_C1 at the N-terminal of FcCB were less conserved than the mature protein, except when compared with Penaeus monodon, Litopenaeus vannamei and Marsupenaeus japonicas, all of which belong to the genus Penaeus. The expression of FcCB in the hepatopancreas was higher than that in the gill. The expression of FcCB in the gill was higher than that in the muscle. A challenge test was performed to reveal the responses of FcCB in different tissues to white spot syndrome virus (WSSV) infection, which causes serious economic losses in the shrimp farming industry. The FcCB gene expressions in the ectoderm, mesoderm and entoderm were not the same prior to WSSV infection, but at 6 h after WSSV challenge, the FcCB expression in the gill, hepatopancreas and muscle was up-regulated, suggesting that FcCB might be involved in the immune response to WSSV. Three single nucleotide polymorphisms (SNPs) were identified in the FcCB gene, involving C/T transitions, which are known as mutation hot spots. Notably, the three SNPs constituted a haplotype that can be used as an indicator of the haplotype block. The SNP genotypes of two groups of shrimps, respectively comprising 96 WSSV-resistant shrimps and 96 WSSV-susceptible shrimps, were obtained using a high-resolution melting (HRM) method. Associated factors, including observed heterozygosity (Ho), expected heterozygosity (He), minor allele frequency (MAF) and P-values for the deviation from Hardy-Weinberg equilibrium (HWE), were obtained. For the association analysis with WSSV resistance, the P-values were calculated using Pearson's chi-square test. In the two groups, the MAFs of all sites were greater than 0.05, and no site departed significantly (P < 0.05) from HWE. The genotype distribution of the C-984T mutation site between the two groups was not significantly different. These results lead to a better understanding of the molecular mechanisms of the host-virus interaction and provide useful information for solving the WSSV problem.

Virus-binding proteins and their roles in shrimp innate immunity.

Disease outbreaks caused by viral pathogens constitute a major limitation to development of the shrimp aquaculture industry. Many research have been conducted to better understand how host shrimp respond to viral infections with the aim of using the gained knowledge to develop better strategies for disease management and control. One approach has been to study the interactions between host and viral proteins, and particularly host virus-binding proteins that might play an important role in the viral infection process. Within the past five years, increasing numbers of virus-binding proteins (VBPs) have been reported in shrimp. Characterization of these molecules has emphasized on their potential therapeutic applications by demonstrating their activities in inhibition of viral replication via in vivo neutralization assay. However, signaling to induce innate antiviral immune responses as a consequence of binding between viral proteins and VBPs remain to be fully elucidated.

Evaluation of immune response and resistance to diseases in tiger shrimp, Penaeus monodon fed with biofilm of Vibrio alginolyticus.

Immune response in juvenile tiger shrimp, Penaeus monodon fed with biofilm (BF) and free cells (FC) of Vibrio alginolyticus was studied by evaluating the hemocyte count, phenoloxidase activity and antibacterial activity. The above immune responses were higher in BF fed shrimp than that in FC fed or control shrimp. Among the different doses of BF of V. alginolyticus tested, 10(9) cfu g(-1) shrimp day(-1) for two weeks could evoke higher immune response. BF fed shrimp were more resistant to injection challenge with V. alginolyticus and whitespot syndrome virus (WSSV) with significantly higher RPS compared to that with FC fed and control shrimp. Better resistance was also reflected by rapid clearance of V. alginolyticus and WSSV from the hemolymph as confirmed by immunodot and histopathology.

Heat shock cognate protein 70 gene is required for prevention of apoptosis induced by WSSV infection.

Here, we show that heat shock cognate protein 70 (Hsc70) in shrimp cells can inhibit apoptosis induced by white spot syndrome virus (WSSV) infection. Caspase-3 protease activity of hemocytes increased significantly, correlating with a reduction in endogenous Hsc70 late in WSSV infection. Hsc70 dsRNA caused a significant increase in caspase-3 activity in the hemocytes of non-infected shrimp and WSSV-infected shrimp. We propose that upregulation of Hsc70 expression early in WSSV infection may also be used to prevent premature apoptotic cell death, and the precipitous downregulation of Hsc70 late in WSSV infection may lead to the timed induction of apoptosis.

Three tetraspanins from Chinese shrimp, Fenneropenaeus chinensis, may play important roles in WSSV infection.

