Characterization of the repetitive DNA elements in the genome of fish lymphocystis disease viruses

The complete DNA nucleotide sequence of the repetitive DNA elements in the genome of fish lymphocystis disease virus (FLDV) isolated from two different species (flounder and dab) was determined. The size of these repetitive DNA elements was found to be 1413 bp which corresponds to the DNA sequences of the 5' terminus of the EcoRI DNA fragment B (0.034 to 0.052 m.u.) and to the EcoRI DNA fragment M (0.718 to 0.736 m.u.) of the FLDV genome causing lymphocystis disease in flounder and plaice. The degree of DNA nucleotide homology between both regions was found to be 99%. The repetitive DNA element in the genome of FLDV isolated from other fish species (dab) was identified and is located within the EcoRI DNA fragment B and J of the viral genome. The DNA nucleotide sequence of one duplicate of this repetition (EcoRI DNA fragment J) was determined (1410 bp) and compared to the DNA nucleotide sequences of the repetitive DNA elements of the genome of FLDV isolated from flounder. It was found that the repetitive DNA elements of the genome of FLDV derived from two different fish species are highly conserved and possess a degree of DNA sequence homology of 94%. The DNA sequences of each strand of the individual repetitive element possess one open reading frame.     

Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus

A defined and complete gene library of the fish lymphocystis disease virus (FLDV) genome was established. FLDV DNA was cleaved with EcoRI, BamHI, EcoRI/BamHI and EcoRI/HindIII and the resulting fragments were inserted into the corresponding sites of the pACYC184 or pAT153 plasmid vectors using T4 DNA ligase. Since FLDV DNA is highly methylated at CpG sequences (Darai et al., 1983; Wagner et al., 1985), an Escherichia coli GC-3 strain was required to amplify the recombinant plasmids harboring the FLDV DNA fragments. Bacterial colonies harboring recombinant plasmids were selected. All cloned fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNA to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned. Using these recombinant plasmids, the physical maps of the genome were constructed for BamHI, EcoRI, BestEII, and PstI restriction endonucleases. Although the FLDV genome is linear, due to circular permutation the restriction maps are circular.     

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 (PvuII 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 PvuII 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 PvuII DNA fragment L. For example, a DNA element (box A, 91 bp) is located at nucleotide positions 1981 to 2072 of the EcoRI CIV DNA fragment H which are complementary (greater than 90%) to the nine regions of the PvuII DNA fragment L (L-boxes 1 to 9). Furthermore heteroduplex mapping revealed the existence of a stem-loop structure (stem, 65 +/- 10 bp 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.     

Identification,mapping and cloning of the thymidine kinase gene of fish lymphocystis disease virus

The thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) was identified by biochemical transformation of 3T3 TK negative (TK⁻) to 3T3 TK positive (TK⁺) cells using specific viral DNA sequences. DNA fragments of the viral genome used in this study were obtained from a defined gene library of FLDV genome containing the complete viral DNA sequences. The selection of the converted cells was carried out under the condition of the HAT selection procedure. The results of these experiments revealed that the EcoRI FLDV DNA fragment C (11.2 kbp; 0.611 to 0.718 map units) is able to transform 3T3 TK⁻ to 3T3 TK⁺ cells. Additional experiments using the subclones of EcoRI DNA fragment C revealed that DNA sequences of 4.1 kbp size between the coordinates 0.669 to 0.718 of the FLDV genome possessed the ability for biochemical transformation, indicating that the TK gene locus is located in this particular region.     

Identification of lymphocystis disease virus from paradise fish Macropodus opercularis (LCDV-PF)

Iridoviruses are large DNA viruses that are subdivided into five genera: Ranavirus, Megalocytivirus, Lymphocystivirus, Chloriridovirus and Iridovirus. The iridovirus lymphocystis disease virus (LCDV) is an important fish pathogen that can infect marine and freshwater fish worldwide. In this study, we have identified the pathogen in paradise fish (Macropodus opercularis) with lymphocystis. On the skin and fins of diseased paradise fish, a large number of nodules were observed. H&E staining showed that the nodules were composed of encapsulated hypertrophied cells. Using electron microscopy, numerous virus particles with a diameter of >210 nm and with hexagonal profiles were observed in the cytoplasm. Phylogenetic analysis based on the major capsid protein (MCP), DNA polymerase and myristylated membrane protein (MMP) genes revealed that LCDV from paradise fish (LCDV-PF) was closely related to lymphocystis disease virus from China (LCDV-C), followed by lymphocystis disease virus 1 (LCDV-1). Taken together, our data provide the first molecular evidence that, in addition to megalocytivirus, LCDV is an important iridoviral pathogen in paradise fish besides megalocytivirus.     

