Summary. Recent evidence from genome sequence analyses demands a substantial revision of the taxonomy and classification of the family Baculoviridae. Comparisons of 29 baculovirus genomes indicated that baculovirus phylogeny followed the classification of the hosts more closely than morphological traits that have previously been used for classification of this virus family. On this basis, dipteran-and hymenopteran-specific nucleopolyhedroviruses (NPV) should be separated from lepidopteran-specific NPVs and accommodated into different genera. We propose a new classification and nomenclature for the genera within the baculovirus family. According to this proposal the updated classification should include four genera: Alphabaculovirus (lepidopteran-specific NPV), Betabaculovirus (lepidopteran-specific Granuloviruses), Gammabaculovirus (hymenopteran-specific NPV) and Deltabaculovirus (dipteran-specific NPV). * The taxonomy and classification of living organisms is itself a living and steadily evolving process. The same holds true for taxonomy and classification of viruses.

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.

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White spot syndrome virus (WSSV) occurs worldwide and causes high mortality and considerable economic damage to the shrimp farming industry. No adequate treatments against this virus are available. It is generally accepted that invertebrates such as shrimp do not have an adaptive immune response system such as that present in vertebrates. As it has been demonstrated that shrimp surviving a WSSV infection have higher survival rates upon subsequent rechallenge, we investigated the potential of oral vaccination of shrimp with subunit vaccines consisting of WSSV virion envelope proteins. Penaeus monodon shrimp were fed food pellets coated with inactivated bacteria overexpressing two WSSV envelope proteins, VP19 and VP28. Vaccination with VP28 showed a significant lower cumulative mortality compared to vaccination with bacteria expressing the empty vectors after challenge via immersion (relative survival, 61%), while vaccination with VP19 provided no protection. To determine the onset and duration of protection, challenges were subsequently performed 3, 7, and 21 days after vaccination. A significantly higher survival was observed both 3 and 7 days postvaccination (relative survival, 64% and 77%, respectively), but the protection was reduced 21 days after the vaccination (relative survival, 29%). This suggests that contrary to current assumptions that invertebrates do not have a true adaptive immune system, a specific immune response and protection can be induced in P. monodon. These experiments open up new ways to benefit the WSSV-hampered shrimp farming industry.

Several phylogenetic methods based on whole genome sequence data were evaluated using data from nine complete baculovirus genomes. The utility of three independent character sets was assessed. The first data set comprised the sequences of the 63 genes common to these viruses. The second set of characters was based on gene order, and phylogenies were inferred using both breakpoint distance analysis and a novel method developed here, termed neighbor pair analysis. The third set recorded gene content by scoring gene presence or absence in each genome. All three data sets yielded phylogenies supporting the separation of the Nucleopolyhedrovirus (NPV) and Granulovirus (GV) genera, the division of the NPVs into groups I and II, and species relationships within group I NPVs. Generation of phylogenies based on the combined sequences of all 63 shared genes proved to be the most effective approach to resolving the relationships among the group II NPVs and the GVs. The history of gene acquisitions and losses that have accompanied baculovirus diversification was visualized by mapping the gene content data onto the phylogenetic tree. This analysis highlighted the fluid nature of baculovirus genomes, with evidence of frequent genome rearrangements and multiple gene content changes during their evolution. Of more than 416 genes identified in the genomes analyzed, only 63 are present in all nine genomes, and 200 genes are found only in a single genome. Despite this fluidity, the whole genome-based methods we describe are sufficiently powerful to recover the underlying phylogeny of the viruses.