Three members of the tetraspanin/TM(4)SF superfamily were cloned from Chinese shrimp, Fenneropenaeus chinensis. The deduced amino acid sequences of the three proteins have typical motifs of the tetraspanin/TM(4)SF superfamily. Phylogenetic analysis of the proteins, together with the known tetraspanins of invertebrates and vertebrates, revealed that they belong to different tetraspanin subfamilies: CD9, CD63 and tetraspanin-3. The three cloned genes of CD9, CD63 and tetraspanin-3 showed apparently different tissue distributions. The CD9 gene (FcCD9) was specifically expressed in the hepatopancreas. While for the CD63 gene (FcCD63), the highest expression was detected in nerves, epidermis and heart, with low expression in haemocytes, ovary, gill, hepatopancreas and stomach and no expression in intestine, muscle and lymphoid organ. Compared with FcCD9 and FcCD63, the tetraspanin-3 gene (FcTetraspanin-3) was more broadly expressed and its highest expression was detected in the intestine. Its expression in nerves was lower than in the intestine, but was higher than in other tissues. Expression in haemocytes, ovary and muscle was much lower than in other tissues. The expression profiles of FcCD9, FcCD63 and FcTetraspanin-3 in different tissues, including haemocytes, lymphoid organ and hepatopancreas, were compared by real-time PCR when shrimp were challenged by live white spot syndrome virus (WSSV) and heat-inactivated WSSV. All three tetraspanins were markedly up-regulated in the live WSSV-challenged shrimp tissues. The data suggested that the three cloned members of TM(4)SF superfamily in Chinese shrimp may play a key role in the route of WSSV infection.

Whispovirus.

During the last two decades, a combination of poor management practices and intensive culturing of penaeid shrimp has led to the outbreak of several viral diseases. White spot disease (WSD) is one of the most devastating and it can cause massive death in cultured shrimp. Following its first appearance in 1992-1993 in Asia, this disease spread globally and caused serious economic losses. The causative agent of WSD is white spot syndrome virus (WSSV), which is a large, nonoccluded, enveloped, rod- or elliptical-shaped, dsDNA virus of approximately 300 kbp. WSSV has a very broad host range among crustaceans. It infects many tissues and multiplies in the nucleus of the target cell. WSSV is a lytic virus, and in the late stage of infection, the infected cells disintegrate, causing the destruction of affected tissues. The WSSV genome contains at least 181 ORFs. Most of these encode proteins that show no homology to known proteins, although a few ORFs encode proteins with identifiable features, and these are mainly involved in nucleotide metabolism and DNA replication. Nine homologous regions with highly repetitive sequences occur in the genome. More than 40 structural protein genes have been identified, and other WSSV genes with known functions include immediate early genes, latency-related genes, ubiquitination-related genes, and anti-apoptosis genes. Based on temporal expression profiles, WSSV genes can be classified as early or late genes, and they are regulated as coordinated cascades under the control of different promoters. Both genetic analyses and morphological features reveal the uniqueness of WSSV, and therefore it was recently classified as the sole species of a new monotypic family called Nimaviridae (genus Whispovirus).

Efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie1 promoter, a novel shuttle promoter between insect cells and mammalian cells.

Recombinant baculoviruses have emerged as a new gene delivery vehicle for mammalian cells. Thus, a shuttle promoter that mediates gene expression in both insect and mammalian cells will facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle. This study described the generation of three recombinant baculoviruses with an EGFP reporter gene under the control of the white spot syndrome virus (WSSV) ie1 promoter, or either of two control promoters, the baculovirus early-to-late (ETL) promoter and polyhedrin promoter. The resulting recombinant baculoviruses were used to infect insect Sf9 cells and transduce several mammalian cell lines to test the expression of EGFP. We found that the WSSV ie1 promoter displayed a strong promoter activity in both insect and mammalian cells, and showed a stronger promoter activity than the ETL promoter in some mammalian cell lines. The activity of the WSSV ie1 promoter, but not the ETL promoter, can be enhanced by sodium butyrate, a histone deacetylase inhibitor. A transient plasmid transfection assay indicated that the WSSV ie1 promoter activity in mammalian cells is independent of baculovirus gene expression, differing from the ETL promoter, which was shown to be baculovirus-dependent. This study demonstrates, for the first time, that the WSSV ie1 promoter can function as a baculovirus-independent shuttle promoter between insect cells and mammalian cells. This novel shuttle promoter will facilitate the application of baculovirus-based vectors in gene expression, gene therapy, and non-replicative vector vaccines.