Chilo Iridescent Virus Research Articles

Characterization of an iridescent virus isolated from Gryllus bimaculatus (Orthoptera: Gryllidae).

We have isolated an iridescent virus from commercially produced colonies of Gryllus bimaculatus in Germany, which showed apparent mortality. Transmission electron microscopy studies on adult cricket specimens revealed the paracrystalline assembly of icosahedral virus particles in the cytoplasm of hypertrophied abdominal fat body cells. The infecting agent could be cultivated in the lepidopteran cell line sf-9, where it caused cytopathogenic effects such as cell hypertrophy, cytoplasmic vacuolization, and cell death within 8 days postinfection. Infection titers of the first virus passage reached 10(7.5) TCID(50)/ml. Negatively stained virus particles (n = 100) had dimensions of 172 +/- 6 nm (apex to apex) and 148 +/- 5 nm (side to side). SDS-polyacrylamide gel electrophoresis of virus proteins showed more than 20 distinct polypeptides with a major species of approximately 50 kDa. Analysis of the restriction fragment length profiles from digestion of purified viral DNA with the endonucleases EcoRI, BamHI, and HindIII showed marked differences from the profiles of iridoviruses of lower vertebrates (genus Ranavirus), e.g., Rana esculenta Iridovirus and Frog virus 3. Restriction enzyme digests with the endonucleases MspI and HpaII indicated the lack of methylation of viral DNA. Polymerase chain reaction led to the amplification of a 420-bp gene fragment with 97% sequence homology to the major capsid protein gene of Chilo iridescent virus. These data indicate that this new isolate, which we propose to be termed Gryllus bimaculatus iridescent virus, belongs to the genus Iridovirus of the family Iridoviridae.

Structure and assembly of large lipid-containing dsDNA viruses.

Structure and assembly of large lipid-containing dsDNA viruses.

CIV, An Iridovirus With a Pseudo T=147 Capsid Structure

Abstract Chilo iridescent virus (CIV), the type species of the genus Iridovirusin the family Iridoviridae,was first isolated from the rice stem borer, Chilo suppressalis,a member of Lepidoptera insect order. ‘2 CIV is being considered as a potential pest control agent because of its wide host range which includes leafhoppers and some arthropods. The virion (∼lxl09 daltons) encapsidates a 209 kbp, dsDNA genome within its fiber-covered, icosahedral shell. The virion is composed of about 26 structural proteins, two (P50 and P'50, 50kDa each) of which are the major capsid proteins. P50 migrates as a monomer in either a reducing or non-reducing SDS-PAGE gel and is exposed to the surface, whereas P'50 exists as a disulfide-bond, stabilized trimer in a non-reducing gel and is only partially exposed. Virions contain 9% lipid present as a lipid bilayer within the protein capsid.

Identification of a gene cluster within the genome of Chilo iridescent virus encoding enzymes involved in viral DNA replication and processing.

The nucleotide sequence of the genome of Chilo iridescent virus (CIV) between the genome coordinates 0.974 and 0.101 comprising 27,079 bp was determined. Computer-assisted analysis of the DNA sequence of this particular region of the CIV genome revealed the presence of 42 potential open reading frames (ORFs) with coding capacities for polypeptides ranging from 50 to 1,273 amino acid residues. The analysis of the amino acid sequences deduced from the individual ORFs resulted in the identification of 10 potential viral genes that show significant homology to functionally characterized proteins of other species. A cluster of five viral genes that encode enzymes involved in the viral DNA replication was identified including the DNA topoisomerase II (A039L,1,132 amino acids (aa)), the DNA polymerase (ORF A031L,1,273 aa), a helicase (ORF A027L, 530 aa), a nucleoside triphosphatase I (ORF A025L, 1,171 aa), and an exonuclease II (ORF A019L, 624aa), all ORFs possessing the same genomic orientation. The DNA polymerase of CIV showed the highest homology (24.8% identity) to the DNA polymerase of lymphocystis disease virus lymphocystis disease virus 1 (LCDV-1), a member of the family Iridoviridae, indicating the close relatedness of the two viruses. In addition, four putative gene products were found to be significantly homologous to previously identified hypothetical proteins of CIV.

