Nucleocapsid protein VP15 is the basic DNA binding protein of white spot syndrome virus of shrimp.

White spot syndrome virus (WSSV) is type species of the genus Whispovirus of the new family Nimaviridae. Despite the elucidation of its genomic sequence, very little is known about the virus as only 6% of its ORFs show homology to known genes. One of the structural virion proteins, VP15, is part of the nucleocapsid of the virus and shows homology to some putative baculovirus DNA binding proteins. These DNA-binding or histone-like proteins are thought to be involved in the condensation and packaging of the genome in the nucleocapsid. Using bacterially expressed VP15 fusion proteins in ELISA and Far-Western experiments showed that VP15 interacts with itself, forming homomultimers, but not with the other major structural proteins of the WSSV virion. Antibodies against phosphorylated proteins revealed that VP15 originating from different sources was not phosphorylated. WSSV VP15 binds non-specifically to double-stranded DNA, but has a clear preference to supercoiled DNA suggesting that VP15 is involved in the packaging of the WSSV genome in the nucleocapsid. This research shed further light on the composition of WSSV virions and the function of one of its nucleocapsid proteins.

Genetic variation among isolates of White spot syndrome virus.

White spot syndrome virus (WSSV), member of a new virus family called Nimaviridae, is a major scourge in worldwide shrimp cultivation. Geographical isolates of WSSV identified so far are very similar in morphology and proteome, and show little difference in restriction fragment length polymorphism (RFLP) pattern. We have mapped the genomic differences between three completely sequenced WSSV isolates, originating from Thailand (WSSV-TH), China (WSSV-CN) and Taiwan (WSSV-TW). Alignment of the genomic sequences of these geographical isolates revealed an overall nucleotide identity of 99.32%. The major difference among the three isolates is a deletion of approximately 13 kb (WSSV-TH) and 1 kb (WSSV-CN), present in the same genomic region, relative to WSSV-TW. A second difference involves a genetically variable region of about 750 bp. All other variations >2 bp between the three isolates are located in repeat regions along the genome. Except for the homologous regions ( hr1, hr3, hr8 and hr9), these variable repeat regions are almost exclusively located in ORFs, of which the genomic repeat regions in ORF75, ORF94 and ORF125 can be used for PCR based classification of WSSV isolates in epidemiological studies. Furthermore, the comparison identified highly invariable genomic loci, which may be used for reliable monitoring of WSSV infections and for shrimp health certification.

White spot syndrome virus envelope protein VP28 is involved in the systemic infection of shrimp.

White spot syndrome virus (WSSV) is a large DNA virus infecting shrimp and other crustaceans. The virus particles contain at least five major virion proteins, of which three (VP26, VP24, and VP15) are present in the rod-shaped nucleocapsid and two (VP28 and VP19) reside in the envelope. The mode of entry and systemic infection of WSSV in the black tiger shrimp, Penaeus monodon, and the role of these proteins in these processes are not known. A specific polyclonal antibody was generated against the major envelope protein VP28 using a baculovirus expression vector system. The VP28 antiserum was able to neutralize WSSV infection of P. monodon in a concentration-dependent manner upon intramuscular injection. This result suggests that VP28 is located on the surface of the virus particle and is likely to play a key role in the initial steps of the systemic WSSV infection in shrimp.

The white spot syndrome virus DNA genome sequence.

White spot syndrome virus (WSSV) is at present a major scourge to worldwide shrimp cultivation. We have determined the entire sequence of the double-stranded, circular DNA genome of WSSV, which contains 292,967 nucleotides encompassing 184 major open reading frames (ORFs). Only 6% of the WSSV ORFs have putative homologues in databases, mainly representing genes encoding enzymes for nucleotide metabolism, DNA replication, and protein modification. The remaining ORFs are mostly unassigned, except for five, which encode structural virion proteins. Unique features of WSSV are the presence of a very long ORF of 18,234 nucleotides, with unknown function, a collagen-like ORF, and nine regions, dispersed along the genome, each containing a variable number of 250-bp tandem repeats. The collective information on WSSV and the phylogenetic analysis on the viral DNA polymerase suggest that WSSV differs profoundly from all presently known viruses and that it is a representative of a new virus family.

Identification and phylogeny of a protein kinase gene of white spot syndrome virus.

White spot syndrome virus (WSSV) is a virus infecting shrimp and other crustaceans, which is unclassified taxonomically. A 2193 bp long open reading frame, encoding a putative protein kinase (PK), was found on a 8.4 kb EcoRI fragment of WSSV proximal to the gene for the major envelope protein (VP28). The identified PK shows a high degree of homology to other viral and eukaryotic PK genes. Homology in the catalytic domains suggests that this PK is a serine/threonine protein kinase. All of the conserved PK domains are present in the WSSV PK gene product and this allowed the alignment with PK proteins from other large DNA viruses, which encode one or more PK proteins. An unrooted parsonimous phylogenetic tree was constructed and indicated that the PK gene is well conserved in all DNA virus families and hence can be used as a phylogenetic marker. Baculoviruses to date contain only a single PK gene, which is present in a separate well bootstrap-supported branch in the tree. The WSSV PK is not present in the baculovirus clade and therefore is clearly separated phylogenetically from the baculovirus PK genes. Furthermore, the WSSV PK gene does not share a most recent common ancestor with any known PK gene from other viruses. This provides further and independent evidence for the unique position of WSSV in a newly proposed genus named Whispovirus.

