Horizontally transmitted parasitoid killing factor shapes insect defense to parasitoids.

Interkingdom competition occurs between hymenopteran parasitoids and insect viruses sharing the same insect hosts. It has been assumed that parasitoid larvae die with the death of the infected host or as result of competition for host resources. Here we describe a gene family, parasitoid killing factor (pkf), that encodes proteins toxic to parasitoids of the Microgastrinae group and determines parasitism success. Pkfs are found in several entomopathogenic DNA virus families and in some lepidopteran genomes. We provide evidence of equivalent and specific toxicity against endoparasites for PKFs found in entomopoxvirus, ascovirus, baculovirus, and Lepidoptera through a mechanism that elicits apoptosis in the cells of susceptible parasitoids. This highlights the evolutionary arms race between parasitoids, viruses, and their insect hosts.

A new cypovirus from the Japanese peppered moth, Biston robustus.

A cypovirus was isolated from larvae of the Japanese peppered moth, Biston robustus. The viral genome is 23,954 bp comprising 10 segmented double-stranded RNAs with a new electropherotype among cypoviruses. Each segment encodes one putative protein and has non-coding regions that contain conserved sequences at their 5' and 3' termini, 5'-AGAA(U/A)U-3' and 5'-UGC-3', respectively. Seven proteins encoded in the genome are homologous to those of other cypoviruses at a cut-off E-value of 1 × 10-5. The maximal sequence identities of these proteins with cypovirus homologs are 24.30%-39.40%. These results indicate that the virus isolated is a novel cypovirus; herein designated as Biston robustus cypovirus 24 (BrCPV-24). This newly isolated BrCPV-24 infects the larvae of the silkworm Bombyx mori.

Complete Genome Sequence of an Alphabaculovirus from Choristoneura diversana.

The genome sequence of a baculovirus isolated from Choristoneura diversana is 122,827 bp long and contains 150 putative open reading frames (ORFs). The virus is closely related to alphabaculoviruses isolated from insect species of the genus Choristoneura.

Five-year postsurgical monitoring of serum p53 antibody for locally advanced esophageal squamous cell carcinoma.

Serum p53 antibody (s-p53-Ab) titers were postoperatively monitored for over 5 years in a 67-year-old man with locally advanced esophageal squamous cell carcinoma. The tumor stage was classified as clinical stage II (cT3N0M0). Serum SCC antigen (s-SCC-Ag; 6.2 ng/mL) and s-p53-Ab (3.83 U/mL) were noted to be positive before surgery. Radical esophagectomy with three-field lymph node dissection was performed without neoadjuvant therapy. Pathological findings of the surgically resected specimens revealed a stage II tumor (pT3N0M0). Postoperatively, the patients did not receive any adjuvant therapy. Although the s-SCC-Ag was found to be negative at 2 months postoperatively, s-SCC-Ag was found to be six times positive despite no signs of recurrence. The s-p53-Ab titers constantly decreased to less than the cutoff value at 6 months postoperatively and continuously decreased over 5 years postoperatively. Finally, s-p53-Ab titers became less than the detection limit value at 60 months postoperatively. No recurrence was observed throughout the postoperative course. This case report is the first to describe the five-year monitoring of postoperative changes in s-p53-Ab titers in a patient with locally advanced esophageal squamous cell carcinoma without recurrence. s-p53-Ab titers seemed to be more useful than s-SCC-Ag for disease monitoring in this case.

A virus carries a gene encoding juvenile hormone acid methyltransferase, a key regulatory enzyme in insect metamorphosis.

Microbial parasitism, infection, and symbiosis in animals often modulate host endocrine systems, resulting in alterations of phenotypic traits of the host that can have profound effects on the ecology and evolution of both the microorganisms and their hosts. Information about the mechanisms and genetic bases of such modulations by animal parasites is available from studies of steroid hormones. However, reports involving other hormones are scarce. We found that an insect virus, a betaentomopoxvirus, encodes a juvenile hormone acid methyltransferase that can synthesize an important insect hormone, the sesquiterpenoid juvenile hormone. Phylogenetic analysis suggested that this gene is of bacterial origin. Our study challenges the conventional view that functional enzymes in the late phase of the juvenile hormone biosynthesis pathway are almost exclusive to insects or arthropods, and shed light on juvenoid hormone synthesis beyond Eukaryota. This striking example demonstrates that even animal parasites having no metabolic pathways for molecules resembling host hormones can nevertheless influence the synthesis of such hormones, and provides a new context for studying animal parasite strategies in diverse systems such as host-parasite, host-symbiont or host-vector-parasite.

