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Transgenic tobacco transformed with the Trichoplusia ni granulovirus enhancin gene has been demonstrated to enhance baculovirus infection in larvae. In this paper we describe the effect of the long-term feeding of lyophilized transgenic tobacco material to Pseudaletia separata and Spodoptera exigua larvae. Our results demonstrated that the baculovirus enhancin gene products have potential for use in insect pest management.

Viral RNA-dependent RNA polymerase (RdRp) plays crucial roles in the genomic replication and subgenomic transcription of Brome mosaic virus (BMV), a positive-stranded RNA plant virus. BMV RdRp is a complex of virus-encoded 1a and 2a proteins and some cellular factors, and associates with the endoplasmic reticulum at an infection-specific structure in the cytoplasm of host cells. In this study, we investigate the gross structure of the active BMV RdRp complex using monoclonal antibodies raised against the 1a and 2a proteins. Immunoprecipitation experiments showed that the intermediate region between the N-terminal methyltransferase-like domain and the C-terminal helicase-like domain of 1a protein, and the N terminus region of 2a protein are exposed on the surface of the solubilized RdRp complex. Inhibition assays for membrane-bound RdRp suggested that the intermediate region between the methyltransferase-like and the helicase-like domains of 1a protein is located at the border of the region buried within a membrane structure or with membrane-associated material.

Brome mosaic virus (BMV), a tripartite RNA plant virus, accumulates RNA3-derived defective RNAs (D-RNAs) in which 477–500 nucleotides (nt) are deleted in the central region of the 3a protein open reading frame (ORF), after prolonged infection in barley. In the present study, six artificial D-RNAs (AD-RNAs), having deletions of the same size as the naturally occurring D-RNA but at different positions in the 3a ORF, were constructed and tested for their amplification and encapsidation in barley protoplasts by coinoculation with BMV RNA1 and 2, or RNA1, 2, and 3. Northern blot analysis of RNA accumulation in total and virion fractions showed that deletions of 492 nt in the 3′-proximal and the 5′-proximal regions of the 3a ORF decreased encapsidation efficiency of the AD-RNAs compared with that of RNA3, whereas deletions in the central region enhanced encapsidation efficiency. The present results also show that deletion positions affect competition with RNA3 in the amplification and encapsidation of AD-RNAs.

Subcellular localization of the Brome mosaic virus replicase-related 1a and 2a proteins, and the 3a movement protein in infected barley leaves was examined by immunogold electron microscopy. The 1a and 2a proteins colocalized at infection-specific electron-dense cytoplasmic inclusions. The 3a protein was also detected in these inclusions. The inclusions were oval or amorphous, and contained electron-lucent regions. Electron microscopic autoradiography showed that the inclusions were sites of [3H]uridine incorporation, suggesting that they are sites of viral RNA synthesis.

Summary We have constructed a new system for inducible high-level expression of mRNA for foreign genes in transgenic plants by introducing a glucocorticoid-inducible transcription system into the previously developed ‘mRNA amplification system’ where target mRNA can be amplified as a subgenomic RNA by the replicase of a plant tripartite RNA virus, Brome mosaic virus (BMV). In the new amplification system, the amplification of mRNA is tightly regulated by the expression of a subunit of the BMV replicase. Transgenic Nicotiana benthamiana plants (designated GVG1 × 2FR) were produced that contained cDNA of BMV RNA1 coding a subunit of replicase under the control of a tightly regulated, glucocorticoid-inducible promoter. In addition GVG1 × 2FR plants contain cDNAs of BMV RNA2 coding another subunit of the replicase, and a replicable engineered BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN) gene under the control of the Cauliflower mosaic virus 35S promoter. When transgenic plants were treated with dexamethasone (DEX), a strong synthetic glucocorticoid, induction of replication and amplification of the 35S-driven FCP2IFN and synthesis of subgenomic mRNA for IFN were observed. Accumulation levels of amplified FCP2IFN were over 300 times higher than those of the 35S-driven FCP2IFN in the GVG1 × 2FR plant without the treatment and those of the mRNA for IFN were 30–230 times higher than in the previous, non-inducible mRNA amplification system. Without DEX treatment, no subgenomic mRNA for IFN was detected in the GVG1 × 2FR plant. The advantages and potential uses of this system are also discussed.

To investigate the functional domains of the coat protein (CP; 189 amino acids) of Brome mosaic virus , a plant RNA virus, 19 alanine-scanning mutants were constructed and tested for their infectivity in barley and Nicotiana benthamiana. Despite its apparent normal replicative competence and CP production, the C-terminal mutant F184A produced no virions. Furthermore, virion-forming C-terminal mutants P178A and D182A failed to move from cell to cell in both plant species, and mutants D181A and V187A showed host-specific movement. These results indicate that the C-terminal region of CP plays some important roles in virus movement and encapsidation. The specificity of certain mutations for viral movement in two different plant species is evidence for the involvement of host-specific factors.