Identification and phylogeny of a non-specific endonuclease gene of white spot syndrome virus of shrimp.

White spot syndrome virus (WSSV) is a taxonomically unclassified virus which causes a disease in shrimps worldwide. A 936 bp long open reading frame (ORF) was found on a 7.2 kb HindIII fragment of the DNA genome of WSSV located adjacent to the ribonucleotide reductase small subunit gene. This putative ORF showed homology to prokaryotic and eukaryotic endonucleases, which contain a non-specific endonuclease motif. Alignment with viral and eukaryotic endonuclease ORFs revealed that most catalytically and structurally important amino acid residues were present in the putative WSSV non-specific endonuclease gene. An unrooted parsimonous phylogenetic tree of non-specific endonucleases indicated that the WSSV ORF was located in a well bootstrap supported clade containing only arthopods, including one of WSSV's natural hosts, Penaeus japonicus. A similar conjunction was found for the only other viral homologue, present in Fowlpox virus, which was also found in a well bootstrap-supported clade with its natural host, Gallus gallus. This clustering of virus and host suggests that both WSSV and Fowlpox virus may have acquired their nuclease genes from their respective natural hosts. Because the motif for non-specific nucleases is found in only two viruses, this gene cannot be used to clarify the taxonomic position of WSSV. However, the presence of this type of nuclease rarely found in viruses adds a novel feature to WSSV.

Transcriptional analysis of the ribonucleotide reductase genes of shrimp white spot syndrome virus.

The causative agent of white spot syndrome (WSS) is a large double-stranded DNA virus, WSSV, which is probably a representative of a new genus, provisionally called Whispovirus. From previously constructed WSSV genomic libraries of a Taiwan WSSV isolate, clones with open reading frames (ORFs) that encode proteins with significant homology to the class I ribonucleotide reductase large (RR1) and small (RR2) subunits were identified. WSSV rr1 and rr2 potentially encode 848 and 413 amino acids, respectively. RNA was isolated from WSSV-infected shrimp at different times after infection and Northern blot analysis with rr1- and rr2-specific riboprobes found major transcripts of 2.8 and 1.4 kb, respectively. 5' RACE showed that the major rr1 transcript started at a position of -84 (C) relative to the ATG translational start, while transcription of the rr2 gene started at nucleotide residue -68 (T). A consensus motif containing the transcriptional start sites for rr1 and rr2 was observed (TCAc/tTC). Northern blotting and RT-PCR showed that the transcription of rr1 and rr2 started 4-6 h after infection and continued for at least 60 h. The rr1 and rr2 genes thus appear to be WSSV "early genes."

Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions.

White spot syndrome is a worldwide disease of penaeid shrimp. The disease agent is a bacilliform, enveloped virus, white spot syndrome virus (WSSV), with a double-stranded DNA genome that probably contains well over 200 kb. Analysis of a 12.3 kb segment of WSSV DNA revealed eight open reading frames (ORFs), including the genes for the large (RR1) and small (RR2) subunits of ribonucleotide reductase. The rr1 and rr2 genes were separated by 5760 bp, containing several putative ORFs and two domains with multiple sequence repeats. The first domain contained six direct repeats of 54 bp and is part of a coding region. The second domain had one partial and two complete direct repeats of 253 bp at an intergenic location. This repeat, located immediately upstream of rr1, has homologues at several other locations on the WSSV genome. Phylogenetic analysis of RR1 and RR2 indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and, further, seems to suggest that WSSV and baculoviruses probably do not share an immediate common ancestor. The present analysis of WSSV favours the view that this virus is either a member of a new genus (Whispovirus) within the Baculoviridae or a member of an entirely new virus family.

Identification of two major virion protein genes of white spot syndrome virus of shrimp.

White Spot Syndrome Virus (WSSV) is an invertebrate virus, causing considerable mortality in shrimp. Two structural proteins of WSSV were identified. WSSV virions are enveloped nucleocapsids with a bacilliform morphology with an approximate size of 275 x 120 nm, and a tail-like extension at one end. The double-stranded viral DNA has an approximate size 290 kb. WSSV virions, isolated from infected shrimps, contained four major proteins: 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), and 19 kDa (VP19) in size, respectively. VP26 and VP24 were found associated with nucleocapsids; the others were associated with the envelope. N-terminal amino acid sequences of nucleocapsid protein VP26 and the envelope protein VP28 were obtained by protein sequencing and used to identify the respective genes (vp26 and vp28) in the WSSV genome. To confirm that the open reading frames of WSSV vp26 (612) and vp28 (612) are coding for the putative major virion proteins, they were expressed in insect cells using baculovirus vectors and analyzed by Western analysis. A polyclonal antiserum against total WSSV virions confirmed the virion origin of VP26 and VP28. Both proteins contained a putative transmembrane domain at their N terminus and many putative N- and O-glycosylation sites. These major viral proteins showed no homology to baculovirus structural proteins, suggesting, together with the lack of DNA sequence homology to other viruses, that WSSV may be a representative of a new virus family, Whispoviridae.