Diversity, distribution, and mobility of bro gene sequences in Bombyx mori nucleopolyhedrovirus.

Genetic differences between strains of a baculovirus are often limited to some restriction sites, short DNA deletions or absence of some nonessential genes. The recently coined bro gene family, represents a new major source of intraspecific variability. A comparison between two bro gene sets of Bombyx mori nucleopolyhedroviruses (NPV) shows that bro genes are distributed in three regions for the -T3 and -SC7 virus strains. In BmNPV T3, five bro genes are distributed in three genome locations, whereas the BmNPV SC7 strain possess a single bro copy in each region. In addition, each of the BmNPV SC7 bro genes belongs to one of the three subfamilies present in BmNPV T3. Analysis of bro copy sequences and of adjacent sequences suggests an active redistribution of sequences due to intraspecific recombination. The maintenance of one allele of each subfamily suggests that they play different roles in the viral cycle, and that they are essential.

Comparative analysis of the granulin regions of the Phthorimaea operculella and Spodoptera littoralis granuloviruses.

The nucleotide sequence of two cloned restriction fragments encompassing the granulin genes from the granuloviruses of the potato tuber moth, Phthorimaea operculella, PhopGV, and the Egyptian cotton leaf worm, Spodoptera littoralis, SpliGV, have been determined. Although both viruses are able to infect the same Ph. operculella cell line, their granulins do not cluster in the same phylogenetic branches. PhopGV ganulin is closely related to Cydia pomonella GV (CpGV) and Cryptophlebia leucotreta GV (ClGV) (95.2 and 94% identity at the aminoacid level), while SpliGV granulin falls close to Trichoplusia ni GV and Xestia c-nigrum GV (91.6 and 92.0% respectively). The gene organization around the granulins reflects this clustering. Upstream the PhopGV granulin, an ORF belonging to the ME53 gene family (as ORF 124R of CpGV and 909 of ClGV) is present, while no equivalent ORF is found in this region in SpliGV. Downstream the granulin, both viruses present a gene homologous to the Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 9 followed by a Protein Kinase (AcMNPV ORF10). The structure of this region seems thus conserved not only among nucleopolyhedroviruses but also in at least some granuloviruses.

Protein requirements for assembly of virus-like particles of Junonia coenia densovirus in insect cells.

The coding sequences of four overlapping polypeptides starting at four different in-frame AUG codons and co-terminating at the stop codon of the cap gene of Junonia coenia densovirus (JcDNV) were inserted under the control of the p10 promoter of Autographa californica nucleopolyhedrovirus (AcMNPV) to generate AcMNPV-VP1 (four polypeptides), AcMNPV-VP2 (three polypeptides), AcMNPV-VP3 (two polypeptides), and AcMNPV-VP4 (one polypeptide) recombinant viruses. In all cases, infection of Spodoptera frugiperda cells (Sf9) by each of the four recombinant viruses resulted in the production of virus-like particles (VLPs) 22-25 nm in diameter. The VLPs produced by the three recombinants AcMNPV-VP2, AcMNPV-VP3 and AcMNPV-VP4 were abundant and contained three, two and one polypeptides, respectively. VP4, the shortest polypeptide, thus appears to be sufficient for assembly of VLPs morphologically similar to those formed with two to four polypeptides. The ratio of VPs did not appear to be critical for assembly of the particles. The polypeptide starting at the first AUG immediately downstream from the p10 promoter was always the most abundantly expressed in infected cells, regardless of the construct. In contrast, plaque-purified AcMNPV-VP1 recombinants were unstable and produced less than one-twentieth of the VLPs produced by the others. All VP transcripts started at the TAAG late motif of the p10 promoter and had a poly(A) tail 14 nt downstream of a poly(A) addition signal located 98 nucleotides downstream of the common stop codon. No significant transcription initiation inside the cap sequence of AcMNPV-VP2, AcMNPV-VP3 and AcMNPV-PV4 was observed.

Two key mutations in the host-range specificity domain of the p143 gene of Autographa californica nucleopolyhedrovirus are required to kill Bombyx mori larvae.

Autographa californica nucleopolyhedrovirus (AcMNPV) does not replicate in Bombyx mori cells (Bm5, BmN). We have shown previously that when a short DNA sequence within AcMNPV ORF95, which encodes the viral helicase P143, is replaced with the colinear region of B. mori nucleopolyhedrovirus (BmNPV), AcMNPV gains the ability to replicate in Bm5 cells. To determine the mutational events in the p143 gene required to allow AcMNPV replication in B. mori cells, AcMNPV recombinants produced in Sf9 cells were screened in vivo in B. mori larvae, which are more permissive to baculovirus infection than B. mori cell lines. Eight combinations of mutations were tested and characterization of viral DNA extracted from dead larvae showed that amino acid changes at position 564 and 577 are required to kill B. mori larvae.

The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene.

Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase. This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells. The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon. The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ. This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells. Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins. Shut-down of host protein synthesis was not abolished in recombinant infection. When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution. This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.

Establishment of a cell line derived from embryos of the potato tuber moth phthorimaea operculella (Zeller).

