Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy.

AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD. CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.

Automated ribotyping using different enzymes to improve discrimination of Listeria monocytogenes isolates, with a particular focus on serotype 4b strains.

To develop improved automated subtyping approaches for Listeria monocytogenes, we characterized the discriminatory power of different restriction enzymes for ribotyping. When 15 different restriction enzymes were used for automated ribotyping of 16 selected L. monocytogenes isolates, the restriction enzymes EcoRI, PvuII, and XhoI showed high discriminatory ability (Simpson's index of discrimination > 0.900) and produced complete and reproducible restriction cut patterns. These three enzymes were thus evaluated for their ability to differentiate among isolates representing the two major serotype 4b epidemic clones, those having ribotype reference pattern DUP-1038 (51 isolates) and those having pattern DUP-1042 (20 isolates). Among these isolates, PvuII provided the highest discrimination for a single enzyme (nine different subtypes; index of discrimination = 0.518). A combination of PvuII and XhoI showed the highest discriminatory ability (index of discrimination = 0.590) for these isolates. A group of 44 DUP-1038 isolates and a group of 12 DUP-1042 isolates were identical to each other even when the combined data for all three enzymes were used. We conclude that automated ribotyping using different enzymes allows improved discrimination of L. monocytogenes isolates, including epidemic serotype 4b strains. We furthermore confirm that most of the isolates representing the genotypes linked to the two major epidemic L. monocytogenes clonal groups form two genetically homogeneous groups.

U94, the human herpesvirus 6 homolog of the parvovirus nonstructural gene, is highly conserved among isolates and is expressed at low mRNA levels as a spliced transcript.

Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B, respectively) encode homologs (U94) of the parvovirus nonstructural gene, ns1 or rep. Here we describe the HHV-6B homolog and analyze its genetic heterogeneity and transcription. U94 nucleotide and amino acid sequences differ by approximately 3.5% and 2.5%, respectively, between HHV-6A and HHV-6B. Among a collection of 17 clinically and geographically disparate HHV-6 isolates, intravariant nucleotide and amino acid sequence divergence was less than 0.6% and 0.2%, respectively; all 13 HHV-6B isolates had identical amino acid sequences. The U94 transcript is spliced to remove a 2.6-kb intron and is expressed at very low levels relative to other HHV-6B genes, reaching approximately 10 copies per cell 3 days after infection. The mRNA has several small AUG-initiated open reading frames upstream of the U94 open reading frame, a hallmark of proteins expressed at low levels. Consistent with this, the U94-encoded protein was immunologically undetectable in HHV-6B-infected cells. The high degree of sequence conservation suggests that the gene function is nearly intolerant of sequence variation. The low abundance of U94 transcripts and the presence of encoded inefficient translation initiation suggest that the U94 protein may be required only in small amounts during infection.

Human herpesvirus 6B genome sequence: coding content and comparison with human herpesvirus 6A.

Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) are closely related viruses that can be readily distinguished by comparison of restriction endonuclease profiles and nucleotide sequences. The viruses are similar with respect to genomic and genetic organization, and their genomes cross-hybridize extensively, but they differ in biological and epidemiologic features. Differences include infectivity of T-cell lines, patterns of reactivity with monoclonal antibodies, and disease associations. Here we report the complete genome sequence of HHV-6B strain Z29 [HHV-6B(Z29)], describe its genetic content, and present an analysis of the relationships between HHV-6A and HHV-6B. As sequenced, the HHV-6B(Z29) genome is 162,114 bp long and is composed of a 144,528-bp unique segment (U) bracketed by 8,793-bp direct repeats (DR). The genomic sequence allows prediction of a total of 119 unique open reading frames (ORFs), 9 of which are present only in HHV-6B. Splicing is predicted in 11 genes, resulting in the 119 ORFs composing 97 unique genes. The overall nucleotide sequence identity between HHV-6A and HHV-6B is 90%. The most divergent regions are DR and the right end of U, spanning ORFs U86 to U100. These regions have 85 and 72% nucleotide sequence identity, respectively. The amino acid sequences of 13 of the 17 ORFs at the right end of U differ by more than 10%, with the notable exception of U94, the adeno-associated virus type 2 rep homolog, which differs by only 2.4%. This region also includes putative cis-acting sequences that are likely to be involved in transcriptional regulation of the major immediate-early locus. The catalog of variant-specific genetic differences resulting from our comparison of the genome sequences adds support to previous data indicating that HHV-6A and HHV-6B are distinct herpesvirus species.

