James E. Maruniak

Sequence Analysis of the Genome of the Neodiprion sertifer Nucleopolyhedrovirus

ABSTRACT The genome of the Neodiprion sertifer nucleopolyhedrovirus (NeseNPV), which infects the European pine sawfly, N. sertifer (Hymenoptera: Diprionidae), was sequenced and analyzed. The genome was 86,462 bp in size. The C+G content of 34% was lower than that of the majority of baculoviruses. A total of 90 methionine-initiated open reading frames (ORFs) with more than 50 amino acids and minimal overlapping were found. From those, 43 ORFs were homologous to other baculovirus ORFs, and 29 of these were from the 30 conserved core genes among all baculoviruses. A NeseNPV homolog to the ld130 gene, which is present in all other baculovirus genomes sequenced to date, could not be identified. Six NeseNPV ORFs were similar to non-baculovirus-related genes, one of which was a trypsin-like gene. Only one iap gene, containing a single BIR motif and a RING finger, was found in NeseNPV. Two NeseNPV ORFs (nese18 and nese19) were duplicates transcribed in opposite orientations from each other. NeseNPV did not have an AcMNPV ORF 2 homolog characterized as the baculovirus repeat ORF (bro). Six homologous regions (hrs) were located within the NeseNPV genome, each containing small palindromes embedded within direct repeats. A phylogenetic analysis was done to root the tree based upon the sequences of DNA polymerase genes of NeseNPV, 23 other baculoviruses, and other phyla. Baculovirus phylogeny was then constructed with 29 conserved genes from 24 baculovirus genomes. Culex nigripalpus nucleopolyhedrovirus (CuniNPV) was the most distantly related baculovirus, branching to the hymenopteran NeseNPV and the lepidopteran nucleopolyhedroviruses and granuloviruses.

STRAIN IDENTIFICATION OF SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE) INSECTS AND CELL LINE: PCR-RFLP OF CYTOCHROME OXIDASE C SUBUNIT I GENE

Abstract A simple method was developed to analyze the two morphologically indistinguishable host-associated strains of the fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith). Total DNA extracted from the FAW corn and rice strains, as well as from the S. frugiperda cell line (SF9) was used to PCR amplify a 569 base pairs region of the mitochondrial gene cytochrome oxidase subunit I (COI). The amplified DNA from the Spodoptera corn strain and the SF9 cell line contained a restriction fragment length polymorphism (RFLP) marker, the MspI recognition site that was not present in the rice strain. This PCR-RFLP method does not require purification of mitochondrial DNA (mtDNA) or the use of radioactive isotopes, and differs from previous methods in that only a few nanograms of total DNA are needed to yield clear and accurate strain identification of individual insects.

Autographa californica nuclear polyhedrosis virus phosphoproteins and synthesis of intracellular proteins after virus infection

Polypeptides and phosphoproteins isolated from nuclear and cytoplasmic fractions of TN-368-10 cells infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) were analyzed by polyacrylamide gel electrophoresis. Likewise, AcMNPV extracellular virus and alkali-released virus were compared. ACMNPV extracellular virus possessed at least nine phosphoproteins while the alkali-released virus had about 14 phosphoproteins. The major structural polypeptide of AcMNPV, polyhedrin, was phosphorylated. Intracellular proteins of infected TN-368-10 cells were pulse-labeled with [35S]methionine and separated into nuclear and cytoplasmic fractions. At least 36 virus-induced polypeptides were detected in infected cells, and several polypeptides were found as early as 5 hr postinfection. The patterns of synthesis and appearance of infected cell polypeptides were complex, but polypeptide differences between nuclear and cytoplasmic fractions were detected. Twenty phosphoproteins labeled with 32P were also detected in the two fractions, and at least six had electrophoretic mobilities similar to virus-associated phosphoproteins.