A cell line from the main insect pest of potatoes in tropical and subtropical areas, Phthorimaea operculella (Zeller), was obtained from embryoculture. These cells were cultured in Grace's modified medium. The cell line, designated ORS-Pop-93, had a heterogeneous population consisting of spherical and spindle cells with great capacity to adhere and a doubling time of 40 h. They were subcultured for more than 60 passages. Their polypeptidic profile was different from profiles of other lepidopteran cell lines. The cell line supports the multiplication of the Autographa californica nuclear polyhedrosis virus.

Nucleotide sequence of the polyhedrin gene of Spodoptera littoralis multiple nucleocapsid nuclear polyhedrosis virus.

1286 nt portion of the 3.1 kbp HindIII-K fragment of the Spodoptera littoralis multiple nucleocapsid nuclear polyhedrosis virus (SIMNPV) DNA genome has been sequenced. The sequence contains the polyhedrin gene preceded by a highly-repeated AGATAA-rich sequence. With 249 amino acids, SIMNPV polyhedrin is the longest known polyhedrin.

Extension of Autographa californica nuclear polyhedrosis virus host range by interspecific replacement of a short DNA sequence in the p143 helicase gene.

Recombinant baculoviruses obtained by coinfection of insect cells with Autographa californica and Bombyx mori nuclear polyhedrosis viruses (AcNPV and BmNPV, respectively) possess a wider in vitro host range than either parent virus. To localize the DNA sequences responsible for this species specificity, we used a two-step method of production and selection of recombinant viruses with altered specificity. Sf9 cells, which are permissive for AcNPV, were first cotransfected with genomic AcNPV DNA and a complete or incomplete set of BmNPV restriction fragments. AcNPV-BmNPV recombinants from the Sf9 supernatant were then selected on the basis of ability to replicate in B. mori Bm5 cells, which are not permissive for AcNPV. Cotransfection of AcNPV DNA with the 7.6-kbp BmNPV Sma I-C fragment was sufficient to produce recombinants able to infect both Sf9 and Bm5 cells. A series of cotransfections with subclones of this fragment defined a 79-nt sequence within the p143 helicase gene capable of extending AcNPV host range in vitro. In this 79-nt region, BmNPV and AcNPV differ at six positions, corresponding to four amino acid substitutions. The involvement of the 79-nt region in species specificity control was confirmed by cotransfecting AcNPV DNA and gel-purified polymerase chain reaction products derived from the BmNPV p143 gene. Replacement in the AcNPV genome of three AcNPV-specific amino acids by the three corresponding BmNPV-specific amino acids at positions 556, 564, and 577 of the p143 protein extends AcNPV host range to B. mori larvae.

Drosophila S virus is a member of the Reoviridae family.

The S character of Drosophila simulans, the absence or malformation or both of bristles and other cuticular structures, was described by Comendador (Drosophila Inf. Serv. 55:26-28, 1980). Its characteristics (maternal transmission, low pathogenicity, and sensitivity to temperature) suggested the existence of a virus as the causative agent. Indeed, reoviruslike particles were found in subcuticular cells of S individuals, and its association with S phenotypic expression was shown. This virus was called Drosophila S virus (DSV) (C. Louis, M. López-Ferber, N. Plus, G. Kuhl, and S. Baker, J. Virol. 62:1266-1270, 1988). We report here the purification and analysis of some properties of DSV particles, the morphology (spherical, 60 nm in diameter with an electron dense central core and less dense shell) and genome composition (double-stranded RNA divided into segments), which classify DSV as a new member of the family Reoviridae.

A physical map of Spodoptera littoralis B-type nuclear polyhedrosis virus genome.

A physical map for the genome of Spodoptera littoralis (Egyptian cottonworm) nuclear polyhedrosis virus of B-type (S1MNPV-B) [Cherry CL, Summers MD (1985) J Invertebr Pathol 46: 289-295] was prepared using the restriction endonucleases SmaI, SfiI, NotI, ApaI, KpnI, BstEII, PstI, SacI, and HindIII. The size of the genome was estimated to 136 kbp for the Morocco isolate (S1MNPV-M2) and to 135 kbp for the Lyon isolate (S1MNPV-L4). HindIII-K was used as the zero-point of the map because it hybridized to EcoRI-I and BamHI-F fragment of Autographa californica (AcMNPV) DNA, and the direction of transcription of the S1MNPV polyhedrin gene was chosen left to right. Hybridization of six AcMNPV cloned DNA fragments with S1MNPV-B genome shows that S1MNPV-B and AcMNPV genomic structures are not aligned.

Insect lysozymes from three species of Lepidoptera: their structural relatedness to the C (chicken) type lysozyme.

Sequence studies of the N-terminal halves of the lysozymes isolated from Bombyx mori, Gallera mellonella and Spodoptera littoralis (Lepidoptera) allow us to classify these enzymes among the c (chicken) type lysozymes.

[Estimation of the molecular weight of the matrix protein of insect baculovirus inclusion bodies (author's transl)]. / Evaluation du poids moléculaire de la protéine des corps k'inclusion de divers Baculovirus d'insectes.

The proteinaceous matrix of 23 insect Baculovirus inclusions consists of a single polypeptide of molecular weight varying from 25,100 to 31,360 daltons depending upon the virus and in correlation wwith taxonomic position of the host.