Identification of a thymidylate synthase gene within the genome of Chilo iridescent virus.

The thymidylate synthase (TS, EC 2.1.1.45) is essential for the de novo synthesis of dTMP in pro- and eucaryotic organisms. Consequently it plays a major role in the replication of the DNA genome of a cell or a DNA virus. The gene encoding the TS of Chilo iridescent virus (CIV) was identified by nucleotide sequence analysis of the viral genome and was mapped within the EcoRI CIV DNA fragments G and R. Computer assisted analysis of the DNA nucleotide sequence between the genome coordinates 0.482 and 0.489 revealed an open reading frame (ORF) of 885 nucleotides. This ORF was found to encode a polypeptide of 295 amino acid residues (33.9 kDa) that showed significant homologies to known TS of different species including mammals, plants, fungi, protozoa, bacteria, and DNA viruses. The highest amino acid homologies were found between the CIV-TS and the TS of herpesvirus ateles (54.0%), Saccharomyces cerevisiae (51.8%), herpesvirus saimiri (51.0%), rhesus monkey rhadinovirus (50.7%), mouse (50.5%), rat (50.2%), varicella-zoster virus (50.2%), equine herpesvirus 2 (50.0%), and the human TS (48.4%). The CIV-TS contains six amino acid domains that are highly conserved in the TS of other species. Within these domains the major amino acid residues are present for which a functional role has been reported. The CIV-TS was found to be more closely related to the TS of eucaryotes than to the TS of procaryotes indicating the phylogenetic origin of the CIV-TS gene. The identification of a TS gene in the genome of CIV is the first report of a viral TS that is not encoded by a herpesvirus or a bacteriophage.

Is the major capsid protein of iridoviruses a suitable target for the study of viral evolution?

Iridoviruses are large cytoplasmic DNA viruses that are specific for different insect or vertebrate hosts. The major structural component of the non-enveloped icosahedral virus particles is the major capsid protein (MCP) which appears to be highly conserved among members of the family Iridoviridae, Phycodnaviridae, and African swine fever virus. The amino acid sequences of the known MCPs were used in comparative analyses to elucidate the phylogenic relationships between different cytoplasmic DNA viruses including three insect iridoviruses (Tipula iridescent virus, Simulium iridescent virus, Chilo iridescent virus), seven vertebrate iridoviruses isolated either from fish (lymphocystis disease virus, rainbow trout virus, European catfish virus, doctor fish virus), amphibians (frog virus 3), or reptiles (turtle virus 3, turtle virus 5), one member of the family Phycodnaviridae (Paramecium bursaria Chlorella virus type 1), and African swine fever virus. These analyses revealed that the amino acid sequence of the MCP is a suitable target for the study of viral evolution since it contains highly conserved domains, but is sufficiently diverse to distinguish closely related iridovirus isolates. Furthermore the results suggest that a substantial revision of the taxonomy of iridoviruses based on molecular phylogeny is required.

The DNA sequence of Chilo iridescent virus between the genome coordinates 0.101 and 0.391; similarities in coding strategy between insect and vertebrate iridoviruses.

Chilo iridescent virus (CIV), the type species of the genus Iridovirus within the family Iridoviridae, is highly pathogenic for larvae of important pest insects. The virions contain a single linear double-stranded DNA molecule (209 kbp) that is circularly permuted and terminally redundant. The nucleotide sequence of the viral genome between the genome coordinates 0.101 and 0.391 (60,170 bp) was determined by automated cycle sequencing. This particular region of the CIV genome contains 112 open reading frames (ORFs) with coding capacities for 50 to 1186 amino acids. The alignment of the deduced amino acid sequences with well-characterized proteins stored in protein databases led to the identification of several genes with significant homologies, such as the largest subunit of the DNA-dependent RNA polymerase, large subunit of the ribonucleoside-diphosphate reductase, endonuclease, protein-tyrosine phosphatase, helicase, global transactivator, two apoptosis inhibitor homologs, antibiotic peptide homolog, and others. The highest homologies were detected between putative viral gene products of CIV and the corresponding viral proteins of lymphocystis disease virus of fish (LCDV), which belongs to the genus Lymphocystivirus within the iridovirus family.