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein (P3) that is indispensable for viral infectivity. Although P3 has been shown to be a prerequisite for CaMV aphid transmission, its role in viral replication remains unknown. We previously showed that P3 forms a tetramer in planta and that P3 tetramer co-sediments with viral coat protein on sucrose gradient centrifugation, suggesting that a tetramer may be the functional form of P3. We presumed that disulfide bonds were involved in tetramer formation because 1) the tetramer was detected by Western blotting after electrophoresis under non-reducing conditions, and 2) the cysteine-X-cysteine motif is well conserved in CaMV P3 and P3 homologues among Caulimoviruses. Therefore we mutated either or both of the cysteine residues of CaMV P3. The mutant viruses were infectious and accumulated to a similar extent as the wild-type. An analysis of mutant proteins confirmed that the wild-type P3 molecules in the tetramer are covalently bound with one another through disulfide bonds. It was also suggested that mutant proteins are less stable than wild-type protein in planta. Furthermore, sedimentation study suggested that the disulfide bonds are involved in stable association of P3 with CaMV virions or virion-like particles, or both. The mutant viruses could be transmitted by aphids. These results suggested that the covalent bonds in P3 tetramer are dispensable for biological activity of P3 under experimental situations and may have some biological significance in natural infection in the field.

A hybrid Cowpea chlorotic mottle virus (CCMV) [CCMV(B3a)] in which the CCMV 3a movement protein gene is replaced by the 3a (B3a) gene of Brome mosaic virus cannot infect cowpea systemically. Previously, analysis of RNA3 cDNA clones constructed from cowpea-adapted mutants derived from CCMV(B3a) revealed that a single codon change in the B3a gene allowed CCMV(B3a) to infect cowpea systemically. In this study, to extend the analysis of the CCMV(B3a) adaptation mechanism, we directly sequenced B3a gene RT-PCR products prepared from 28 cowpea plants in which cowpea-adapted mutants appeared, and found seven patterns of a codon change localized at five specific positions in the central region (Ser 118 , Glu 132 , Glu 138 , Gln 178 , and Ser 180 ). All of the patterns involved an amino acid change to Lys or Arg. Mutational analysis of the B3a gene demonstrated that a single codon change resulting in either Lys or Arg at any of the five positions was sufficient for the adaptation of CCMV(B3a) to cowpea. In contrast, CCMV(B3a) variants with a codon change resulting in Lys or Arg at three other positions (137, 155, and 161) in the B3a gene not only showed lack of systemic infection of cowpea but also showed weakened initial cell-to-cell movement in the inoculated leaves and diminished B3a accumulation in protoplasts. These results suggest that adaptive changes in the B3a gene are site-specifically selected in cowpea plants.

Six cell clones were derived from the SSN-1 cell line, which is composed of a mixed cell population and persistently infected with a C-type retrovirus (SnRV). These clones were susceptible to 4 piscine nodavirus strains belonging to different genotypes (SJNNV, RGNNV, TPNNV and *BFNNV [striped jack, redspotted grouper, tiger puffer and barfin flounder nervous necrosis viruses]). Three clones, designated A-6, E-9, and E-11, were highly permissive to nodavirus infection and production. The virus-induced cytopathic effects appeared as cytoplasmic vacuoles and intensive disintegration at 3 to 5 d post-incubation. These observations were highly reproducible and formed the basis for a successful virus titration system. Quantitative analysis using the cloned E-11 cell line clearly revealed differences in the optimal growth temperatures among the 4 genotypic variants: 25 to 30 degrees C for strain SGWak97 (RGNNV), 20 to 25 degrees C for strain SJNag93 (SJNNV), 20 degrees C for strain TPKag93 (TPNNV), and 15 to 20 degrees C for strain JFIwa98 (BFNNV). Electron microscopy demonstrated SnRV retrovirus particles only in A-6 and E-9 cells, but PCR amplification for the pol gene and LTR region of the proviral DNA indicated the presence of the retrovirus in the other clones, including E-11. The cell clones obtained in the present study will be more useful for qualitative and quantitative analyses of piscine nodaviruses than the SSN-1 cell line.

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.

A baculovirus virulence-associated protein, enhancin, was engineered for production in a baculovirus-insect cell expression system and used for transformation of tobacco plants. A recombinant Autographa californica nucleopolyhedrovirus (AcNPV) with the enhancin gene from Trichoplusia ni granulovirus, AcEnh26, was propagated in Sf9 cells. The infected cultured cells combined with either AcNPV occlusion bodies (OBs) or Spodoptera exigua NPV (SeNPV) OBs were fed to 3rd stadium larvae of S. exigua. Feeding larvae with AcEnh26-infected cells resulted in a 21-fold and 10-fold enhancement of infection by AcNPV and SeNPV, respectively, compared to controls. When a similar feeding assay was performed with engineered tobacco plants, a 10-fold enhancement of AcNPV infection was observed, but a marked enhancement of SeNPV infection was not observed. Thus, the engineering and expression of the baculovirus enhancin gene in homologous and heterologous organisms allowed us to analyze its effects on NPV infection of larvae which demonstrated its viral enhancement function.