Acquired resistance to a nucleopolyhedrovirus in the smaller tea tortrix Adoxophyes honmai (Lepidoptera: Tortricidae) after selection by serial viral administration.

A laboratory colony of Adoxophyes honmai was selected for resistance over 156 generations by feeding neonate larvae of every generation with the LC60 or LC70 of its nucleopolyhedrovirus, Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV). A significant difference in LC50 values between the selected (R-strain) and unselected (S1- and S2-strain) strains was first observed after three generations of selection, and the resistance level then increased continuously. The highest degree of acquired resistance, based on the ratio of the LC50 values of R- and S1-strains, was more than 400,000-fold. After selection was stopped at either the 21st or the 149th generation, LC50 values did not decrease significantly, suggesting that resistance of the R-strain to AdhoNPV was stable. To assess which of the two routes of baculovirus infection is affected by resistance to AdhoNPV, 5th instar larvae of the R-strain were inoculated orally and intrahemocoelically with AdhoNPV and their susceptibility was compared to that of S-strain. The ratio of the LC25 values of selected and unselected strains was 91-fold when budded viruses were injected into 5th instar larvae, but was 107,000-fold after oral inoculation. These results indicate that the resistance mechanism of the R-strain of A. honmai disrupts both midgut primary infection and hemocoelic secondary infection.

Genetic and phenotypic comparisons of viral genotypes from two nucleopolyhedroviruses interacting with a common host species, Spodoptera litura (Lepidoptera: Noctuidae).

Nucleopolyhedroviruses (NPVs) are known to be highly variable, both genetically and phenotypically, at several scales such as different geographic locations or a single host. A previous study using several geographic isolates indicated that two types of NPV, Spodoptera littoralis NPV (SpliNPV) and S. litura NPV (SpltNPV) types, were isolated from the common cutworm, Spodoptera litura (Fabricius), a polyphagous insect that causes serious damage to many forage crops and vegetables. That study also indicated that the SpliNPV type was widely distributed in Japan. Here, we investigated the genotypic and phenotypic variation of cloned NPVs that infect S. litura; such variation is an important resource for biological control agents, and may represent the genetic diversity of an NPV species. Eighteen genotypically distinct NPVs were cloned from four field-collected NPV isolates using an in vivo cloning technique. They were divided into two virus types according to the similarity of banding patterns of DNA fragments generated by restriction endonucleases, and Southern hybridization analysis. Partial polyhedrin gene sequences revealed that the two types corresponded to SpliNPV and SpltNPV. Bioassays seem to suggest that the SpliNPV virus type was, overall, more infectious and killed S. litura larvae faster, but yielded fewer viral occlusion bodies, than the SpltNPV type. These data provide a basis for explaining the distribution pattern of SpliNPV and SpltNPV types in S. litura populations in Japan.

Lymantria mathura nucleopolyhedrovirus: Identification, occurrence and genetic diversity in Iwate Prefecture, Japan.

A high prevalence of nuclear polyhedrosis has been observed in larval outbreaks of Lymantria mathura in Iwate Prefecture, Japan. However, the virus responsible has not been identified. Here the virus was designated Lymantria mathura nucleopolyhedrovirus (LymaNPV), based on partial sequence results of the lef-8, lef-9, and polh genes and transmission electron microscopic observations. Diagnosis by polymerase chain reaction targeting of the partial polh gene and a subsequent restriction fragment length polymorphism (RFLP) analysis indicated that LymaNPV was an exclusive causative agent for the nuclear polyhedrosis of the L. mathura larvae. LymaNPV was also detected from a very small fraction of L. dispar larvae that co-occurred with L. mathura larvae where the prevalence of LymaNPV-caused disease was high. A bioassay using LymaNPV against L. dispar larvae produced infection with the inoculated virus. This finding was consistent with the results of the field sample analysis. LymaNPV contained six genome types in the L. mathura populations sampled, as determined by RFLP of LymaNPV genomic DNA and southern blot hybridization analyses. None of the genome types was unique to any sampling site, indicating that some flow had occurred among sites. However, genome-type composition seemed to differ among sites. This study provides basic information about the interaction between L. mathura and LymaNPV.

Entomopoxvirus infection induces changes in both juvenile hormone and ecdysteroid levels in larval Mythimna separata.