Identification and properties of the largest subunit of the DNA-dependent RNA polymerase of fish lymphocystis disease virus: dramatic difference in the domain organization in the family Iridoviridae

Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes. The amino acid sequences of the known largest subunits of DdRPs from different species contain highly conserved regions. Oligonucleotide primers, deduced from two conserved domains (RQP[T/S]LH and NADFDGDE) were used for detecting the corresponding gene of fish lymphocystis disease virus (FLCDV), a member of the family Iridoviridae, which replicates in the cytoplasm of infected cells of flatfish. The gene coding for the largest subunit of the DdRP was identified using a PCR-derived probe. The screening of the complete EcoRI gene library of the viral genome led to the identification of the gene locus of the largest subunit of the DdRP within the EcoRI DNA fragment B (12.4 kbp, 0.034 to 0.165 map units). The nucleotide sequence of a part (8334 bp) of the EcoRI DNA fragment B was determined and a large ORF on the lower strand (ATG = 5787; TAA = 2190) was detected which encodes a protein of 1199 amino acids. Comparison of the amino acid sequences of the largest subunits of the DdRP (RPO1) of FLCDV and Chilo iridescent virus (CIV) revealed a dramatic difference in their domain organization. Unlike the 1051 aa RPO1 of CIV, which lacks the C-terminal domain conserved in eukaryotic, eubacterial and other viral RNA polymerases, the 1199 aa RPO1 of FLCDV is fully collinear with its cellular and viral homologues. Despite this difference, comparative analysis of the amino acid sequences of viral and cellular RNA polymerases suggests a common origin for the largest RNA polymerase subunits of FLCDV and CIV.

Insect iridescent virus type 6 encodes a polypeptide related to the largest subunit of eukaryotic RNA polymerase II.

Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes. The amino acid sequences of known large subunits of DdRPs contain highly conserved regions. Oligonucleotide primers, deduced from two conserved domains [RQP(T/S)LH and NADFDGDE] were used in PCR experiments for the detection of the corresponding gene of the genome of insect iridescent virus type 6, also known as Chilo iridescent virus (CIV). A specific DNA product of about 150 bp could be amplified and was used as a hybridization probe against the CIV gene library to identify the corresponding gene. The gene encoding the DdRP was identified within the EcoRI fragments M (7099 bp) and L (7400 bp) of CIV DNA, between map units 0.310 and 0.347 (7990 bp). The DNA nucleotide sequence (3153 bp) of the gene encoding the largest subunit of DdRP (RPO1) was determined. Northern blot hybridization revealed the presence of a 3.4 kb RNA transcript in CIV-infected cells that hybridized to the CIV DdRP gene. This predicted viral protein consists of 1051 amino acid residues (120K) and showed considerably higher similarity to the largest subunit of eukaryotic RNA polymerase II than to the homologous proteins of vaccinia virus and African swine fever virus. Phylogenetic analysis suggested that the putative RPO1 of CIV could have evolved from RNA polymerase II after the divergence of the three types of eukaryotic RNA polymerases. The putative RPO1 of CIV lacked the C-terminal domain that is conserved in eukaryotic, eubacterial and other viral RNA polymerases and in this respect was analogous to the RNA polymerases of Archaea. It is hypothesized that the equivalent of the C-terminal domain may reside in another subunit of CIV DdRP encoded by an unidentified viral gene.