Aggregations of 27 nm virus-like particles were observed in electron microscopy images of sectioned Varroa destructor mite tissue. The scattered occurrence of individual particles and accumulation of the virions in lattices in the cytoplasm gave an apparent indication that the virus replicates in the mite. Sequence analysis of the RNA of the purified virus revealed a genome organization with high similarity to that of members of the genus Iflavirus. Phylogenetic analysis of the polymerase showed that the virus was related most closely to Deformed wing virus (DWV) and Kakugo virus (KV) of bees. The virus has a genome of 10 112 nt without the poly(A) tail, with an overall RNA genome identity of 84 % to those of DWV and KV and has one large ORF, translated into a 2893 aa polyprotein with an amino acid identity of 95 % to those of DWV and KV. The first 1455 nt of the ORF encoding the lower molecular mass structural proteins shows the greatest diversion from those of DWV and KV, with an RNA identity of 79 %, and translates to a polypeptide of 485 aa with an identity of 90 %. The name proposed for this virus is Varroa destructor virus 1 (VDV-1). To determine whether VDV-1 replicates in mites, a selective RT-PCR was done to detect the presence of the negative-sense RNA strand. The virus isolate and the closely related DWV could be discriminated by two primer sets, each specific to one virus. Both viruses replicated in the population of the mite species studied.

Chikungunya virus nonstructural protein nsP3 has an essential but unknown role in alphavirus replication and interacts with Ras-GAP SH3 domain-binding protein (G3BP). Here we describe the first known function of nsP3, to inhibit stress granule assembly by recruiting G3BP into cytoplasmic foci. A conserved SH3 domain-binding motif in nsP3 is essential for both nsP3-G3BP interactions and viral RNA replication. This study reveals a novel role for nsP3 as a regulator of the cellular stress response.

By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa calafornica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2, and PE38 stimulate DNA replication. No stimulation by the AcMNPVpcna gene, encoding a protein with sequence homology to proliferating-cell nuclear antigen, was observed. A pattern of amino acids found in a number of single-stranded-DNA-binding proteins was identified in the carboxyl-terminal region of IE-1.The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) is the type species of the Baculoviridae, a large family of insect viruses, and has a circular, supercoiled DNA genome of -134 kb (1, 2). It has been extensively exploited for the overexpression of eukaryotic genes and is being engineered for possible use as a viral insecticide. Despite its widespread use, little is known about the mechanism by which AcMNPV DNA replicates. Eight regions distributed around the genome have been identified as putative origins of DNA replication (3-6). Seven of these origins (ori) are located within homologous regions (hr) (Fig. 1), which contain repeats of closely related imperfect palindromes (8). One origin is located within the HindIII-K fragment, which does not contain a hr (9).To date only a putative helicase gene (10) and a putative DNA polymerase gene (11,12) have been identified as essential for baculovirus DNA replication. In addition, a gene (pcna) encoding a protein resembling proliferating-cell nuclear antigen (PCNA), which is a DNA polymerase processivity factor in other systems, has been identified in Ac-MNPV (13), but its role in DNA replication has not been determined.With a transient replication assay, six large regions of the AcMNPV genome were identified that contain one or more genes involved in DNA replication (7). In this report, this assay was used for the identification of six genes encoding proteins essential for AcMNPV DNA replication and three genes whose products stimulate DNA replication.MATERIALS AND METHODS Cells and Virus. Spodoptera frugiperda Sf9 cells (14) were cultured in TNM-FH medium (15), supplemented with 10% fetal bovine serum (FBS). The E2 strain ofAcMNPV (16) was used as wild-type virus. Routine cell culture maintenance and virus infection procedures were carried out as described (17).Plasmid Constructs. The nine replication genes were identified within six regions previously shown to be essential for DNA replication (ref. 7; see also Fig. 1). Subclones of each region were tested for their ability to substitute for the larger parental clone. The following clones were constructed. lef-) is located on the EcoRI-O fragment (18) and was cloned as an Nru I-EcoRI fragment (m.u. 7.5-8.7, ref. 2) into plasmid pUC19. lef-2 is on EcoRI-I (19, 20) and was cloned as an Mlu I fragment (m.u. 1.9-2.6) with Mlu I-Bgl II linkers into the BamHI site of pUC19. The DNA polym...