Complete nucleotide sequence of the cucumber necrosis virus genome

The complete nucleotide sequence of the cucumber necrosis virus (CNV) genome has been determined. The genome is 4701 nucleotides in length and contains five long open reading frames (ORF). ORF1 begins at the first AUG codon at the 5' terminus and terminates at an amber codon. The predicted molecular weight of the polyprotein encoded by ORF1 is 33 kilodaltons (kDa). Readthrough of the ORF1 amber codon would yield a protein with a molecular weight of 92 kDa. Comparison of the amino acid sequence of the 92-kDa protein with the putative replicases of carnation mottle virus (CarMV) and barley yellow dwarf virus (BYDV) shows extensive sequence similarity. This suggests that the CNV 92-kDa protein is the viral replicase and, furthermore, suggests a close evolutionary relationship between CNV, CarMV, and BYDV, members of the Tombus-, Carmo-, and Luteovirus groups, respectively. Immediately following the 92-kDa protein is ORF3 which can encode a 40-kDa protein. It is identified as the coat protein based on its similarity in amino acid composition to the previously determined CNV coat protein sequence (J. H. Tremaine, 1972, Virology 48, 582-590) and on its amino acid sequence similarity with the tomato bushy stunt virus coat protein. Two nested ORFs (ORF4 and -5), in different frames, follow the coat protein gene. Although it is not known if both ORFs are expressed, they would encode proteins with predicted molecular weights of 21 and 20 kDa, respectively.     

Identification of the gene encoding the DNA (cytosine-5) methyltransferase of lymphocystis disease virus

The gene encoding the DNA (cytosine-5) methyltransferase (m5C-MTase) of lymphocystis disease virus (flounder isolate, LCDV-1) has been identified by polymerase chain reaction (PCR) using oligonucleotide primers synthesized corresponding to different regions of the m5C-MTase gene of frog virus 3 (FV3). A DNA fragment of 487 bp was amplified using oligonucleotide primers L3 and R4 which correspond to the nucleotide positions 87 to 109 and 530 to 550 of the m5C-MTase gene of FV3, respectively. The DNA nucleotide sequence of the PCR product was determined by direct cycle sequencing. The alignment of the deduced amino acid sequence derived from the PCR product and the m5C-MTase protein of FV3 revealed a homology of 55.4% identity and 29.1% similarity. The amino acid sequence which was found to be significantly homologous to the amino acid sequence deduced from the nucleotide sequence of the PCR product was located at the amino acid position 37 to 175 of the m5C-MTase of FV3 indicating the specificity of the amplified PCR product. The DNA nucleotide sequence of the LCDV-1 genome corresponding to the 5 and 3 termini of the m5C-MTase gene was determined by primer walking. The locus of the m5C-MTase gene of LCDV-1 was identified within the EcoRI DNA fragment G of LCDV-1 (7.9 kbp; 0.947 to 0.034 map units). The m5C-MTase gene of LCDV-1 comprises 684 nucleotides coding for a putative protein of 228 amino acid residues. A high degree of amino acid sequence homology (53.3% identity and 25.8% similarity) was detected between the m5C-MTases of LCDV-1 and FV3.     

Partial nucleotide sequence of the Japanese encephalitis virus genome

Approximately 10 kb of the estimated 10.9-kb genome of the Japanese encephalitis virus (JE; Nakayama strain) has been cloned as cDNA; the uncloned portion includes 430 bases at the 5'-terminus and 450 bases at the 3'-end. A map of the genome has been developed through nucleotide sequencing and in vivo expression with the Escherichia coli expression vector lambda gt11 and immunological identification. Sequence results for 4320 nucleotides suggest the JE genome organization is very similar to those of three other flaviviruses for which sequence information is available. Like the other flaviviruses, the JE proteins are encoded by a single open reading frame that continues uninterrupted throughout the region sequenced. Considerable homology exists between the JE RNA and protein sequences and those of the other characterized flaviviruses. Comparative nucleotide and (amino acid) homology values for the M-E-NS1-ns2 segment of JE are approximately MVE, 70% (80%), WN, 68% (76%), and YF, 50% (45%). Even greater homology is suggested when the protein hydrophobicity profiles are compared. The molecular relationships are consistent with the established serological relationships among JE, MVE, and WN viruses and argue that these flaviviruses may have been derived from a common evolutionary ancestor.     

