Molecular identification of Campylobacter concisus

A 1.6-kb DNA fragment isolated from a Campylobacter concisus genomic library gave C. concisus-specific restriction fragment length patterns when it was used as a probe in hybridization studies. All of the strains tested, including type strains and clinical isolates, contained a 0.5-kb HindIII fragment that hybridized to the probe. DNA sequencing of the 1.6-kb fragment identified three open reading frames (ORFs). One of the ORFs encodes the carboxy terminus of GyrB, and the translational products of ORF2 and ORF3 showed similarity to hypothetical proteins, previously identified in Campylobacter jejuni. DNA-DNA hybridization studies with a fragment internal to ORF3 showed that this sequence was responsible for the signal observed with the 0.5-kb HindIII fragment. A rapid PCR assay was developed and evaluated. Primers that annealed to the extremities of the 1.6-kb fragment were used to obtain an amplicon of the correct size from both reference and clinical strains of C. concisus.     

Characterization of the gene encoding glycoprotein C of duck enteritis virus

A total of 2,718 bp of DNA fragment was amplified from the C-KCE strain of duck enteritis virus (DEV) genome using thermal asymmetric interlaced PCR. This newly identified viral DNA fragment contained two non-overlapping open reading frames (ORFs) oriented from the 5′ to 3′ direction. The first ORF was comprised of 43.5% G + C and contained the full-length genomic sequence of the UL44 gene (1,296 bp) encoding 431 amino acid residues of DEV glycoprotein C (gC). The second ORF encoded a partial peptide of the UL43 gene. The sequences of DNA and deduced amino acids of the DEV gC gene shared high homology with other members of known herpesviruses, supporting the classification of DEV. Phylogenetic analysis of the DEV gC gene revealed that the gC gene had a close evolutionary relationship with the subfamily of Alphaherpesvirinae.     

Map location of lactate dehydrogenase-elevating virus (LDV) capsid protein (Vp1) gene

Lactate dehydrogenase-elevating virus (LDV) is currently classified within the Togaviridae family. In an effort to obtain further information on the characteristics of this virus, we have begun to sequence the viral RNA genome and to map the virion structural protein genes. A sequence of 1064 nucleotides, which represents the 3' terminal end of the genome, was obtained from LDV cDNA clones. A 3' noncoding region of 80 nucleotides followed by two complete open reading frames (ORFs) were found within this sequence. The two ORFs were in different reading frames and overlapped each other by 11 nucleotides. One ORF encoded a protein of 170 amino acids and the other ORF, located adjacent to the 3' noncoding region of the viral genome, encoded a 114 amino acid protein. Thirty-three N-terminal residues were sequenced directly from purified LDV capsid protein, Vp1, and this amino acid sequence mapped to the ORF adjacent to the 3' noncoding region. The presence of overlapping ORFs and the 3' terminal map position of Vp1 indicate that LDV differs significantly from the prototype alpha togaviruses.     

Resistance of vaccinia virus to rifampicin conferred by a single nucleotide substitution near the predicted NH2 terminus of a gene encoding an Mr 62,000 polypeptide

Marker transfer procedures were used to locate the site of mutation in the genome of a previously characterized (B. Moss, E. N. Rosenblum, and P. Grimley, 1971), Virology 45, 135-148) rifampicin-resistant (RifR) vaccinia virus isolate. Starting with a cosmid library prepared from the mutant genome, recombination with successively smaller DNA fragments was shown to transfer drug resistance to wild-type vaccinia virus. In this manner, the mutation was mapped within a 485-bp DNA segment in the central region of the genome at the extreme right end of the HindIII D fragment. Nucleotide sequencing indicated that this DNA segment differed from the homologous region of wild-type DNA by a single C/G----A/T substitution. Sequencing of the flanking 2195 bp revealed two tandem nonoverlapping open reading frames (ORFs) encoding putative polypeptides of Mr 16,908 and 61,840. The RifR mutation resulted in a predicted glutamine----lysine change only 27 amino acids from the NH2 terminus of the longer ORF. A predicted asparagine to aspartic acid substitution, found in another RifR vaccinia virus mutant by J. Tartaglia and E. Paoletti (Virology 147, 394-404, 1985), mapped near the carboxyl terminus of the same ORF. These data suggest a model in which head-to-tail interaction between Mr 61,840 polypeptides occurs and in which rifampicin blocks virus assembly by preventing this association.     