Physical maps of SfMNPV baculovirus DNA and its genomic variants

Spodoptera frugiperda MNPV was plaque-purified, and the viral DNA from the plaque-purified isolates was analyzed with restriction endonuclease enzymes. Seven distinct variants were identified when the DNA of the isolates were analyzed by EcoRI and HindIII. The DNAs of the SfMNPV predominant type (prototype) and the variants were mapped with BamHI, BglII, BstEII, EcoRI, HindIII, KpnI, and PstI by multiple enzyme digestion and blot hybridization. The cleavage sites generated by the seven restriction enzymes were ordered, and the sites were assigned map coordinates using a least-squares procedure. Since Autographa californica MNPV-E2 EcoRI fragment I, which contains the polyhedrin gene, hybridized with SfMNPV EcoRI fragment P, the physical map of SfMNPV was oriented with EcoRI P on the left, with site 1 being the EcoRI site between fragments F and P. The calculated genome size was 121.76 kilobase pairs or 80.36 X 10(6) Da. The DNA from each variant was compared to the DNA of the prototype for insertions, deletions, and new restriction sites. Physical maps were generated for each of the variants. The differences between the variant and the prototype were confined to four regions in the SfMNPV genome representing less than 16% of the prototype genome.

A variable region of Anticarsia gemmatalis nuclear polyhedrosis virus contains tandemly repeated DNA sequences

A variable region (PstI-T fragment) of two genotypic variants of the Anticarsia gemmatalis multiple nucleocapsid nuclear polyhedrosis virus (AgMNPV) was sequenced and compared. This region, which is known to have deletions and duplications in AgMNPV variants, was shown to be formed of units of a 127 bp tandemly repeated sequence containing two 30 bp imperfect palindromes. Southern blot experiments showed that the PstI-T fragment contained one of the four homologous regions mapped interspersed in the AgMNPV genome. The comparison of the nucleotide sequences of the two variants, AgMNPV-2D and AgMNPV-D7, showed that the difference between these two variants in one of these regions (hr4) was caused by the addition or elimination of 381 bp corresponding exactly to three of the 127 bp repeated sequences. An analysis of the sequence showed homology to the homologous regions (hr) of other baculoviruses. The sequence upstream of the repetitive region contained a sequence homologous to the N-terminal portion of the Autographa californica MNPV and Choristoneura fumiferana MNPV p74 gene.

The Anticarsia gemmatalis nuclear polyhedrosis virus polyhedrin gene region: sequence analysis, gene product and structural comparisons

The genomic region of the Anticarsia gemmatalis multiple nucleocapsid nuclear polyhedrosis virus (AgMNPV) strain 2D encoding the polyhedrin gene was cloned and mapped, and a 2085 bp SphI-PstI fragment containing the gene was sequenced. The polyhedrin polypeptide of the parental isolate AgMNPV was manually sequenced, and the amino acid sequence obtained agreed with that deduced from the DNA coding region sequence. AgMNPV and Orgyia pseudotsugata MNPV (OpMNPV) are similar in terms of promoter structure and polyhedrin primary sequence, and the polyhedrin gene of both viruses is transcribed in the anti-clockwise direction in relation to their physical maps. The region upstream from the polyhedrin gene of AgMNPV, OpMNPV, Bombyx mori NPV and Autographa californica MNPV (AcMNPV) was compared and this showed that the open reading frame (ORF) common to all four viruses (ORF 5) has sequence homology with the AcMNPV 25K gene. The sequences between ORF 5 and the polyhedrin gene were found to be variable among the polyhedrin gene loci compared. Additionally, conserved elements in the promoters of the very late genes encoding polyhedrin and granulin, and those encoding two p10 proteins were found to share sequence homology and positional similarity with consensus regions in the conserved boxes A and C, responsible for binding transcription factors to eukaryotic 5S ribosomal RNA genes, and to box C of tRNA genes.

Physical Map of Anticarsia gemmatalis Nuclear Polyhedrosis Virus (AgMNPV-2) DNA

Summary A physical map for a plaque-purified isolate of a baculovirus which infects Anticarsia gemmatalis Hubner (velvetbean caterpillar), was constructed. By applying multiple-enzyme digestion and DNA-DNA hybridization techniques to the viral DNA restriction fragments, a total of 51 restriction sites were mapped. This baculovirus was found to possess a 133 kb circular dsDNA genome typical of members of baculovirus subgroup A.