Cucumber mosaic cucumovirus (CMV) and brome mosaic bromovirus (BMV) have many similarities, including the three-dimensional structure of virions, genome organizations, and requirement of the coat protein (CP) for cell-to-cell movement. We have shown that a chimeric BMV with the CMV 3a movement protein (MP) gene instead of its own cannot move from cell to cell in Chenopodium quinoa, a common permissive host for both BMV and CMV. Another chimeric BMV was constructed by replacing both MP and CP genes of BMV with those of CMV (MP/CP-chimera) and tested for its infectivity in C. quinoa, to determine whether the CMV CP has some functions required for the CMV MP-mediated cell-to-cell movement and to exhibit functional difference between CPs of BMV and CMV. Cell-to-cell movement of the MP/CP-chimera occurred, and small local lesions were induced on the inoculated leaves. A frameshift mutation introduced in the CMV CP gene of the MP/CP-chimera resulted in a lack of cell-to-cell movement of the chimeric virus. These results indicate that the viral movement mediated by the CMV MP requires its cognate CP. Deletion of the amino-terminal region in CMV CP, which is not obligatory for CMV movement, also abolished cell-to-cell movement of the MP/CP-chimera. This may suggest some differences in cell-to-cell movement of the MP/CP-chimera and CMV. On the other hand, the sole replacement of BMV CP gene with that of CMV abolished viral cell-to-cell movement, suggesting a possibility that the viral movement mediated by the BMV MP may also require its cognate CP. Functional compatibility between MP and CP in viral cell-to-cell movement is discussed.

Bromoviruses are a group of spherical plant RNA viruses. Their genome consists of approximately 8, 000 nucleotides in total and contains only four genes: 1a and 2a genes necessary for viral RNA replication, 3a gene required for viral cell-to-cell movement and the coat protein gene. Bromoviruses produce a large number of progeny virus particles in host plants, but never induce death. Therefore, we suppose that these viruses are adapted to host plant cells in various ways. Here, I describe molecular mechanisms of bromovirus adaptation to host plant cells and non-host plants.

Biosynthesis of melanin by Colletotrichum lagenarium is essential for appressorial penetration of its host plants. Carpropamid is a novel melanin-inhibiting fungicide for control of rice blast caused by Magnaporthe grisea. In C. lagenarium, appressorial and colonial melanization were inhibited by carpropamid. Accumulation of melanin intermediates, scytalone, was also shown in the culture medium containing carpropamid. One of the enzymes involved in the melanin biosynthesis, scytalone dehydratase catalyzes the conversion of scytalone to 1,3,8-trihydroxynaphthalene. We previously cloned SCD1 gene coding for scytalone dehydratase of C. lagenarium. In this study, a recombinant SCD1 expression vector was constructed using pMAL system and the recombinant scytalone dehydratase was purified from transformed Escherichia coli. Recombinant SCD1 showed scytalone dehydratase activity in an in vitro reaction using scytalone as substrate. The activity was assayed by measuring the production of 1,3,8-trihydroxynaphthalene or disappearance of scytalone, which was detectable by reverse-phase high-pressure liquid chromatography. Dehydration of scytalone to 1,3,8-trihydroxynaphthalene was inhibited by carpropamid in the in vitro reaction.

The movement protein (MP) gene of brome mosaic virus (BMV) was precisely replaced with that of cucumber mosaic virus (CMV). Infectivity tests of the chimeric BMV on Chenopodium quinoa, a permissive host for cell-to-cell movement of both BMV and CMV, showed that the chimeric BMV failed to move from cell to cell even though it replicated in protoplasts. A spontaneous mutant of the chimeric BMV that displayed cell-to-cell movement was subsequently obtained from a local lesion during one of the experiments. A cloned cDNA representing the genomic RNA encoding the MP of the chimeric BMV mutant was analyzed and found to contain a mutation in the CMV MP gene resulting in deletion of the C-terminal 33 amino acids of the MP. Directed mutagenesis of the CMV MP gene showed that the C-terminal deletion was responsible for the movement capability of the mutant. When the mutation was introduced into CMV, the CMV mutant moved from cell to cell in C. quinoa, though the movement was less efficient than that of the wild-type CMV. These results indicate that the CMV MP, except the C-terminal 33 amino acids, potentiates cell-to-cell movement of both BMV and CMV in C. quinoa. In addition, since C. quinoa is a common host for both BMV and CMV, these results suggest that the CMV MP has specificity for the viral genomes during cell-to-cell movement of the virus and that the C-terminal 33 amino acids of the CMV MP are involved in that specificity.

