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During plaque purification of Spodoptera littoralis nucleopolyhedrovirus in S. littoralis Sl52 cell culture, a deletion mutant virus was isolated. Analysis of the biological properties of this mutant virus revealed an absence of per os infectivity of the occluded virus. Infectivity by injection of the non-occluded (budded) virus is not different between the wild-type and the deleted virus. Restriction analysis of the mutant virus genome revealed a 4·5 kb deletion within the NotI D fragment. The observed phenotype was mapped to the deleted region by rescue experiments. The deletion was characterized and the equivalent DNA fragment on the wild-type virus was sequenced. By co-transfecting the DNA of the deleted virus with plasmids derived from the wild-type virus, it was possible to determine that ORF 7 in this fragment is responsible for the observed phenotype. ORF 7, called pif (per os infectivity factor), is homologous to ORF 119 of Autographa californica nucleopolyhedrovirus. Similar ORFs are present in all sequenced baculoviruses. The product of this gene is an occlusion body-derived virion structural protein required only for the first steps of larva infection, as viruses being produced in cells expressing the gene but not containing it in their genomes are able to produce successful infections.

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.

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.

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.

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.

Autographa californica nucleopolyhedrovirus (AcMNPV) lef-4 gene [ORF 90; Ayres et al. (1994) Virology 202, 586–605] is involved in both late and very late gene expression [Passarelli and Miller (1993) Virology 197, 704–714]. The transcriptional properties of this gene have been analyzed. It is transcribed as a single 1.6-kb mRNA and transcripts were first detected 3 h postinfection (pi). The extremities of the transcript have been mapped by primer extension and 3′ RACE–PCR to positions −56 from the translation start codon and +96 downstream of the stop codon. A rabbit polyclonal antiserum has been raised against an internal polypeptide of LEF-4. A 55-kDa protein was observed by Western blot analysis from 5 h pi. LEF-4 localizes preferentially in the nucleus of infected cells and is associated with the virogenic stroma.

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.

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.

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.

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.

Trois isolats de polyedres nucleaires deSpodoptera littoralisBoisduval (isolats Egypte, Maroc et Lyon) sont compares entre eux et avec des preparations de polyedres nucleaires des lepidopteresBombyx mori L.,Galleria mellonella L.,Lymantria dispar L. etAutographa californicaSpeyer. L'analyse porte a la fois sur la composition peptidique des virions, sur le poids moleclaire des proteines des polyedres et sur les parentes antigeniques entre ces proteines.

Une polyedrose nucleaire de la tordeuse de la pelure,Pandemis heparanaDenis & Schiffermuller (Lep.: Tortricidae) due a un baculovirus isole dans un verger de pommiers en France est etudiee. Le baculovirus, uniquement actif sur les larves nouvelles nees, induit un arret de developpement des larves au stade L3 et une mortalite ulterieue des larves aux stades L5 et L6. L'etude en microscopie optique et en microscopie electronique des larves L5 et L6 presentant des symptomes tres prononces de la viroise (debut de paralysie et couleur blanchâtre des segments abdominaux) montre que c'est essentiellement le tissu adipeux et accessoirement certains hemocytes et quelques cellules de l'hypoderme qui sont le siege de la multiplication virale. La morphogenese virale dans les cellules du tissu adipeux est decrite en detail. Certains aspects de l'etude ultrastructurale lies a la forme en massue des virions, au processus de cristallisation des polyedres et a l'accumulation de masses fibrillaires viro-induites sont discutes.

Le poids moleculaire de la proteine constituant la trame des corps d'inclusion des Baculovirus a ete estime pour 33 virus affectant trois ordres differents d'Insectes. Ce poids varie entre 25.100 et 31.360 daltons.

Trois Baculovirus isoles de larves deSpodoptera littoralis atteintes de polyedroses nucleaires au Maroc, en Egypte et dans un elevage a Lyon ont ete compares par analyse de leurs genomes avec des enzymes de restriction. Les variants genetiques contenus dans les populations virales sont obtenus apres isolement de clones en culture cellulaire. Sur 27 clones analyses par les endonucleases on observe 11 variants avecEco RI, 3 variants avecBam HI et avecSac I et un type unique avecHin dIII. Les 3 virus Maroc, Egypte et Lyon sont differents, mais appartiennent a l'espece B definie parCherry & Summers (1985).