Chilo iridescent virus encodes a putative helicase belonging to a distinct family within the “Dead/H” superfamily: Implications for the evolution of large DNA viruses

The complete nucleotide sequence of the EcoRI DNA fragment M (7099 bp; 0.310-0.345 map units) of the genome of insect iridescent virus type 6--Chilo iridescent virus (CIV)--was determined. A 606 codon open reading frame located in this region encoded a protein (p69) related to a distinct family of putative DNA and/or RNA helicases belonging to the "DEAD/H" superfamily. Unique sequence signatures were derived that allowed selective retrieval of the putative helicases of the new family from amino acid sequence databases. The family includes yeast, Drosophila, mammalian, and bacterial proteins involved in transcription regulation and in repair of damaged DNA. It is hypothesized that p69 of CIV may be a DNA or RNA helicase possibly involved in viral transcription. A distant relationship was observed to exist between this family of helicases and another group of proteins that consists of putative helicases of poxviruses, African swine fever virus, and yeast mitochondrial plasmids. It is shown that p69 of CIV is much more closely related to cellular helicases than any of the other known viral helicases. Phylogenetic analysis suggested an independent origin for the p69 gene and the genes encoding other viral helicases.

Identification of the primary structure and the coding capacity of the genome of insect iridescent virus type 6 between the genome coordinates 0.310 and 0.347 (7990 bp).

The primary structure and the coding capacity of the insect iridescent virus type 6--Chilo iridescent virus (CIV)--were determined between the genome coordinates 0.310 (EcoRI site) and 0.347 (ClaI site). The EcoRI CIV DNA fragment M (7.1 kb; 0.310-0.345 map units) harbors one out of at least six loci of DNA replication origins which is located at nucleotide position 485-513. The identification of the structural properties and the coding capacity of the EcoRI CIV DNA fragment M was carried out by DNA nucleotide sequencing, computer-aided sequence analysis and DNA/RNA hybridization. The EcoRI CIV DNA fragment M (7,099 bp; 71.14% A+T and 28.86% G+C) possesses two clusters of five tandemly organized repetitive DNA elements with complex structural arrangements (R1-R5) which are located between nucleotide positions 3272-3350 and 3403-3414. The analysis of the DNA sequences of the EcoRI CIV DNA fragment M revealed the presence of six open reading frames (ORFs 1-6). Two out of six detected putative proteins are of particular interest. ORF-2 was found to be terminated at nucleotide position 366 (TAA) within the DNA sequence of the EcoRI CIV DNA fragment L (0.345-0.381 map units; 7.4 kb). The analysis of ORF-2 (1,051 amino acids; 120 kD) revealed homologies to several DNA-directed RNA polymerases. ORF-6 encodes a protein (606 amino acids; 69 kD) which is related to a group of yeast, Drosophila and mammalian proteins of a distinct family of putative DNA and/or RNA helicases belonging to the 'DEAD/H' superfamily. The transcriptional activity of the EcoRI CIV DNA fragment M was determined by DNA/RNA hybridization experiments. These analyses revealed the existence of three RNA transcripts of about 3.4 kb (t1), 1.8 kb (t2) and 1.2 kb (t3) which agree with the predicted size of the expected RNA transcripts from ORF-2 (1,051 amino acids; 3.1 kb) and ORF-6 (606 amino acids; 1.8 kb).

Identification of genes encoding zinc finger proteins, non-histone chromosomal HMG protein homologue, and a putative GTP phosphohydrolase in the genome of Chilo iridescent virus.

Five RNA transcripts of about 1.2 to 1.7 kilobases were mapped to a part of the genome of insect iridescent virus type 6 (Chilo iridescent virus; CIV) between genome coordinates 0.832 and 0.856 within the EcoRI DNA fragment F. The nucleotide sequence of this particular region (5702 base pairs) of the CIV genome was determined. The DNA sequence contains a number of perfect direct, inverted, and palindromic repeats including three clusters of tandemly organized repetitive DNA elements located between the nucleotide positions 1534 to 1566, 3720 to 3780, and 4350 to 4450. Eight long open reading frames (ORFs; EF1 to 8) were detected in the sequenced region of the CIV genome. ORF EF1 encodes a putative protein of 221 amino acid residues (aa) that is closely related to eukaryotic nonhistone chromosomal proteins of the high mobility group (HMG) superfamily. Virus encoded homologues of HMG proteins have not been reported so far. The EF2 gene product (145 aa) contains a specific zinc finger motif and belongs to a distinct group of identified and putative zinc finger proteins including a second putative protein (239 aa) of CIV encoded in the EcoRI DNA fragment Y (1984 bp; 0.381 to 0.391 viral map units). The product of EF6 (127 aa) is related to D250 ORF product of African swine fever virus (ASFV) and belongs to the recently described protein family sharing a highly conserved sequence motif with bacterial antimutator GTP phosphohydrolase MutT. Thus the sequenced region of the CIV genome encodes three putative proteins which may be directly involved in the replication and/or transcription of the viral DNA.