Colletotrichum lagenarium and Alternaria alternata produce a dark pigment, melanin. The C. lagenarium PKS1 and A. alternata ALM genes are involved in polyketide synthesis in the melanin biosynthesis pathway. PKS1 encodes a type 1 polyketide synthase. For functional comparison of the ALM gene with the PKS1 gene, we examined whether the A. alternata ALM gene could restore melanin synthesis in C. lagenarium albino mutant (Pks1-). The ALM gene transformed the albino mutant (Pks1-) to melanin-producing phenotypes, designated CAL transformants. The pigment intensity of both melanized colonies and appressoria of CAL transformants was weaker than that of the wild type. Ultrastructural studies of the cell walls of appressoria demonstrated that CAL transformants formed an outer melanized layer, as did the wild type. On the other hand, the thin inner and middle layers were less electron-dense than those of the wild type. CAL transformants were able to penetrate cellulose membranes as effectively as the wild type. By contrast, the penetration frequency of CAL transformants on cucumber cotyledons was remarkably reduced compared to that of the wild type. During conidial germination, the PKS1 transcript accumulated de novo in both the wild-type and CAL transformants after the start of conidial incubation. On the other hand, ALM transcript accumulated in conidia of CAL transformants before the start of conidial incubation.

A phytopathogenic fungus, Colletotrichum lagenarium, produces melanized appressoria that display temperature-sensitive differentiation. Conidia incubated in water at 24(deg)C germinated, and germ tubes differentiated into melanized appressoria. On the other hand, conidia incubated in water at 32(deg)C germinated and produced germ tubes that elongated without appressorium differentiation. Conidia in 0.1% yeast extract solution at 32(deg)C germinated and developed into vegetative hyphae. In this study, we investigated the temporal transcriptional pattern of cloned melanin biosynthesis genes, PKS1, SCD1, and THR1, in these differentiating and nondifferentiating conidia. During appressorium differentiation, de novo transcripts of the three melanin biosynthesis genes accumulated 1 to 2 h after the start of conidial incubation at 24(deg)C, and the amount of transcripts began to decrease at 6 h. In conidia germinating in water at 32(deg)C, the transcriptional pattern of the PKS1, SCD1, and THR1 genes was similar to that of these genes in appressorium-forming conidia, although no appressoria were formed. However, in conidia in 0.1% yeast extract solution at 32(deg)C, transcripts of the three melanin biosynthesis genes hardly accumulated.

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes, but distinct host ranges: BMV systemically infects the monocot barley, while CCMV systemically infects the dicot cowpea. We have previously shown that in approximately 10% of inoculated cowpea plants, a CCMV hybrid [CCMV(B3a)] with the 3a cell-to-cell movement protein gene replaced by that of cowpea-nonadapted BMV directs systemic infections, which are caused by secondary mutation(s) of the hybrid virus. Here, to further analyze the role of RNA3 in adaptation to a new host, RNA3 cDNA clones were constructed from total RNA recovered from the uninoculated upper leaves of systemically infected cowpea plants inoculated with CCMV(B3a). Sequence and mutational analysis of two such RNA3 clones revealed that a single codon change (A776 → C) in a conserved motif of the 3a movement protein gene conferred compatibility for systemic infection of a new host, cowpea, suggesting that this site in the 3a gene is directly or indirectly involved in crucial host interactions associated with host-range specificity. The adaptive hybrid viruses carrying this mutation induced exacerbated symptoms, while wt CCMV appeared nearly symptomless, showing that the bromovirus 3a movement protein gene can significantly contribute to regulating symptom development. However, introducing this cowpea-adaptive mutation into the BMV genome had little effect on the ability of BMV to systemically infect barley.

The effects of some chemical disinfectants, organic solvents, hydrogen ions, heat, ultraviolet (UV) irradiation and ozone on the inactivation of striped jack nervous necrosis virus (SJNNV) were investigated. SJNNV was inactivated by contact with a final concentration of 50 ppm of sodium hypochlorite, calcium hypochlorite, benzalkonium chloride and iodine for 10 min at 20 °C. Cresol concentrations of more than 10 000 ppm were required to inactivate SJNNV, and no inactivation of SJNNV by formalin was detected at any concentration tested. The effective concentrations of ethanol and methanol were 60% and 50%, respectively, but SJNNV was resistant to ether and chloroform. SJNNV was inactivated by high alkalinity, pH 12 for 10 min at 20 °C, and also inactivated by heat treatment at 60 °C for 30 min. Inactivation of SJNNV by UV irradiation was observed at an intensity of 410 μW cm−2 for 244 s. Ozone at 0.1 μg ml−1 as a total residual oxidant was required to inactivate SJNNV for 2.5 min. Washing fertilized eggs and the treatment of sea water with ozone decreased the rate of occurrence of VNN.

