Douglas J. Leisy

Variation in Cydia pomonella Granulosis Virus Isolates and Physical Maps of the DNA from Three Variants

Summary Cydia pomonella granulosis viruses (CpGV) from seven different sources in Europe, America and New Zealand were compared by restriction enzyme analysis. Most samples were indistinguishable from the Mexican isolate (CpGV-M). Isolates from Russia (CpGV-R) and England (CpGV-E) showed small genotypic differences. CpGV-E was shown to be a mixture of two variants, E1 and E2. CpGV-E1 was indistinguishable from CpGV-M. A physical map of CpGV-M was constructed for the enzymes EcoRI, BamHI, HindIII, SmaI and ApaI. A comparison of fragment profiles allowed construction of maps for CpGV-R and CpGV-E2. Relative to CpGV-M, CpGV-R had a single deletion of 2.4 kbp and CpGV-E2 was modified in one area resulting in an additional EcoRI site, a shift in a BamHI site and in total about 1 kbp more DNA. The map was orientated by locating the granulin gene using the cloned granulin gene from Trichoplusia ni GV as a probe. There was no significant difference between the infectivities of the Mexican, Russian and English isolates for neonate larvae.

Comparison of the p26 gene region of two baculoviruses.

A 1.1-kb region of DNA containing the p26 gene of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was sequenced, transcriptionally mapped, and compared to the same region in the MNPV of Autographa californica (AcMNPV). The mRNA start site of the p26 gene occurs about 22 nucleotides downstream from an A/T-rich putative promoter sequence that is highly conserved between AcMNPV and OpMNPV. The p26 mRNA is transcribed through the p26 gene and coterminates with the p10 gene resulting in a mRNA containing copies of both genes. The reading frames of the OpMNPV and AcMNPV p26 genes showed 47% amino acid sequence homology which is clustered in six regions with over 65% amino acid homology. There was a distinct bias toward incorporation of G/C-rich codons in the OpMNPV p26 gene. No DNA homology was observed between the region upstream of the p26 gene in AcMNPV and OpMNPV. In AcMNPV, this region contains the homologous repeated (hr) sequence hr5. Hybridization of a plasmid containing an AcMNPV-repeated sequence (hr5) to Southern blots of the OpMNPV genome indicated that this repeated sequence is lacking in OpMNPV.

Identification, cloning, and R-Loop mapping of the polyhedrin gene from the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata

Polyadenylated RNA was isolated from Orgyia pseudotsugata larvae 8-10 days postinfection with the multicapsid nuclear polyhedrosis virus. This RNA was centrifuged through a sucrose gradient and fractions enriched for polyhedrin mRNA were identified by in vitro translation. Complementary DNA made to this RNA hybridized predominantly to a 5-kb fragment of XhoI-digested viral DNA. This fragment was cloned into the plasmid pACYC177 and mapped with restriction endonucleases. A SalI subclone with a 2.5-kb insert derived from the cloned XhoI fragment was found to select by hybridization only polyhedrin mRNA as determined by the size of the in vitro translation product and its precipitation by anti-polyhedrin antibodies. The orientation of the polyhedrin gene and the region of the insert encoding the N terminus of the polyhedrin protein were determined by DNA sequencing. R-Loop mapping indicated polyhedrin mRNA is 980 +/- 75 bases long and contains about 250 nucleotides not represented in the final protein. The polyhedrin gene had no observable intervening sequences.

Physical map of Anagrapha falcifera multinucleocapsid nuclear polyhedrosis virus

A physical map of Anagrapha falcifera multinucleocapsid nuclear polyhedrosis virus (AfMNPV) DNA was constructed for restriction endonucleases EcoRI, HindIII, PstI and XhoI. The genome size was estimated to be 130 kbp. Ordering of the restriction fragments was accomplished by cross-blot hybridization, double digestion and DNA-DNA hybridization. The polyhedrin gene and homologous repeat (hr) regions were located by hybridization to the Autographa californica MNPV (AcMNPV) polyhedrin gene and hr4, respectively. Restriction pattern comparison and Southern blot analysis suggest that AfMNPV is closely related to AcMNPV.