Genetic identification of novel poxviruses of cetaceans and pinnipeds

Summary.Novel poxviruses were identified in skin lesions of several species of cetaceans and pinnipeds using polymerase chain reaction targeting DNA polymerase and DNA topoisomerase I genes of members of the subfamily Chordopoxvirinae. With the exception of parapoxviruses, no molecular data of marine mammal poxviruses were available to infer genetic and evolutionary relatedness to terrestrial vertebrate poxviruses. Viruses were assigned to a cetacean poxvirus 1 (CPV-1) group based on nucleotide and amino acid identities of gene fragments amplified from skin lesions of Asian bottlenose (Tursiops aduncus), Atlantic bottlenose (Tursiops truncatus), rough-toothed (Steno bredanensis), and striped (Stenella coeruleoalba) dolphins. A different poxvirus was detected in skin lesions of a bowhead whale (Balaena mysticetus) and provisionally assigned to a CPV-2 group. These viruses showed highest identity to terrestrial poxviruses of the genera Orthopoxvirus and Suipoxvirus. A novel species-specific poxvirus was also identified in skin lesions of Steller sea lions (Eumetopias jubatus). None of these poxviruses were found to have amplifiable hemagglutinin gene sequences. Novel parapoxviruses were also identified in skin lesions of Steller sea lions and spotted seals (Phoca largha). A significant degree of divergence was observed in sequences of Steller sea lion parapoxviruses, while those of spotted seals and harbor seals (Phoca vitulina) were highly conserved.

Detection and identification of multiple baculoviruses using the polymerase chain reaction (PCR) and restriction endonuclease analysis

A technique using the polymerase chain reaction (PCR) and restriction analysis was developed for the simultaneous detection of eight baculoviruses. The baculoviruses detected by this technique were Autographa californica multiple-embedded nuclear polyhedrosis virus (MNPV). Anticarsia gemmatalis MNPV, Bombyx mori MNPV, Orgyia pseudotsugata MNPV. Spodoptera frugiperda MNPV, S. exigua MNPV, Anagrapha falcifera MNPV, Heliothis zea single-embedded nuclear polyhedrosis virus (SNPV). A highly conserved DNA sequence within the coding region of the polyhedrin gene was targeted for amplification. One pair of degenerate primers was designed, and PCR conditions were optimized to produce 575 base pair fragments for all eight baculoviruses. Restriction analysis of the PCR products resulted in distinct profiles for each virus. This technique would be useful in monitoring the release of wild type as well as genetically engineered baculoviruses.

Two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica are related and form a distinct phylogenetic clade.

Glossina pallidipes and Musca domestica salivary gland hypertrophy viruses (GpSGHV and MdSGHV) replicate in the nucleus of salivary gland cells causing distinct tissue hypertrophy and reduction of host fertility. They share general characteristics with the non-occluded insect nudiviruses, such as being insect-pathogenic, having enveloped, rod-shaped virions, and large circular double-stranded DNA genomes. MdSGHV measures 65x550 nm and contains a 124 279 bp genome (approximately 44 mol% G+C content) that codes for 108 putative open reading frames (ORFs). GpSGHV, measuring 50x1000 nm, contains a 190 032 bp genome (28 mol% G+C content) with 160 putative ORFs. Comparative genomic analysis demonstrates that 37 MdSGHV ORFs have homology to 42 GpSGHV ORFs, as some MdSGHV ORFs have homology to two different GpSGHV ORFs. Nine genes with known functions (dnapol, ts, pif-1, pif-2, pif-3, mmp, p74, odv-e66 and helicase-2), a homologue of the conserved baculovirus gene Ac81 and at least 13 virion proteins are present in both SGHVs. The amino acid identity ranged from 19 to 39 % among ORFs. An (A/T/G)TAAG motif, similar to the baculovirus late promoter motif, was enriched 100 bp upstream of the ORF transcription initiation sites of both viruses. Six and seven putative microRNA sequences were found in MdSGHV and GpSGHV genomes, respectively. There was genome. Collinearity between the two SGHVs, but not between the SGHVs and the nudiviruses. Phylogenetic analysis of conserved genes clustered both SGHVs in a single clade separated from the nudiviruses and baculoviruses. Although MdSGHV and GpSGHV are different viruses, their pathology, host range and genome composition indicate that they are related.