Herpesvirus ICP18.5 and DNA-binding protein genes are conserved in equine herpesvirus-1

The genome of equine herpesvirus-1 (EHV-1) contained three open reading frames (ORFs) in a 3.9 kbpBamHI-SmaI fragment at 0.38–0.41 map units in the long unique region. The most 5′ ORF encoded the carboxy terminus of a protein with 45–55 percent amino acid homology to the DNA-binding proteins (ICP8-DBP) of four other alphaherpesviruses. The middle ORF translated to a polypeptide of 775 residues with 43–55% homology to the ICP18.5 proteins. The most 3′ ORF encoded the EHV-1 glycoprotein B (gB) gene. Three mRNAs of 4.3, 4.4–4.8, and 3.5–3.9 kb (corresponding to the three sequenced ORFs) were all transcribed from the same strand. The gene order of this group was conserved in all herpesviruses examined.     

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, 624 aa), 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.     

Tandemly repeated sequences are present at the ends of the DNA of raccoonpox virus

The DNA of raccoonpox virus (RCN) has been characterized by restriction enzyme analysis. DNA hybridization studies showed that all HindIII fragments of the 215-kbp RCN DNA share some nucleotide sequence similarity with fragments of the DNA of cowpox virus (CPV). This information was used to construct a HindIII restriction map of the RCN DNA. The nucleotide sequence of the 2.2-kbp Sal 1 end fragment of the RCN DNA has been determined from a cloned copy of the HindIII O fragment. Of this 2.2-kb region 75% consists of short, tandemly repeated sequences. It does not contain any open reading frames capable of encoding polypeptide chains of more than 62 amino acids. There are six related types of repeated sequence, and these are arranged into two separate sets, each flanked by nonrepeated sequences. The nucleotide sequences of both repeated and nonrepeated sequences within this Sal 1 fragment are extremely similar to those of the Sal 1-generated end fragments of the DNas of CPV and vaccinia virus. The arrangements of the repeated and nonrepeated sequences are also similar in the DNAs of these three viruses. In contrast, the remainder of the RCN DNA is markedly different from the DNAs of other orthopoxviruses. The high degree of similarity between the ends of the RCN DNA and the ends of the other orthopoxvirus DNAs suggest that the complex arrays of repeated and nonrepeated sequences have been conserved because they have a role in virus multiplication.     

Identification and DNA sequence of the large subunit of the capping enzyme from Shope fibroma virus.

A 3.6-kb region of the Shope fibroma virus (SFV) BamHI D fragment located in the central region of the viral genome was sequenced. Three open reading frames (ORFs) were identified, D3R, D4L, and D5R. Each of these ORFs have a counterpart organized identically within the HindIII fragment D of the vaccinia virus genome (D1R, D2L, and D3R). Homology scores and assays of viral cores indicate that SFV D3R encodes the large subunit of the SFV mRNA capping enzyme.     

Open reading frames encoding a protein kinase, homolog of glycoprotein gX of pseudorabies virus, and a novel glycoprotein map within the unique short segment of equine herpesvirus type 1.

DNA sequence analysis of the unique short (Us) segment of the genome of equine herpesvirus type 1 Kentucky A strain (EHV-1) by our laboratory and strains Kentucky D and AB1 by other workers identifies a total of nine open reading frames (ORF). In this report, we present the DNA sequence of three of these newly identified ORFs, designated EUS 2, EUS 3, and EUS 4. The EUS 2 ORF is 1146 nucleotides (nt) in length and encodes a potential protein of 382 amino acids. Cis-regulatory sequences upstream of the putative ATG start codon include a G/C box 112 nt upstream and two potential TATA-like elements located between 15 and 90 nt before the ATG. The EUS 2 translation product exhibits significant homology to Ser/Thr protein kinases encoded within the Us segments of other herpesviruses, such as herpes simplex virus (26% homology) and pseudorabies virus (PRV), (45% homology), and possesses sequence domains conserved in protein kinases of cellular and viral origin. The EUS 3 ORF begins 127 nt downstream from the EUS 2 stop codon and ends at a stop codon 1119 nt further downstream. A single TATA-like element maps 61 nt upstream of the ORF. This ORF encodes a potential protein of 373 amino acids and is a homolog of glycoprotein gX of PRV, as judged by overall homology of amino acid residues, cysteine displacement, and presence of potential glycosylation sites and signal sequence. Interestingly, the EUS 4 ORF encodes a potential membrane glycoprotein that does not exhibit homology to any reported protein sequence. The EUS 4 ORF encodes a 383 amino acid polypeptide with a sequence indicative of a signal sequence at its amino terminal end, glycosylation sites for N-linked oligosaccharides, and a transmembrane domain near its carboxyl terminus. Several cis-acting regulatory sequences lie upstream of this ORF. These findings support the observation that the short region of alphaherpesviruses show considerable variation in their genetic content and gene organization.     