Identification of the gene encoding the major capsid protein of fish lymphocystis disease virus

The gene encoding the major capsid protein (MCP) of fish lymphocystis disease virus (flounder isolate; FLCDV-f) has been identified by PCR using oligonucleotide primers corresponding to different regions of the MCP of Tipula iridescent virus (TIV), iridescent virus 22 (IV22) and Chilo iridescent virus (CIV). DNA fragments of 0.4 kbp, 0.5 kbp and 0.27 kbp in size were amplified using oligonucleotide primers corresponding to amino acids (aa) 146 to 153 (primer 1) and 274 to 268 (primer 6), or aa 146 to 153 (primer 1) and 313 to 304 (primer 8), or aa 304 to 312 (primer 7) and 385 to 381 (primer 9) of the MCP of TIV, respectively. The PCR products were used as hybridization probes for screening the gene library of FLCDV-f. The MCP gene of FLCDV-f(1377 bp; 459 aa; 51.4K) was identified within the DNA sequence of the EcoRI FLCDV-f DNA fragment C (11.2 kbp; 0.611 to 0.718 map units). A high degree of aa sequence identity/similarity was detected between the MCP of FLCDV-f and TIV (50.3%/33.8%), IV22 (49.1%/34.2%). CIV (53%/29.5%) and African swine fever virus (16%/38.1%).

Identification of the gene encoding the major capsid protein of insect iridescent virus type 6 by polymerase chain reaction.

The gene encoding the major capsid protein of Chilo iridescent virus (CIV) has been identified by PCR using oligonucleotide primers corresponding to different regions of the major capsid proteins of Tipula iridescent virus (TIV) and iridescent virus 22 (IV22). A DNA fragment of 0.5 kbp was amplified using two oligonucleotide primers corresponding to the amino acid positions 146 to 153 and 304 to 313 of the major capsid protein of TIV, respectively. The radioactively labelled DNA fragment derived from PCR was hybridized to a CIV gene library. This analysis revealed that only the EcoRI CIV DNA fragment X [2.85 kbp; 0.589 to 0.603 viral map units (m.u.)] hybridized to the amplified DNA fragment. An RNA transcript of about 1.5 kb was identified when the PCR product was used as a hybridization probe. The same RNA transcript was detected when the EcoRI fragments X and Q (5.9 kbp; 0.603 to 0.631 viral m.u.) were used as probes. This indicates that the expected gene is located within map coordinates 0.589 to 0.631 and harbours part of the DNA sequences of fragments Q and X. The analysis of the DNA sequences of this particular region of the CIV genome revealed the presence of one open reding frame of 1401 bp. The DNA sequences of this region encode a protein of 467 amino acid residues with an M(r) of 51.4K. A high degree (64.7%) of amino acid sequence identity was detected between the major capsid protein of TIV and/or IV22 and the amino acid composition of the identified CIV protein.

Prokaryotic Members of a New Family of Putative Helicases with Similarity to Transcription Activator SNF2

Cloning and sequence analysis of a new open reading frame from Bacillus cereus reveals the relationship to a recently identified family of putative eukaryotic transcription activators similar to the yeast SNF2 gene product. As a result of comparative analysis of sequence features conserved in all members of this family, a gene from a chilo iridescent virus, as well as a putative helicase from Escherichia coli (hepA), can also grouped into this family. The unexpected presence of prokaryotic and viral sequences in the previously purely eukaryotic SNF2 family suggests a defined subgroup of DNA helicases present in all species, with specific function in transcription activation.

