Peter J. Krell

Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2015)

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed.

Sequence and Organization of the Neodiprion lecontei Nucleopolyhedrovirus Genome

ABSTRACT All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopolyhedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and characterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the redheaded pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content of only 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified by conserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity of homologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. The conserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA binding protein, late expression factor 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40, and p45, but lacks 21 others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k, odv-e25, protein kinase-1, fibroblast growth factor, and ubiquitin. The lack of identified baculovirus homologues may be due to difficulties in identification, differences in host-virus interactions, or other genes performing similar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, and phylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirus and granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteran baculoviruses may be necessary.

Nonstructural protein 1α subunit-based inhibition of NF-κB activation and suppression of interferon-β production by porcine reproductive and respiratory syndrome virus

Induction of type I interferon (IFN-α/β) is an early antiviral response of the host, and porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to downregulate the IFN response during infection in cells and pigs. We report that the PRRSV nonstructural protein 1α (Nsp1α) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-β production that is mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1α suppressed the activation of nuclear factor (NF)-κB when stimulated with dsRNA or tumor necrosis factor (TNF)-α, and NF-κB suppression was RIG-I-dependent. The suppression of NF-κB activation was associated with the poor production of IFN-β during PRRSV infection. The C-terminal 14 amino acids of the Nsp1α subunit were critical in maintaining immunosuppressive activity of Nsp1α for both IFN-β and NF-κB, suggesting that the newly identified zinc finger configuration comprising of Met180 may be crucial for inhibitory activities. Nsp1α inhibited IκB phosphorylation and as a consequence NF-κB translocation to the nucleus was blocked, leading to the inhibition of NF-κB stimulated gene expression. Our results suggest that PRRSV Nsp1α is a multifunctional nuclear protein participating in the modulation of the host IFN system.

Baculovirus DNA Replication

Baculovirus replication initiates a cascade of gene expression that ultimately results in the production of progeny virus. This cascade is regulated at different points during the replication cycle: early gene expression is regulated by the interaction of cis-acting elements, viral trans-acting factors, and host factors (see Chapter 6, this volume), and late/very late gene expression is regulated by a combination of viral DNA replication, cis-acting elements as well as early and late viral factors (see Chapter 8, this volume).

Four genotypic variants of a Spodoptera exigua Nucleopolyhedrovirus (Se-SP2) are distinguishable by a hypervariable genomic region

Four genotypes named SP2A, SP2B, SP2C and SP2D were obtained in vivo by infecting S. exigua larvae with limiting dilutions of the Spanish field isolate Spodoptera exigua Nucleopolyhedrovirus (Se-SP2) of SeMNPV. The cloning of variants SP2A, SP2B and SP2C took 1, 6, and 3 passages, respectively, before the DNA profiles showed all bands in equimolar concentrations, and they remained constant for at least six further passages indicating the stability of their genotypes. The SP2D variant isolation took over ten passages and it was genetically less stable. Physical maps of their genomes were constructed for the restriction enzymes BamHI, BglII, PstI, and XbaI. The region between 8-10 m.u. was highly variable and characteristic of each cloned genotype and, hence, can be used as RFLP markers for all four genotypic variants. This region, included in the PstI-MB fragment, was cloned and sequenced showing that all the Se-SP2 variants contained a homologous region (hr) with a variable number of 98 bp sequences tandemly repeated, which were used to distinguish genotypic variants from each other. The biological activity of the genotypic variants SP2A, SP2B, and SP2C when compared in terms of LD50 and LT50, were not significantly different. However, the SP2D genotypic variant was found to be significantly less infective (higher LD50). The emergence of new genotypes in the Se-SP2 field populations is discussed.

Studies on two ecdysone receptor isoforms of the spruce budworm, Choristoneura fumiferana.

A full-length cDNA clone corresponding to the Choristoneura fumiferana ecdysone receptor-A isoform (CfEcR-A) was isolated. The deduced amino acid sequence of CfEcR-A differed from CfEcR-B in the NH2-terminal region of the A/B domain. The CfEcR-A-specific region showed high amino acid identity with EcR-A isoforms of Manduca sexta, Bombyx mori, Drosophila melanogaster and Tenebrio molitor. Isoform-specific probes were used to study the expression of EcR-A and EcR-B mRNAs. Both probes detected 6 kb mRNAs that were present in second-sixth larval instars and in the pupae. Both EcR-A and EcR-B mRNA levels increased during the molting periods. In the sixth instar larvae, the increase in EcR-A and EcR-B mRNA levels were more pronounced in the midgut than in epidermis and fat body. Both EcR-A and EcR-B mRNAs were induced in CF-203 cells (a cell line developed from C. fumiferana midgut) grown in the presence of 4 x 10(-6) M 20E. EcR-B specific mRNAs were induced within 1 h of exposure to 20E, but EcR-A specific mRNAs were induced only after 3 h of exposure to 20E. Induction of mRNAs for both isoforms was unaffected by the presence of a protein synthesis inhibitor, cyclohexamide, in the culture medium. RH-5992, a stable ecdysone agonist, caused a similar induction pattern of EcR-A and EcR-B mRNAs in the midgut, epidermis and fat body of sixth instar larvae. In vitro translated CfEcR-A, CfEcR-B and CfUSP proteins were used to study the DNA binding and ligand binding properties of EcR-A/USP and EcR-B/USP protein complexes. The Kd values indicated that both complexes have similar binding affinities for ecdysone response elements and ponasterone A.