Transcription and translation mapping of the 13 genes in the vaccinia virus HindIII D fragment

The vaccinia virus HindIII D fragment is 160,060 bp in length and encodes 13 complete open reading frames [Niles et al. (1986) Virology 153, 96-112; S. L. Weinrich and D. E. Hruby (1986). Nucleic Acids Res. 14, 3003-3016]. We have employed a two-step Northern hybridization protocol using single-stranded DNA probes from M13 recombinants in order to identify the mRNA products from the 13 genes. Six of these genes are expressed only at early times after infection; six are transcribed only at late times; one gene is expressed at both early and late times after virus infection. The D11 gene is transcribed into two late mRNA species, one full-length and the other derived from the 3' one-third of the coding sequence. Translation of hybrid-selected mRNA was carried out in an attempt to identify the protein products encoded by each mRNA. Protein products were found for each early gene but translation was successful for only two of the eight late mRNAs. With the completion of the physical map it is apparent that the early and late genes in the HindIII D fragment are arranged in order to minimize potential interference caused by the expression of closely packed viral genes.     

Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus

Summary The 3-portion of the genome of a U.S. isolate of the porcine reproductive and respiratory syndrome (PRRS) virus, ATCC VR-2332, was cloned and sequenced. The resultant 3358 nucleotides contain 6 open reading frames (ORFs) with homologies to ORFs 2 through 7 of the European strain of the PRRS virus and other members of the free-standing genus of arteriviruses. Both VR-2332 and the European isolate (called the Lelystad virus) have been identified as infectious agents responsible for the swine disease called PRRS. Comparative sequence analysis indicates that there are degrees of amino acid identity to the Lelystad virus open reading frames ranging from 55% in ORF 5 to 79% in ORF 6. Hydropathy profiles indicate that the ORFs of VR-2332 and Lelystad virus correspond structurally despite significant sequence differences. These results are consistent with the biological similarities but distinct serological properties of North American and European isolates of the virus.     

Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant

The principal objectives of this study were to analyze the structure and coding potential of a long segment of DNA missing from a previously isolated (B. Moss, E. Winters, and J. A. Cooper (1981) J. Virol. 40, 387-395) attenuated variant of vaccinia virus strain WR and to examine the precise changes in the genome accompanying the deletion. The sequences of a 14.5-kbp region located at the left end of the standard vaccinia virus genome, extending from within the inverted terminal repetition (ITR) of the HindIII C fragment to the end of the HindIII N fragment, and of a 3-kbp segment from a corresponding region of the variant genome were determined. A comparison of these sequences revealed that the variant contained a deletion of 12 kbp and an insertion of 2.1 kbp. The origin of the inserted DNA was traced to the HindIII B region by using oligonucleotide probes indicating that a transposition of unique DNA located adjacent to the right ITR had occurred. Structural analysis indicated no extensive homologies, nucleotide substitutions, additions, or deletions at the boundaries of the transposed DNA. Examination of the right end of the variant genome indicated that a copy of the transposed DNA was still present and, therefore, the length of the ITR had been increased by 2.1 kbp. The variant genome could have formed by a mechanism that resulted in the replacement of a 22-kbp left-terminal fragment with a 12-kbp right-terminal fragment. The DNA missing from the variant and contained within the standard vaccinia virus WR genome contains 17 contiguous open reading frames (ORFs), all of which are directed leftward and apparently not required for replication in cultured cells. One deleted ORF has a 60% sequence similarity to another gene encoding a 42,000-Da protein present within the ITR suggesting that duplications have previously occurred during the evolution of vaccinia virus. Another deleted ORF has a 39% sequence similarity to a complement 4b binding protein. The transposed DNA contains two complete ORFs one of which has a 40% identity to a cowpox gene and a 30% identity to a family of plasma serine protease inhibitors.     

Nucleotide sequence and genome organization of a member of a new and distinct <Emphasis Type="Italic">Caulimovirus</Emphasis> species from dahlia

A distinct caulimovirus, associated with dahlia mosaic, was cloned and sequenced. The caulimovirus, tentatively designated as dahlia common mosaic virus (DCMV), had a double-stranded DNA genome of ca. 8 kb. The genome organization of DCMV was found to be typical of members of the genus Caulimovirus and consisted of six major open reading frames (ORFs), ORFs I–VI, and one minor ORF, ORF VII. Sequence comparisons with the DNA genomes of two known caulimoviruses isolated from dahlia, Dahlia mosaic virus (DMV) and an endogenous caulimovirus, DMV-D10, showed that DCMV is a member of a distinct caulimovirus species, with sequence identities among various ORFs ranging from 25 to 80%. Sequences reported here are available in GenBank under the following accessions: EU090952, EU090953, EU090954, EU090955, EU090956, EU090957.