Expression strategy of densonucleosis virus from Mythimna loreyi.

The genome of Mythimna loreyi densovirus (MlDNV) was cloned into the pEMBL(19)+ vector. This clone was infectious upon transfection, both in LD cells and larvae. The genome possessed ITRs of 543 nucleotides of which the distal 126 nucleotides could form a hairpin. The nonstructural (NS) and structural (VP) genes were located on the 5'-halves of the complementary strands and their transcripts started 27 nts downstream of the ITRs. These transcripts had an overlap of 57 nucleotides in middle of the genome. The NS cassette consisted of three genes with NS1 and the overlapping NS2 downstream of NS3. The NS3 gene was spliced out from a fraction of the NS transcripts to allow leaky scanning translation of the downstream bicistronic NS1 and NS2 genes. The four VPs were similarly generated by leaky scanning translation of unspliced mRNA. The 5'-untranslated region of the VP transcript was only seven nucleotides long.

Lack of infection of vertebrate cells by the densovirus from the maize worm Mythimna loreyi (MlDNV).

Several densoviruses have been used successfully in biological control of pests in the tropics. The densovirus from Mythimna loreyi (MlDNV) could also be an important tool in biological control of important pests. However, safety concerns remain as previous reports suggested that densoviruses may infect and transform L cells (from mouse). In this study, we show using molecular-biology tools that neither L nor other vertebrate cells support replication or transcription of densovirus, either after infection or after transfection. Quantitative PCR indicated no increase of viral DNA due to replication in vertebrate cells, in contrast to that in insect LD652 cells. After transfection, both the NS and VP mRNAs could be detected in LD652 cells but not in L cells. Moreover, the viral genome was excised from the plasmid after transfection of the infectious clone in LD652 cells, indicative of viral NS protein production, in contrast to L cells. The viral genome was able to integrate in the host chromosome of L cells after transfection, but not after infection. However, no viral transcription could be detected after integration.

Genome organization of Casphalia extranea densovirus, a new iteravirus.

The viral genome of Casphalia extranea densovirus (CeDNV) has been cloned and sequenced. It was 5002 nucleotides long and contained inverted terminal repeats of 230 nucleotides. Their distal 159 nucleotides formed imperfect palindromes in two orientations. Three large open reading frames (ORFs) were identified on the same strand, two in the left-hand half and one in the right-hand half. Each of the five structural proteins, expressed from the right-hand ORF in the baculovirus system, autoassembled into capsids. The two left-hand ORFs overlapped and code for nonstructural (NS) proteins. NS1 protein was shown to contain replicator protein and helicase/ATPase motifs. The PGY region in VP1 capsid protein is conserved among most parvoviruses and contained a phospholipase A(2) motif, a novel viral enzyme. This domain was expressed and its enzyme activity was demonstrated. The approximate 75% sequence identity between the DNAs from CeDNV and BmDNV-1 and identical genome organization indicated that CeDNV should be classified in the Iteravirus genus.

Genome organization of the densovirus from Bombyx mori (BmDNV-1) and enzyme activity of its capsid.

Bombyx mori densovirus (BmDNV-1), on the basis of the previously reported genome sequence, constitutes by itself a separate genus (Iteravirus) within the Densovirinae subfamily of parvoviruses. Inconsistencies in the genome organization, however, necessitated its reassessment. The genome sequence of new clones was determined and resulted in a completely different genome organization. The corrected sequence also contained conserved sequence motifs found in other parvoviruses. Some amino acids in the highly conserved domain in the unique region of VP1 were shared by critical amino acids in the catalytic site and Ca(2+)-binding loop of secreted phospholipase A2, such as from snake and bee venoms. Expression of this domain and determination of enzyme activity demonstrated that capsids have a phospholipase A2 activity thus far unknown to occur in viruses. This viral phospholipase A2, which is required shortly after entry into the cell, showed a substrate preference for phosphatidylethanolamine and phosphatidylcholine over phosphatidylinositol.

A new small RNA virus persistently infecting an established cell line of Galleria mellonella, induced by a heterologous infection.

A persistent infection in a Galleria mellonella cell line was revealed when infected with a maize stem borer picorna-like virus isolated on Sesamia cretica (MSBV). The new virus, completely different from the MSBV, is designated as G. mellonella cell line virus (GmclV), induces spectacular cytopathic effects, and is also considered efficient in vivo. The GmclV is a 29-nm-diameter isometric virus, with single-strand RNA of 2.9 x 10(6) Da molecular weight with a poly(A) tract. Its capsid is constituted of only two major polypeptides, of 34,500 and 32,500 Da, and no minor bands could be detected. The characteristics of the GmclV do not permit us to classify it with assurance. Even though it has not yet been identified as a picornavirus, it can be classified in the small RNA virus group of the Picornaviridae. G. mellonella represents a very interesting model, owing to the fact that two different persistent viruses belonging to the same family were isolated in vivo and in vitro, to further the understanding of the general phenomenon of persistency and induction.

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.

Restriction map of the Casphalia extranea densovirus genome.

A physical map of the Casphalia extranea densovirus genome (CeDNV) was constructed. The size of the intact viral genome was estimated to be 4.9 kilobases or 1.6 MDa (single strand). The double-stranded CeDNV genomic DNA was cleaved with 26 restriction endonucleases and 20 restriction sites were mapped on the genome. The CeDNV DNA restriction map was compared to those of other densoviruses. Southern blotting hybridization experiments failed to reveal any homology between the genomes of CeDNV and Junoniacoenia densovirus (JcDNV).