Insect viruses are among the most important pathogens of lepidopteran insects. Virus-infected larvae often show developmental defects including a prolonged larval period and a failure to pupate, but the mechanisms by which insect viruses regulate host development need further investigation. In this study, the regulation of host endocrinology by a lepidopteran entomopoxvirus (EPV), Mythimna separata EPV (MySEV), was examined. When fourth instar M. separata were inoculated with MySEV occlusion bodies, pupation was prevented and the insects died during the final (sixth) larval instar. Liquid chromatography-MS analysis revealed that juvenile hormone (JH) titres in the haemolymph of MySEV-infected sixth instars were higher than those in mock-infected larvae. JH esterase (JHE) activity was also examined by kinetic assay using a colorimetric substrate. The level of JHE activity in the haemolymph of MySEV-infected larvae was generally lower than that found in mock-infected larvae. In contrast, ecdysteroid titre in the haemolymph of final-instar MySEV-infected larvae was lower than that found in mock-infected larvae when measured by radioimmunoassay. A statistically significant difference in the release of ecdysteroids from prothoracic glands (PGs) that were dissected from MySEV- or mock-infected sixth instar Day 3 larvae was not found following prothoracicotropic hormone (PTTH) exposure. Our results indicate that the release of ecdysteroids was reduced not by infection of the PGs by MySEV, but by reduced PTTH production from the brain. Taken together our study suggests that EPVs retard host development by both reducing ecdysone titre and maintaining status quo levels of JH by preventing its metabolism.

Gene acquisition convergence between entomopoxviruses and baculoviruses.

Organisms from diverse phylogenetic origins can thrive within the same ecological niches. They might be induced to evolve convergent adaptations in response to a similar landscape of selective pressures. Their genomes should bear the signature of this process. The study of unrelated virus lineages infecting the same host panels guarantees a clear identification of phyletically independent convergent adaptation. Here, we investigate the evolutionary history of genes in the accessory genome shared by unrelated insect large dsDNA viruses: the entomopoxviruses (EPVs, Poxviridae) and the baculoviruses (BVs). EPVs and BVs have overlapping ecological niches and have independently evolved similar infection processes. They are, in theory, subjected to the same selective pressures from their host's immune responses. Their accessory genomes might, therefore, bear analogous genomic signatures of convergent adaption and could point out key genomic mechanisms of adaptation hitherto undetected in viruses. We uncovered 32 homologous, yet independent acquisitions of genes originating from insect hosts, different eukaryotes, bacteria and viruses. We showed different evolutionary levels of gene acquisition convergence in these viruses, underlining a continuous evolutionary process. We found both recent and ancient gene acquisitions possibly involved to the adaptation to both specific and distantly related hosts. Multidirectional and multipartite gene exchange networks appear to constantly drive exogenous gene assimilations, bringing key adaptive innovations and shaping the life histories of large DNA viruses. This evolutionary process might lead to genome level adaptive convergence.

Replication of Mythimna separata entomopoxvirus in High Five™ cells and the construction of a recombinant.

Mythimna separata entomopoxvirus (MySEV), of the genus Betaentomopoxvirus, was found to replicate in High Five™ cells. The infected cells produced many occlusion bodies and were hypertrophied but did not lyse. Following infection at a multiplicity of infection of 0.1, titers of extracellular virus reached a plateau 3-4days post infection at 25°C and were estimated at ca. 3×10(5) plaque-forming units per ml in TC-100 or TMN-FH media, both of which contained fetal bovine serum (FBS). Serum free medium, Express Five® SFM, also supported virus replication in High Five™ cells, but the titers were approximately one-tenth of those grown in TC-100 or TMN-FH media containing FBS. Using High Five™ cells, a recombinant MySEV was successfully constructed using homologous recombination. This study opens an avenue to the evaluation of entomopoxvirus gene functions using reverse genetic approaches with in vitro and in vivo hosts.

New insights into the evolution of Entomopoxvirinae from the complete genome sequences of four entomopoxviruses infecting Adoxophyes honmai, Choristoneura biennis, Choristoneura rosaceana, and Mythimna separata.