Viral proteins and adenosine triphosphate phosphohydrolase activity of fish lymphocystis disease virus

Fish lymphocystis disease virus (FLDV) was isolated from papilloma-like lesions of various flatfish species, flounder, dab, and plaice. FLDV particles purified by density gradient centrifugation were denatured and analyzed by polyacrylamide gel electrophoresis under denaturing conditions. At least 33 FLDV polypeptides ranging in molecular weight from 220 to 14 K were detectable after staining with Coomassie blue. The patterns of structural polypeptides from different fish species were similar although some slight and distinct differences were found. In addition, polypeptide patterns of FLDV-infected flatfish cells were analyzed and compared with those of uninfected fish cells. A drastic change in the pattern of host cell proteins is observed and new, virus-induced proteins are synthesized as a result of an in vivo infection by FLDV. A nucleoside triphosphate phosphohydrolase activity is associated with FLDV. The enzymatic activity hydrolyzes the γ-phosphate residue of ATP and GTP with a high preference for ATP. The requirements for the ATPase activity and the reaction products are described.     

Restriction mapping of genome and nucleotide sequence of Aujeszky's disease virus strains isolated in CIS

Auesku disease virus--suid herpesvirus type I (SHV-1) genome is analyzed by the restriction fragment pattern analysis. The analyzed strains isolated in CIS countries in 1962-1992 were compared with the European strains. Genomes of field strains Arsk, Turkmenia, and Geneva differed and even lacked the restriction fragments typical of vaccine strains MK-25 or UNIIEV-18S, which agrees with the results of the neutralization test. Immunobiological characteristics of SHV-1 strains are discussed.     

The primary structure of the thymidine kinase gene of fish lymphocystis disease virus

The DNA nucleotide sequence of the thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) which has been localized between the coordinates 0.678 to 0.688 of the viral genome was determined. The analysis of the DNA nucleotide sequence located between the recognition sites of HindIII (0.669 map unit; nucleotide position 1) and AccI (nucleotide position 2032) revealed the presence of an open reading frame of 954 bp on the lower strand of this region between nucleotide positions 1868 (ATG) and 915 (TAA). It encodes for a protein of 318 amino acid residues. The evolutionary relationships of the TK gene of FLDV to the other known TK genes was investigated using the method of progressive sequence alignment. These analyses revealed a high degree of diversity between the protein sequence of FLDV TK gene and the amino acid composition of other TKs tested. However, significant conservations were detected at several regions of amino acid residues of the FLDV TK protein when compared to the amino acid sequence of TKs of African swine fever virus, fowlpox virus, shope fibroma virus, and vaccinia virus and to the amino acid sequences of the cellular cytoplasmic TK of chicken, mouse, and man.     

Complete nucleotide sequence of the Japanese encephalitis virus genome RNA

The complete nucleotide sequence of the Japanese encephalitis virus (JEV) genome RNA was determined. The JEV genome contains 10,976 nucleotides and encodes a single long open reading frame (ORF) of 10,296 nucleotides corresponding to 3432 amino acid residues. This long polypeptide is thought to be cleaved into three structural proteins and several nonstructural proteins of the virus. The genetic location of the three structural proteins was determined by comparing the deduced amino acid sequence from the nucleotide sequence with the N-terminal amino acid sequences that were determined from the three purified structural proteins. The C-terminal region of the ORF may encode a RNA-dependent RNA polymerase which has significant sequence homology with those of other RNA viruses.     

Complete nucleotide sequence and genome organization of butterbur mosaic virus

Butterbur mosaic virus (ButMV), a member of the genus Carlavirus, was isolated from Japanese butterbur. Here we report the complete nucleotide sequence and genome organization of ButMV. The genome of ButMV consists of 8,662 nucleotides in length and is predicted to contain six ORFs. The ButMV replicase and CP genes share 46.4–54.9 and 43.2–62.1% nucleotide and 38.6–46.6 and 31.3–65.0% amino acid sequence identities, respectively, with those of other carlaviruses. Based on phylogenetic analysis, we suggested that ButMV replicase and CP is most closely related to coleus vein necrosis virus and carnation latent virus, respectively. Together, our results demonstrate that ButMV was a distinct species of the genus Carlavirus.     