Les consequences possibles sur le profil des motifs de recherches afferents auxBaculovirus sont evoquees, en faisant etat de la mise en evidence et de la caracterisation deBaculovirus dans des ordres d'insectes non encore concernes, notamment les homopteres ainsi que chez les arachnides et les crustaces. En dehors de l'accroissement d'interet en virologie comparee a cause de la diversification des types, sont soulignes: la possibilite de l'extension de l'application de la lutte virologique a des ravageurs non encore concernes et a des insectes vecteurs, l'etude de la coexistence de virus transmis avec des infections virales propres aux vecteurs, les nouvelles precautions a prendre en matiere d'innocuite et de diagnostic et l'evolution de l'importance economique en epizootiologie des invertebres utiles en milieu marin.

L’introduction de corps etrangers dans les larves de lepidopteres entraine une augmentation parfois considerable du taux de lysozyme dans l’hemolymphe. L’efficacite inductrice de diverses substances varie dans de larges mesures. L’action de l’Actinomycine D et de la Cycloheximide sur la production du lysozyme induit a ete recherchee chez des larves deGalleria mellonella L. recevant un stimulus peu specifique (chlorure de sodium) ou plus specifique (Micrococcus luteus vivants). L’action de ces inhibiteurs sur la quantite de lysozyme presente dans l’hemolymphe 24 h apres induction, indique que les variations du taux de l’enzyme sont sous le controle d’un mecanisme unique independant de la specificite des stimuli inducteurs. L’augmentation du taux de lysozyme chez les larves deG. mellonella induites, fait suite a une synthese proteique et non a la liberation d’enzyme preforme.

On the basis of their buoyant densities in CsCl and their capsid polypeptides, three viruses isolated from Drosophila spp. which were originally described as serotypes, are now classified as distinct viruses. The biochemical properties of each virus suggest that it has several key features in common with the mammalian picornaviruses.

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.

Abol Ela Saïd, Croizier Guy. Influence des infections virales sur la diapause chez les Insectes. In: Bulletin de la Société entomologique de France, volume 89 (1-4), Janvier-avril 1984. Livre du Cent Cinquantenaire. Premier congrès international des entomologistes d'expression française. Paris, 6-9 juillet 1982. Comptes rendus des travaux. V. pp. 701-710.

L'evolution du Baculovirus deMamestra brassicae origine France a ete suivie au cours de passages successifs sur des chenilles du chou infecteesper os au stade L5. Le genome viral d'un poids moleculaire de 148 kpb comporte en debut d'experience 82 sites de restriction reconnus par 7 endonucleases. 81 sites sont conserves au cours des passages repetes. Un site separant les fragmentsHin dIII G etHin dIII J disparait progressivement des populations virales par deletion d'un fragment d'ADN de 2,7 kpb localise entre les positions 0,25 et 0,37 sur la carte de restriction deWiegers & Vlak (1984). Cette modification du genome n'a pas de consequence appreciable sur la virulence des preparations virales successives dont les DL50 pour les larves L1 PM passent de 11 a 17,9 polyedres par mm2 d'aliment contamine en 25 multiplications.

Summary Cotransfection of Rachiplusia ou nuclear polyhedrosis virus (RoMNPV) and BamHI or SmaI fragments from the genome of Autographa californica nuclear polyhedrosis virus (AcMNPV) yielded a high frequency of virus recombinants in Galleria mellonella larvae. When larvae were cotransfected either with the entire genome of AcMNPV and RoMNPV restriction fragments or with a mixture of the two viruses, only AcMNPV was produced. In the virus recombinants, the prevalence of certain regions from the genome of the donor virus (AcMNPV) varied considerably, e.g. from 0% in the 35 to 41 map unit region area to a high level in the 20 to 23 map unit region. The AcMNPV polyhedrin gene was shown to be inherited preferentially.

Croizier Guy. Les apports de la biologie moléculaire à la connaissance des Baculovirus et leurs conséquences sur la lutte virologique contre les Insectes nuisibles. In: Bulletin de la Société entomologique de France, volume 89 (1-4), Janvier-avril 1984. Livre du Cent Cinquantenaire. Premier congrès international des entomologistes d'expression française. Paris, 6-9 juillet 1982. Comptes rendus des travaux. V. pp. 807-816.