Transgenic tobacco plants (V123 plants) expressing a set of full-length brome mosaic virus (BMV) genomic RNAs from the cauliflower mosaic virus 35S promoter were produced. The accumulation level of BMV RNAs in V123 plant cells was approximately 1% of that in non-transgenic tobacco protoplasts inoculated with BMV RNAs. The level of BMV RNA in V123 protoplasts did not increase after inoculating the protoplasts with BMV RNAs, whereas V123 protoplasts supported the accumulation of cucumber mosaic virus (CMV) RNAs to a level similar to that in non-transgenic tobacco protoplasts after inoculation with CMV RNA. Such BMV-specific resistance was also observed in protoplasts from V12 plants expressing full-length BMV RNA1 and RNA2, both of which are required and sufficient for BMV RNA replication. On the other hand, protoplasts from M12 plants, expressing truncated BMV RNA1 and RNA2 in which the 3′ 200 nucleotides required for BMV RNA replication were deleted, exhibited weaker resistance to infection with BMV RNA than V12 protoplasts, although the accumulation level of truncated BMV RNA1 and RNA2 in M12 protoplasts was higher than that of BMV RNA1 and RNA2 and V12 protoplasts. These results suggest that expression of BMV RNA replicons is involved in the induction of resistance, rather than high-level accumulation of BMV RNAs and/or their encoded proteins.

Striped jack nervous necrosis virus (SJNNV), a nodavirus, is the causative agent of viral nervous necrosis (VNN) in larval striped jack fish. In the present study, the SJNNV coat protein gene was sequenced and compared with that of four known insect nodaviruses and with four other fish nodaviruses causing VNN. The SJNNV coat protein gene was 1410 bases in length and contained a single ORF of 1023 bases encoding a protein of 340 amino acids. The sequence similarities between the coat protein gene of SJNNV and four insect nodaviruses were 28.6% or less at the nucleotide level and 10.6% or less at the amino acid level. A portion of the coat protein gene from four additional fish VNN viruses was amplified by PCR using primers designed for SJNNV and the amplified fragments (870-876 bases) were sequenced. The sequence similarities among SJNNV and the four VNN viruses were 75.8% or greater at the nucleotide level and 80.9% or greater at the amino acid level. In the fish nodaviruses a highly conserved region of 134 amino acids with sequence similarity of 92.5% or greater was detected. This conserved sequence was not found in the coat protein of insect nodaviruses. These results indicate that the fish nodaviruses that cause VNN are closely related to each other but are quite different from insect nodaviruses.

Many particles of a non-occluded baculovirus with a membrane 70 × 330 nm in size were observed in the nuclei of the mid-gut glands of larval Kuruma shrimp ( Penaeus japonicus ) with mid-gut necrosis using an electron microscope. Nucleotides of the baculovirus were isolated from the diseased larvae in the presence of Triton X-100. The nucleocapsid was a bacilliform virion, approximately 50 nm in width and 260 nm in length. From the purified preparations of nucleocapsids of the virus, two major proteins with molecular weights of 35 and 14 kDa were identified by SDS-polyacrylamide gel electrophoretic analysis. The viral genomic DNA extracted from the nucleocapsids was digested with Bam HI and Sau 3AI, but not with Eco RI, Pst I, Xho I and Sal I. The molecular weight of the DNA was estimated to be approximately 85.1 × 10 6 from the agarose gel electrophoretic pattern of the Bam HI fragments. A part of the Bam HI fragments was cloned, labeled with 32 P-CTP and used for Southern blot analysis as a probe. The fragment hybridized to DNA isolated from the diseased larvae, but not to DNA isolated from healthy larvae.

The polymerase chain reaction (PCR) was used to amplify a portion of the coat protein gene (RNA2) of striped jack nervous necrosis vlrus (SJNNV), the causative agent of viral nervous necrosis (VNN) of larval striped jack Pseudocaranx dentex. Based on the sequence data of SJNNV RNA2, 2 forward (F1 and F2) and 3 reverse (RI. R2 and R3) PCR primers were synthesized and the 5 potential target regions were amplified with a combination of these primers. After reverse transcription of genomic RNA extracted from SJNNV and 25 cycles of PCR amplification, products of the expected size were detected separately on agarose gels stained with ethidium bromide. Southern blot hybridization confirmed that all of the amplified products were specific to cDNA of SJNNV RNA2. Two primer sets, F1-R2 and F2-R3, produced the specified 180 bp and 430 bp products. The PCR system, using the F2-R3 primer set, was able to detect 100 fg of SJNNV RNA after 25 cycles and was also able to efficiently amplify the target region of SJNNV gene in the total nucleic acids extracted from larval striped jack affected with VNN.