Characterization of the third origin of DNA replication of the genome of insect iridescent virus type 6.

The structure of the third origin of DNA replication (CIV-ori-M) of the genome (209 kbp) of Chilo iridescent virus (CIV) was determined by DNA nucleotide sequence analysis. The CIV-ori-M is located within the DNA sequences of the EcoRI CIV DNA fragment M (7 kbp; 0.310-0.345 viral map units) between the genome coordinates 0.310 (EcoRI site) and 0.317 (NcoI site). The DNA nucleotide sequence of the EcoRI/NcoI CIV DNA fragment (1601 bp) was determined for identifying the DNA sequence of the corresponding origin of DNA replication. The analysis of the DNA sequences of this region revealed the presence of a 12-mer inverted repeat at nucleotide positions 485-496 and 503-513 (485-AGATATTTGACT-496-TATGT-503-AGTCAAATATCT-513) that are able to form a hairpin-loop structure. A double-stranded DNA fragment was synthesized that corresponds to the nucleotide positions 485-513 that were cloned into the phages M13mp18 and M13mp19, and were screened for their ability to be amplified in CF-124 cell cultures infected with CIV. The successful amplification of the DNA sequence of the CIV-ori-M is strong evidence that this particular region of the CIV genome indeed serves as the origin of DNA replication.

Identification and mapping of origins of DNA replication within the DNA sequences of the genome of insect iridescent virus type 6.

The origins of DNA replication of the genome (209 kbp) of Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, were identified. The defined genomic library of CIV, which represents 100% of DNA sequences of the viral genome (e.g., all 32 EcoRI CIV DNA fragments), was used for transfection of Choristoneura fumiferana insect cell cultures (CF-124) that were previously infected with CIV. The plasmid rescue experiments were carried out to select those recombinant plasmids that were amplified during viral replication in CIV-infected cell cultures. It was found that six recombinant plasmids harboring the EcoRI DNA fragments C [13.5 kbp, 0.909-0.974 map units (m.u.)], H (9.8 kbp, 0.535-0.582 m.u.), M (7.25 kbp, 0.310-0.345 m.u.), O (6.5 kbp, 0.196-0.228 m.u.), Q (5.9 kbp, 0.603-0.631 m.u.), and Y (2.0 kbp, 0.381-0.391 m.u.) were able to be amplified under the conditions used. This indicates that the CIV genome possesses six DNA replication origins. Subclones of the EcoRI CIV DNA fragments C and H were screened under the same conditions. It was found that DNA sequences within the EcoRI DNA fragments C and H at the genome coordinates 0.924-0.930 and 0.535-0.548, respectively, contain origins of viral DNA replication. The DNA nucleotide sequences of the EcoRI CIV DNA fragment Y (1986 bp) were determined for identifying the DNA sequence of the corresponding origin of DNA replication. The computer-aided analysis revealed the presence of a 15-mer inverted repeat at nucleotide positions 661-675 and 677-691 (661-TAAATTTAATGAGAA-G-TTCTCATTAAATTTA-692). The analysis of the DNA sequence of the EcoRI DNA fragment H corresponding to the particular region at the genome coordinates 0.535-0.548 (1) showed that this region contains a 16-mer inverted repeat at the nucleotide positions 1315 and 1332 (1315-TAAATTTTAATGGTTA-A-TAACCATTAAAATTTA-1347), which is very similar to the inverted repetition found within the EcoRI DNA fragment Y. The successful recognition and amplification of the single-stranded synthetic DNA sequences of both strands of CIV-ori-Y (nucleotide position 661-691) using phage M13 system in CIV-infected cells is strong evidence that the CIV-ori-Y is bidirectionally active, and this DNA sequence is considered to be the origin of DNA replication within the EcoRI CIV DNA fragment Y.