Molecular cloning and nucleotide sequence of the genome of hog cholera virus

A cDNA clone derived from genomic RNA of hog cholera virus (HCV) was identified using an oligonucleotide complementary to the RNA encoding a hexapeptide from the putative RNA-dependent RNA polymerase of the closely related bovine viral diarrhea virus (BVDV). This clone served as a probe for screening different size-selected cDNA libraries. After molecular cloning and nucleotide sequencing the HCV genome was shown to consist of 12,284 nucleotides containing one long open reading frame. Sequence comparison revealed a high degree of homology between HCV and BVDV genomic RNAs. With respect to HCV the genome of BVDV contains an insertion coding for 90 amino acids.     

Identification and mapping of single nucleotide polymorphisms in the varicella-zoster virus genome

We have analysed the expression and cellular localisation of the matrix protein VP40 from Ebola virus. Full-length VP40 and an N-terminal truncated construct missing the first 31 residues [VP40(31-326)] both locate to the plasma membrane of 293T cells when expressed transiently, while a C-terminal truncation of residues 213 to 326 [VP40(31-212)] shows only expression in the cytoplasm, when analysed by indirect immunofluorescence and plasma membrane preparations. In addition, we find that full-length VP40 [VP40(1-326)] and VP40(31-326) are both released into the cell culture supernatant and float up in sucrose gradients. The efficiency of their release, however, is dependent on the presence of the N-terminal 31 residues. VP40 that is released into the supernatant is resistant to trypsin digestion, a finding that is consistent with the formation of viruslike particles detected by electron microscopy. Together, these results provide strong evidence that Ebola virus VP40 is sufficient for virus assembly and budding from the plasma membrane.     

The nucleotide sequence and genome organization of Sclerophthora macrospora virus B

Sclerophthora macrospora Virus B (SmV B) found in S. macrospora, the pathogenic fungus responsible for downy mildew in gramineous plants, is a small icosahedral, monopartite virus containing a positive-strand ssRNA genome. In the present study, the complete nucleotide sequence of the SmV B genome was determined. The viral genome consists of 5533 nucleotides and has two large open reading frames (ORFs). ORF1 encodes a putative polyprotein containing the motifs of chymotrypsin-related serine protease and RNA-directed RNA polymerase. ORF2 encodes a capsid protein. The deduced amino acid sequence shows some similarity to those of certain positive-strand RNA viruses, but the genome organization is characteristic and distinct from those of other known fungal RNA viruses. These results suggest that SmV B should be classified into a new group of mycoviruses.     

The nucleotide sequence and genome organization of Magnaporthe oryzae virus 1

Summary Magnaporthe oryzae virus 1 (MoV1) found in Magnaporthe oryzae, the pathogenic fungus responsible for rice blast, is a small icosahedral virus with a nonsegmented double-stranded RNA genome. The viral genome has two open reading frames (ORF 1 and 2). The deduced amino acid sequences of both ORF 1 and ORF 2 show a significant similarity to those of capsid protein and RdRp, respectively, of members of the family Totiviridae. Both a comparison of genome organization and phylogenic analysis have indicated that MoV1 is closely related to some of the totiviruses that infect filamentous fungi. These results suggest that MoV1 belongs to the family Totiviridae.     

The complete nucleotide sequence and genome organization of tomato chlorosis virus

Summary. The crinivirus tomato chlorosis virus (ToCV) was discovered initially in diseased tomato and has since been identified as a serious problem for tomato production in many parts of the world, particularly in the United States, Europe and Southeast Asia. The complete nucleotide sequence of ToCV was determined and compared with related crinivirus species. RNA 1 is organized into four open reading frames (ORFs), and encodes proteins involved in replication, based on homology to other viral replication factors. RNA 2 is composed of nine ORFs including genes that encode a HSP70 homolog and two proteins involved in encapsidation of viral RNA, referred to as the coat protein and minor coat protein. Sequence homology between ToCV and other criniviruses varies throughout the viral genome. The minor coat protein (CPm) of ToCV, which forms part of the “rattlesnake tail” of virions and may be involved in determining the unique, broad vector transmissibility of ToCV, is larger than the CPm of lettuce infectious yellows virus (LIYV) by 217 amino acids. Among sequenced criniviruses, considerable variability exists in the size of some viral proteins. Analysis of these differences with respect to biological function may provide insight into the role crinivirus proteins play in virus infection and transmission.