PCR法によるシマアジ親魚(生殖巣)からのウイルス性神経壊死症(VNN)原因ウイルス(SJNNV)遺伝子の検出結果と仔魚でのVNN発生との関係を検討した。その結果, VNNはウイルス陰性親魚群からの仔魚には発生せず, ウイルス陽性魚を含む親魚群の仔魚に発生した。また後者の親魚群からウイルス陽性魚を除去して産卵させて得た仔魚にはVNNは発生しなかった。従って, PCR法によるウイルス遺伝子の検出はVNN防除のための親魚選別に有効であると考えられた。

The complete nucleotide sequence of a set of cDNA clones designated KU1 strain, from which infectious in vitro transcripts can be transcribed, of the ATCC66 strain of brome mosaic virus (BMV) was determined and compared with the Russian strain of BMV in both nucleotide and the deduced amino acid sequence. Each corresponding genomic RNA in the KU1 strain and the Russian strain was homologous by more than 97% in nucleotide sequence. Base substitution of the transition type was more frequent than that of the transversion type in all three genomic RNAs. Amino acid changes in three regions where significant amino acid sequence homology is shown among the Sindbis-like plant RNA viruses were mostly substitutions for amino acids categorized to similar groups. The nucleotide sequence of the 5′ noncoding region in RNA1 and RNA3 was identical in the corresponding RNAs of the two strains while seven base changes were observed in RNA2. Any base change in the 3′ noncoding region did not disrupt a predicted secondary structure. Two adjacent adenine residues were absent in the noncoding leader sequence of the coat protein gene of both KU1 strain and ATCC66 strain. The intercistronic oligo(A) tract in RNA3 of the ATCC66 strain was heterogeneous in length in which more than two guanine residues were observed by direct RNA sequencing, while one guanine residue was present in the RNA3 of the cDNA clone of the KU1 strain.

シマアジのウイルス性神経壊死症（VNN）は孵化後2～20日目の仔魚に発生し, 病魚は摂餌をせず, 鰾の膨ら張, 脊索の湾曲, 水面に浮上して斃死するなどの症状が認められた. 孵化後10日目以前に発病した仔魚は数日以内に全滅したが, 11日目以降で発病した場合は, 一部が生き残る例もあった. ELISA 法で測定したウイルス抗原量（SJNNV）の上昇は, 孵化後6日目以前の仔魚では極めて早かったが, 11日目以降の仔魚では比較的遅かった. 同一の親魚群では産卵回数が増す程 VNN の発生率が高くなる傾向が認められた.

We succeeded in infecting turnip protoplasts with a cloned cauliflower mosaic virus (CaMV) DNA, pCa122, which contains 1.2 copy of CaMV genomic DNA and a plasmid expressing open reading frame (ORF) VI products (pEXP6) using polyethylene glycol. Fluorescent antibody staining showed that up to 50% of protoplasts were infected. It was difficult to detect the progeny DNA and the viral protein in protoplasts inoculated with pCa122 alone. Co-transfection with the plasmid pEXP6 produced larger fluorescing specks in each infected protoplasts and increased the accumulation of the progeny DNA and some other viral proteins to detectable levels. Using this protoplast system, three CaMV ORF I insertional mutants which were not infectious on turnip plants were tested for their infectivity on turnip protoplasts. Viral DNA and products accumulated in infected protoplasts to the same extent of the wild type DNA-infected protoplasts. These results indicate that ORF I product is not required for multiplication of CaMV in protoplasts, but is indispensable for infection on whole plants, strongly supporting that ORF I product is involved in cell-to-cell movement of CaMV.

We have constructed transgenic tobacco plants (M1x2-FCP2IFN plants) expressing viral RNA replication genes of brome mosaic virus (BMV) and BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN-gamma) gene. In M1x2-FCP2IFN plants the RNA3 derivative expressed from the integrated cDNA was replicated and subgenomic RNA (i.e. mRNA of IFN-gamma) was produced by BMV replicase. The accumulation level of the mRNA of IFN-gamma was approximately 5-fold higher than that by the cauliflower mosaic virus (CaMV) 35S RNA promoter. In addition IFN-gamma accumulated in M1x2-FCP2IFN plants.

The synthesis and accumulation of aphid transmission factor protein (p18) in cauliflower mosaic virus (CaMV)-infected turnip protoplasts were examined in time course and pulse-labelling experiments, comparing an aphid-non-transmissible isolate (CM1841) with an in vitro recombinant aphid-transmissible CaMV (CMBX) generated from the CM1841 isolate. There was little difference in the synthesis and accumulation of p18 between CM1841- and CMBX-infected protoplasts. When the accumulation of p18 in infected leaves was monitored from 3 to 28 days post-symptom emergence (p.e.) by Western blotting, the amount of p18 accumulated in CM1841-infected leaves continuously decreased from 3 days p.e. throughout the experimental period, whereas the amount of p18 in CMBX-infected leaves was lowest at 3 days p.e. and increased thereafter. These results suggested that CM1841 differed from CMBX not in the synthesis of p18 but in the stability of p18 in infected leaves.