Synthesis of heat shock proteins in Cf124 cells: Effect of virus infection

The transient synthesis of a class of proteins known as heat shock or stress response proteins was induced when Cf124 cells were incubated at high temperature. When cells were infected with Chilo iridescent virus and simultaneously heat shocked, heat shock protein (hsp) synthesis was delayed, and the shut-off of hsp synthesis was suppressed. In previously heat shocked cells, inhibition of hsp synthesis was dependent upon the multiplicity of infection, however, when infection preceded heat shock, the synthesis of hsp started immediately after heat shock. In all cases, hsp synthesis was dependent upon newly synthesized messenger RNA.

Infectivity of vesicles prepared from chilo iridescent virus inner membrane: evidence for recombination between associated DNA fragments

Treatment of CIV particles with octylglucoside at high ionic strength leads to the solubilization of the inner viral membrane. Incubation of permissive cells (Cf124 cells) with vesicles obtained after dialysis of the detergent shows that this fraction is infectious. This infectivity, which is very low, could only be detected after two serial passages on permissive cells. This phenomenon is, however, reproducible. Isopycnic centrifugation analysis shows that some DNA cosediments with the vesicles. Extraction and purification of this DNA confirm the presence of a large DNA fragment of about 50 · 10 6 Da. Digestion with restriction endonucleases demonstrated that this DNA did not correspond to a particular fragment but to a population of DNA fragments of homogeneous size arising from various regions of the viral genome. Purified viral DNA was not infectious, the presence of DNA in the vesicles could not account therefore for their infectivity. Experiments of non-genetic reactivation of purified CIV DNA by UV-irradiated virus suggest that one (or several) structural component(s) of CIV particles must be involved in the first stages of the viral replication cycle. In addition, transfection of cells with large overlapping DNA fragments could generate infectious particles when the cells were superinfected with UV-irradiated virus. It can be supposed that the vesicle suspensions, which probably contain the reactivating factor, are composed of a population of vesicles which are all different in their DNA content. Infectivity of such suspensions would be the consequence of a recombination between large overlapping DNA fragments.

Identification and characterization of the repetitive DNA element in the genome of insect iridescent virus type 6

The genome of the Chilo iridescent virus (CIV) was analyzed for existence of repetitive DNA sequences by DNA-DNA hybridization using a defined and complete gene library of the viral genome (209 kbp) and by heteroduplex mapping. These experiments revealed the presence of repetitive DNA elements in the CIV genome, which are located in the EcoRI fragment H and in the EcoRI DNA fragment C at the coordinates 0.535 to 0.548 ( EcoRI/ Pstl DNA fragment, 2.7 kbp) and 0.920 to 0.944 ( Pvull CIV DNA fragment L, 5.1 kbp), respectively. The DNA nucleotide sequence (2708 bp) of the EcoRI/ Pstl subfragment was determined. The comparative analysis of the DNA sequences of this particular region of the viral genome with the DNA sequences of the Pvull DNA fragment L (5064 bp) revealed the presence of several DNA sequences within the EcoRI/ Pstl subfragment of the EcoRI CIV DNA fragment H which show homology to DNA sequences of the Pvull DNA fragment L. For example, a DNA element (☐ A, 91 bp) is located at nucleotide positions 1981 to 2072 of the EcoRI CIV DNA fragment H which are complementary (>90%) to the nine regions of the Pvull DNA fragment L (L-☐es 1 to 9). Furthermore heteroduplex mapping revealed the existence of a stem-loop structure (stem, 65 ± 10 by and loop, 652 ± 80 bp) at the genome coordinates 0.571 to 0.582 (2.5 kbp, HindIII/ EcoRI subfragment of the EcoRI CIV DNA fragment H). This indicates that an inverted repeat sequence is located at this region of the viral genome. The DNA nucleotide sequence of this subfragment was determined (2555 bp) which confirmed the data obtained from electron microscopy. An inverted repeat DNA sequence located at nucleotide positions 304 and 1011 is able to form this type of stem-loop structure.