Poxviruses are nucleocytoplasmic large DNA viruses encompassing two subfamilies, the Chordopoxvirinae and the Entomopoxvirinae, infecting vertebrates and insects, respectively. While chordopoxvirus genomics have been widely studied, only two entomopoxvirus (EPV) genomes have been entirely sequenced. We report the genome sequences of four EPVs of the Betaentomopoxvirus genus infecting the Lepidoptera: Adoxophyes honmai EPV (AHEV), Choristoneura biennis EPV (CBEV), Choristoneura rosaceana EPV (CREV), and Mythimna separata EPV (MySEV). The genomes are 80% AT rich, are 228 to 307 kbp long, and contain 247 to 334 open reading frames (ORFs). Most genes are homologous to those of Amsacta moorei entomopoxvirus and encode several protein families repeated in tandem in terminal regions. Some genomes also encode proteins of unknown functions with similarity to those of other insect viruses. Comparative genomic analyses highlight a high colinearity among the lepidopteran EPV genomes and little gene order conservation with other poxvirus genomes. As with previously sequenced EPVs, the genomes include a relatively conserved central region flanked by inverted terminal repeats. Protein clustering identified 104 core EPV genes. Among betaentomopoxviruses, 148 core genes were found in relatively high synteny, pointing to low genomic diversity. Whole-genome and spheroidin gene phylogenetic analyses showed that the lepidopteran EPVs group closely in a monophyletic lineage, corroborating their affiliation with the Betaentomopoxvirus genus as well as a clear division of the EPVs according to the orders of insect hosts (Lepidoptera, Coleoptera, and Orthoptera). This suggests an ancient coevolution of EPVs with their insect hosts and the need to revise the current EPV taxonomy to separate orthopteran EPVs from the lepidopteran-specific betaentomopoxviruses so as to form a new genus.

Characterization of Paecilomycescinnamomeus from the camellia whitefly, Aleurocanthus camelliae (Hemiptera: Aleyrodidae), infesting tea in Japan.

The whitefly, Aleurocanthus camelliae Kanmiya and Kasai (Hemiptera: Aleyrodidae), is an invasive species in Japan that was first discovered in 2004 on tea in Kyoto. Soon after its arrival epizootics of an entomopathogenic fungus were observed in populations of the whitefly in many tea-growing regions. Here we identify this fungus as Paecilomyces cinnamomeus (Petch) Samson and W. Gams (Hypocreales: Clavicipitaceae) based on morphological characteristics and molecular analyses. This is the first record of P. cinnamomeus in Japan and also the first time it has been recorded from the genus Aleurocanthus. A isolate of P. cinnamomeus caused greater than 50% and 90% infection in whitefly nymphs at 1×10(6) and 1×10(7)conidia/ml respectively, while the commercial mycoinsecticides Preferd® (Isaria fumosorosea) and Mycotal® (Lecanicillium muscarium) caused <10% infection at their recommended field rates (5×10(6) and 9×10(6)conidia/ml, respectively), suggesting that P. cinnamomeus may be more useful as a control agent than the currently available mycoinsecticides. Optimum and upper limit temperatures for in vitro growth of P. cinnamomeus isolates were 22.5-25°C and 32.5°C, respectively. At field rates, the fungicide thiophanate-methyl caused some inhibition of in vitro growth of P. cinnamomeus isolates, and the bactericide copper oxychloride and the insecticides tolfenpyrad and methidathion were strongly inhibitory. The findings obtained in this study will be useful in the development of microbial control programs using P. cinnamomeus against A. camelliae.

Fitness-related traits of entomopoxviruses isolated from Adoxophyes honmai (Lepidoptera: Tortricidae) at three localities in Japan.

Three entomopoxviruses (EPVs) isolated from diseased Adoxophyes honmai larvae at different localities (Tsukuba, Itsukaichi, and Miyazaki) in Japan were compared for biochemical identity and key parameters of virus fitness, fatal infection, speed of kill, and virus yield. When the structural peptides of occlusion bodies (OBs) and occlusion-derived viral particles were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no difference in banding patterns was observed. However, DNA restriction endonuclease analysis showed that the three isolates were genotypically different, but many commonly sized DNA fragments were observed. Five tortricid species, A. honmai, Adoxophyes orana, Adoxophyesdubia, Homona magnanima, and Archips insulanus were susceptible to all isolates. No significant differences in the key viral fitness parameters were detected among the isolates in A. orana. However, the Miyazaki isolate had a different effect on H. magnanima; it allowed infected insects to survive longer and develop to a larger size, but had a lower yield of OBs per larva at any given time to death. OB yields per unit cadaver weight for the Miyazaki isolate, which indicate the conversion rate of the insect to virus, were lower over time compared to the other two isolates. The implications for selecting a candidate isolate to control tortricid pests are discussed.

Characterization of a nucleopolyhedrovirus of Epinotia granitalis (Lepidoptera: Tortricidae).