The pathogenicity of the agent causing viral nervous necrosis (VNN) of striped jack, Pseudocaranx dentex (Bloch & Schneider), was examined in striped jack and other selected marine fish species. Fish were exposed to purified striped jack nervous necrosis virus (SJNNV) (0·1–100 ng ml−1) or homogenates of diseased striped jack larvae. Striped jack larvae (3·5 and 4·4 mm total length) were susceptible to the virus, but juveniles (78 mm) were not. The viral antigens were detected by indirect ELISA and the characteristic pathological changes, i.e. vacuolation in the retina and brain, were reproduced in the affected larvae. The infection was also established in healthy larvae by cohabitation with the diseased larvae. Larvae of red sea bream, Pagrus major Temminck & Schlegel, yellowtail, Seriola quinqueradiata Temminck & Schlegel, and goldstriped amberjack, Seriola lalandi Valenciennes, were not susceptible to SJNNV.

We succeeded in producing human gamma interferon (IFN-gamma) in tobacco protoplasts in quantity using genetically engineered brome mosaic virus (BMV strain ATCC66). This strain of BMV produces two types of coat protein, a full-length coat protein (20K) and a truncated coat protein (19K) which are translated from the first and second initiation codons, respectively. We replaced the truncated coat protein gene with the IFN-gamma gene and synthesized BMV-IFN-gamma chimera RNAs using an in vitro transcription system. The BMV-IFN-gamma chimera RNAs were used to inoculate tobacco protoplasts together with BMV RNA 1 and RNA 2 and produced IFN-gamma to a level of 5 to 10% of total extracted proteins per infected protoplast after 24 h of incubation. The efficient production of IFN-gamma was attributed to the high translation activity of the BMV-IFN-gamma chimera RNA. We demonstrate that 24 nucleotides coding for the N-terminal amino acids of the full-length coat protein were probably involved in the high translation activity of the BMV-IFN-gamma chimera RNA.

Rice plants fixed in glutalaldehyde were cut into 30μm thickness with Automatic MT-2 (Nihon-Ika). The cross sections were immersed in the fluorescent antibody labelled with FITC against Pseudomonas glumae and observed under a fluorescent microscope. Specific fluorescences derived from the bacteria were found in and on an upper and lower epidermis, in a parenchyma and in a sclerenchyma of palea and lemma of grain. They were found in an upper and lower epidermis and a vascular bundle sheath of a flag leaf blade, and around a vascular bundle and a lysigenous aerenchyma of a flag leaf sheath. These results suggest that this immunofluorescent antibody technique is useful for detecting P. glumae on rice plants.

1,8-Dihydroxynaphthalene (1,8-DHN) was isolated from Cochliobolus heterostrophus pale green mutant M13BL19 ( Alb1 + Brn1 + Sal1 + Pgr1-1 ) as the active substance which restored melanization of HE1AS73 ( Alb1-8 Brn1-2 Sal1-1 Pgr1 + ). HE1AS73 oxidized 1,8-DHN, 1-naphthol, N,N -dimethyl- p -phenylenediamine, tetramethylbenzidine, syringaldazine, and 3-(3,4′-dihydroxyphenyl)alanine, but did not oxidize p -cresol and l -tyrosine. None of these compounds was oxidized by HE5R13 ( Alb1-8 Brn1-2 Sal1-1 Pgr1-1 ). The syringaldazine oxidation activity of HE1AS73 was inhibited by diethyldithiocarbamate, cetyltrimethylammonium bromide, and dithiopyrimidine. These results indicate that the Pgr1 + strain contained p -diphenol oxidase which was absent from the Pgr1 − strain. We consider from these results that p -diphenol oxidase, in which Pgr1 is involved, plays a role in melanin synthesis of C. heterostrophus and 1,8-DHN is a natural substrate of this enzyme.

It has been indicated that parental spawners were a source of infection of a viral disease, “viral nervous necrosis", in larval striped jack Pseudocaranx dentex. In this study, the prevalence of antibodies against the causative virus (SJNNV) was examined among brood stocks of striped jack by indirect ELISA. Indirect ELISA using purified SJNNV, rabbit anti-SJNNV serum, and enzyme-conjugated goat anti-rabbit IgG antibody was employed for antibody detection from the plasma. The plasma IgM partially purified by ion-exchange chromatography was used for ELISA detection. The antibody to SJNNV was detected at high frequency (65%) in plasma samples collected from brood stocks reared at various facilities regardless of their sex or origin (wild or domestic). This indicates that the virus is prevalent among cultured populations of this fish species.