A molt-associated chitinase cDNA from the spruce budworm, Choristoneura fumiferana.

Chitinase (CfChitinase) cDNA from the spruce budworm, Choristoneura fumiferana, was cloned using reverse transcription PCR and cDNA library screening. The CfChitinase cDNA was determined to be 2856 nucleotides long with the longest open reading frame made up of 1671 nucleotides that encoded a protein that was 557 amino acid long with a predicted molecular mass of 62 kDa. The deduced amino acid sequence showed 76-79% identity with other lepidopteran chitinases. Northern blots revealed that transcripts of CfChitinase appeared prior to each molt and peaked on the day of ecdysis from the second instar to the pupal stage but disappeared immediately after the molt. No transcripts could be detected in the early first instar prior to the spinning of the hibernaculum or in the diapausing second instars or during the intermolt periods of the other instars. Western blot analysis revealed that the protein appeared 12 h prior to ecdysis and disappeared 12 h after ecdysis from the sixth instar to pupal stage. The 20-hydroxyecdysone analog, tebufenozide (RH5992), induced expression of CfChitinase in the early stage of the sixth instar and caused a precocious and incomplete molt into an extra larval stage. During the sixth instar to the pupal molt, transcripts could be detected only in the epidermis and fat bodies, but not in the midgut. Western blots showed that the protein was present in the epidermis and midgut, but not in the fat bodies. The recombinant protein expressed in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) showed high levels of chitinolytic activity with an optimal pH range 6-9. Glycosylation appeared to be necessary for the chitinolytic activity and secretion of the recombinant protein.

The ultraspiracle gene of the Spruce Budworm, Choristoneura fumiferana: Cloning of cDNA and developmental expression of mRNA†

Cloning and characterization of a Choristoneura fumiferana ultraspiracle (Cfusp) cDNA are described. First, a PCR fragment and then a cDNA clone (4.4 kb) were isolated from spruce budworm cDNA libraries. Comparison of the deduced amino acid sequence of this cDNA with the sequences in Genbank showed that this sequence had high homology with the ultraspiracle cDNAs cloned from Drosophila melanogaster (Dmusp), Bombyx mori (Bmusp), Manduca sexta (Msusp), and Aedes aegypti (Aausp). The Cfusp cDNA contained all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNA binding domain or C region was the most conserved sequence among all the usps. The A/B, D, and E regions also showed high amino acid identity with the amino acid sequences of Dmusp, Msusp, Bmusp, and Aausp. The Cfusp 4.5-kb mRNA was present in the embryos, in all larval stages, and in the pupae. The Cfusp mRNA levels in the midgut increased during the sixth-instar larval development and reached peak levels during the ecdysteroid raises for the pupal molt. However, Cfusp mRNA levels remained unchanged in the midgut of fifth-instar larvae, and in the epidermis and fat body of sixth-instar larvae indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression.

Growth characteristics of fowl adenovirus type 8 in a chicken hepatoma cell line

Fowl adenoviruses, many of which appear to be non-pathogenic, are ubiquitous in birds. In addition, the genome of these viruses is large, making them ideal candidates for construction as vectors for foreign genes. Current methods to cultivate fowl adenoviruses use primary cell cultures derived from embryonated chicken eggs. In order to provide a more suitable culture method, the growth of fowl adenovirus type 8 (FAdV-8) was investigated in CH-SAH, a continuous hepatoma cell line. A one step growth curve demonstrated release of extracellular virus beginning by 18 h p.i. and with a final yield about 100 fold higher than that in chicken embryo liver cells. Viral DNA synthesis was first detected 8 h prior to this. The CH-SAH cell line supported the production of progeny viruses similar to the wild-type virus after being transfected with purified FAdV-8 DNA. This study demonstrated that the continuous hepatoma cell line is an appropriate in vitro host for FAdV-8.

Basis for selective action of a synthetic molting hormone agonist, RH-5992 on lepidopteran insects

The non-steroidal ecdysone agonist, RH-5992, induces a precocious incomplete molt in lepidopteran insects but is refractory to insects of other orders. We used two lepidopteran cell lines, FPMI-CF-203 (CF-203) and IPRI-MD-66 (MD-66) and two dipteran cell lines, DM-2 and Kc, to investigate the lepidopteran specificity of RH-5992. The mRNAs for hormone receptor 3 homologues cloned from Drosophila (DHR3) and Choristoneura (CHR3) are directly induced by 20-hydroxyecdysone (20E) and serve as suitable markers for studying ecdysone action. Dose response experiments showed that 10(-7) M 20E induced CHR3 mRNA in CF-203 cell and DHR3 mRNA in DM-2 cells. Concentrations of RH-5992 as low as 10(-10) M induced CHR3 mRNA in CF-203 cells, whereas concentrations as high as 10(-6) M induced only very low levels of DHR3 mRNA in DM-2 cells. Studies using 14C-RH-5992 revealed that lepidopteran cell lines (CF-203 and MD-66) retained more of this compound within the cells than the dipteran cell lines (DM-2 and Kc). The clearance of RH-5992 from DM-2 cells was temperature dependent and was blocked by 10(-5) M ouabain, an inhibitor of Na+, K(+)-ATPase suggesting that the efflux was due to active transport.