A nucleopolyhedrovirus (NPV) from the cypress bark moth Epinotia granitalis Butler was partially characterized. Electron microscopy indicated that E. granitalis NPV (EpgrNPV) is a singly embedded nucleocapsid NPV with a relatively small polyhedron, ranging from 480 to 1100nm in diameter. Phylogenetic analysis revealed that EpgrNPV is a Group II NPV that is related to but distinct from Adoxophyes honmai NPV. The LC(50) against third instar E. granitalis larvae was 10(5.5) polyhedral occlusion bodies per gram (OBs/g diet), and the average time to death was 14 days. E. granitalis was the only EpgrNPV-susceptible species in a series of infection tests using insect species belonging to the family Tortricidae. Other test species, including Eucoenogenes ancyrota, A. honmai, Adoxophyes dubia, Pandemis heparana, Homona magnanima, and Archips insulanus, were not susceptible to the infection.

Up-regulation of hnRNP A1 gene in sporadic human colorectal cancers.

We have previously reported that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a major hnRNP, binds to G-rich repetitive sequences and quadruplex (G4') structures in DNA, including the 5'-TTAGGG-3' telomere repeat and 5'-GGCAG-3' short-tandem-repeat. DNA synthesis arrest at the (GGG) sites within these repeats in vitro was retrieved by the addition of the hnRNP A1 protein or its N-terminal proteolytic product, UP1, in a dose-dependent manner. Therefore, functional perturbation of hnRNP A1 may abrogate the genomic stability of telomere repeats and other G-rich sequences, independent of its major role in transcriptional and translational regulation. In the present study, we conducted genetic and expression analysis of the hnRNP A1 gene in sporadic human colorectal cancers to clarify its possible involvement in human carcinogenesis. Of 30 lesions, one harbored a mutation at the -11 position from the translation initiation site, but none in the coding region. A single nucleotide polymorphism, an A or G-allele, was found in the 5' upstream promoter region of the gene. Quantitative gene expression analysis revealed that 60% (18/30) of cases showed over-expression of hnRNP A1 in cancer tissues by 2-fold or greater, compared to their normal colon tissues, with values of 78, 64 and 40% for clinicopathological stages II, III and IV, respectively. Although the biological consequences of hnRNP A1 overexpression in colorectal cancers remain to be clarified, it could contribute to maintenance of telomere repeats in cancer cells with enhanced cell proliferation. Alternatively, since the variations in the stoichiometry of hnRNP family proteins are considered to affect cell-specific gene expression, quantitative alteration of hnRNP A1 could result in facilitation of transformation of colon epithelial cells as a consequence of transcriptional and translational perturbation.

Characterization of Adoxophyes honmai single-nucleocapsid nucleopolyhedrovirus: morphology, structure, and effects on larvae.

A nucleopolyhedrovirus (NPV) was isolated from a diseased larva of the smaller tea tortrix, Adoxophyes honmai, collected from a tea field in Tsukuba, Ibaraki, Japan. Electron microscopic observations confirmed that A. honmai NPV (AdhoNPV) was a single-nucleocapsid type virus. The genome size of AdhoNPV was estimated to be 111.6 +/- 0.9kb (mean +/- SE) by restriction endonuclease analysis. AdhoNPV was also infectious to two other Adoxophyes species, the summer fruit tortrix Adoxophyes orana and Adoxophyes dubia. The LD50 values for neonatal, second, third, fourth, and fifth (final) instar larvae of A. honmai were determined as 61, 107, 688, 1,961, and 4,085 occlusion bodies/insect, respectively. Most of the infected larvae died 5-9 days after molting to the final instar, regardless of the timing of inoculation. However, when neonates were exposed to extremely high doses of AdhoNPV (greater than 100 x LD90), larval development was prevented and most of the larvae died in the first instar.

Reconstruction after distal gastrectomy by interposition of a double-jejunal pouch using a triangulating stapling technique.

Sustaining good nutrition and preventing postgastrectomy syndrome are important for increasing the quality of life after distal gastrectomy. Many surgeons have proposed surgical methods designed to enhance long-term patient quality of life. An immediate, safe method based on current physiologic reconstructive principles shown to reduce postoperative patient complaints is presented. A reconstructive method using a modified interpositioned double-jejunal pouch after distal gastrectomy in 18 cancer patients was reviewed. This method uses a triangulating stapling technique with wide end-to-side anastomosis between the residual stomach and the pouch. In all patients, the anastomosis site was without leakage or stenosis, and there were no episodes of severe reflux esophagitis, residual gastritis, or dumping syndrome. The mean pooling rate was 44.2%, and emptying half-time was 73.0 minutes. After 2 years the body weight was 91.3% of the preoperative weight, the food volume was 89.2% of normal intake, and meal frequency was 3.0 per day. This method of reconstruction is useful for immediate and safe creation of a wide anastomosis between the residual stomach and the double-jejunal pouch after distal gastrectomy and in the prevention of esophagitis and residual gastritis.