The present paper describes the detection of striped jack nervous necrosis virus (SJNNV), the causative agent of viral nervous necrosis, in eggs, larvae, and brood stocks of hatchery-reared and captured striped jack Pseudocaranx dentex by indirect ELISA. An appropriate condition for the ELISA was determined using purified SJNNV and the detection limit of virus antigens was approximately 5 ng/well. All samples of larvae supposed to be affected with the disease were proved positive for the presence of SJNNV antigens in this ELISA test. The viral antigens were also detected in some samples of fertilized eggs and ovaries of spawners but not in the testis or brain. Thus, it was indicated that spawners were an inoculum source of this viral disease in striped jack.

We introduced replicase genes of brome mosaic virus (BMV) to Nicotiana tabacum cv. Petit Habana (SR1) using two different types of transformation vectors containing cDNAs of BMV RNA 1 and RNA 2. One type (V type) contains cDNA from which complete viral RNAs are transcribed. These RNAs can function as templates for viral replicase. The other type (M type) contains cDNA from which viral RNAs without their 3' non-coding regions are transcribed; these RNAs can only function as mRNA. Viral replicase expressed from the integrated cDNAs in both V and M type transgenic plants can complement an infection by BMV RNA 3.

Lipoxygenase (LOX) and lipid hydroperoxide-decomposing activity (LHDA) markedly increased in the fifth leaves of rice (Oryza sativa cv Aichiasahi) after infection with the rice blast fungus, Magnaporthe grisea. The increases in the enzyme activities were significantly higher in response to infection with an incompatible strain (race 131) compared with infection with a compatible strain (race 007) of the fungus. Using ion-exchange chromatography, we isolated three LOX activities (leaf LOX-1, -2, -3) from both uninoculated and infected leaves. The activity of leaf LOX-3, in particular, increased in the incompatible race-infected leaves. The leaf LOX-3 had a pH optimum of 5.0 and produced preferentially 13-l-hydroperoxy-9,11 (Z,E)-octadecadienoic acid (13-HPODD) from linoleic acid. 13-HPODD and 13-l-hydroxy-9,11 (Z,E)-octadecadienoic acid, one of the reaction products from 13-HPODD by LHDA, were highly inhibitory to the germination of conidia of the fungus. The present study provides correlative evidence for important roles of LOX and LHDA in the resistance response of rice against the blast fungus.

Full-length biologically active cDNAs of brome mosaic virus genomic RNAs 1, 2 and 3 were constructed by joining cDNA fragments. The cDNAs were constructed so that, at the 5' ends, unique SnaBI sites were present at the site of initiation of transcription. The cDNAs were inserted between a modified cauliflower mosaic virus (CaMV) 35S RNA promoter and terminator regions derived from CaMV DNA, and cloned into pUC18. When a mixture of the plasmid DNAs was inoculated onto Chenopodium hybridum leaves, local lesions appeared 5 to 6 days later. However, no symptoms appeared in similarly inoculated barley plants. Plasmid cDNAs with extra sequences at the 5' end were infectious but RNAs transcribed from cDNAs with similar sequences were not.

Spraying cucumber leaves with salicylic acid (SA), 7-methoxycarbonyl benzo-1, 2, 3-thiadiazol and 2-chlorethylphosphate (ethephon) (abiotic inducers) reduced the diseased area caused by Pseudoperonospora cubensis by more than 50% in the sprayed first leavesand also in the upper second leaves provided challenge inoculation was made 3 to 6 days but not one to 24hr after treatment. Localized infection of cotyledons with P. cubensis (biotic inducer) also reduced the diseased area caused by the same pathogen by more than 50% in the upper leaves challenge-inoculated 6 days after inducer inoculation. Plants acquired systemic resistance when inoculated cotyledons remained attached for at least 3 days after inoculation. Protection by both abiotic and biotic inducers was more prominent in the second leaves which expanded after induction than in the first leaves which expanded before induction. The present results show that systemic resistance induced in cucumber plants by either biotic or abiotic inducers was effective in controlling infection by P. cubensis whose cell walls contain no chitin. Electrophoretic analysis of extracted proteins on polyacrylamide gel showed that both the SA treatment and localized infection with P. cubensis induced several novel acid soluble proteins in the treated and the upper untreated leaves in correlation with induced resistance.

Aphid transmissible Japanese isolate of caulifiower mosaic virus M (CaMV-M) was cloned into pUC19 and its ORF II region was sequenced. Comparison of this sequence with that of CM1841 (non-aphid transmissible isolate of CaMV) showed that there were nine base changes, four of which gave rise to alterations in the predicted amino acid sequence. Characterization of in vitro recombined hybrid viruses between CaMV-M and CM1841 showed that 5' half of ORF II was responsible for the lack of aphid transmissibility in CM1841 and this region influenced the amount of accumulation of ORF II product in the infected leaves.