Genetic content of a 20.9 kb segment of human herpesvirus 6B strain Z29 spanning the homologs of human herpesvirus 6A genes U40-57 and containing the origin of DNA replication.

A continuous 20.9 kb sequence from human herpesvirus 6 variant B (HHV-6B) strain Z29 (GenBank accession number L16947) is genetically colinear with a discrete segment of the human cytomegalovirus (HCMV) UL region and with HHV-6 variant A (HHV-6A). Short nucleotide sequence determinations at multiple sites within an 8.5 kb region immediately 3' to the 20.9 kb contig revealed additional colinearity between HHV-6B, HCMV and HHV-6A. Homology studies with the predicted peptide sequences from 11 complete and 12 partial HHV-6B open reading frames (ORFs) revealed that most encode proteins conserved to varying degrees in all previously sequenced primate herpesviruses. HHV-6B homologs were identified for the HSV-1 ICP18.5, ICP8, UL52, UL24, UL25 and major capsid protein. Several HHV-6B proteins had limited amino acid similarity to their positional homologs in other herpesviruses. Each gene identified is highly homologous to its HHV-6A counterpart, including two unique HHV-6 genes predicted to encode membrane-associated glycoproteins. However, two regions of substantial divergence were noted, one spanning the origin of replication and the other encoding one of the putative HHV-6-specific glycoprotein genes. Substitutions in the latter region lead to predicted differences in reading frames and protein lengths among HHV-6 isolates.

Comparison of a 20 kb region of human herpesvirus 6B with other human beta herpesviruses reveals conserved replication genes and adjacent divergent open reading frames.

The sequence of a 20.15 kb region from human herpesvirus 6 variant B (HHV-6B) strain Z29 is described (GenBank accession number L14772). Determinations of protein homologies for seventeen predicted gene products revealed HHV-6B homologs of six proteins well-conserved both in genetic context and amino acid sequence throughout the alpha-, beta-, and gammaherpesvirus subfamilies. These include proteins involved in viral DNA replication, packaging and nucleotide metabolism, and conserved proteins of undefined function. The close evolutionary relationship of the human betaherpesviruses, HHV-6B, HHV-6A, HHV-7 and human cytomegalovirus (HCMV) was confirmed by identification of several protein sequences encoded only by these viruses, including homologs of the HCMV early phosphoprotein family and a series of HCMV open reading frames predicted to encode glycoprotein exons. Homologs of essential HSV-1 replication proteins, UL8 and UL9, were also identified. Downstream from the conserved replication locus, each betaherpesvirus contains a region of divergent, small open reading frames. The evolution of this region and its potential use in the development of a viral vector system are discussed.

Restriction endonuclease mapping and molecular cloning of the human herpesvirus 6 variant B strain Z29 genome.

Human herpesvirus 6(HHV-6) variants A and B differ in cell tropism, reactivity with monoclonal antibodies, restriction endonuclease profiles, and epidemiology. Nonetheless, comparative nucleotide and amino acid sequences from several genes indicate that the viruses are very highly conserved genetically, The B variant is the major etiologic agent of exanthem subitum and is frequently isolated from children with febrile illness; no disease has been etiologically associated with HHV-6A. One HHV-6A strain has been cloned and sequenced, but similar information and reagents are not available for HHV-6B. We report here the determination of maps of the restriction endonuclease cleavage sites for BamHI, C1aI, HindIII, KpnI, and Sa1I, and the cloning in plasmids and bacteriophages of fragments representing over 95% of the HHV-6B strain Z29 [HHV-6B(Z29)] genome. Hybridization experiments and orientation of several blocks of nucleotide sequence information onto the genomic map indicate that HHV-6A and HHV-6B genomes are colinear.

Intragenomic linear amplification of human herpesvirus 6B oriLyt suggests acquisition of oriLyt by transposition.

We identified some passage lineages of human herpesvirus 6 variant B (HHV-6B) strain Z29 that contain as many as 12 tandem copies of a genomic segment that corresponds almost precisely to a previously identified minimal efficient origin of lytic replication (oriLyt). Analysis of nucleotide sequences in the vicinity of the amplified segment suggests that the amplification occurred as a two-step process, with the first step being a rare sequence duplication mediated through directly repeated sequences located near the termini of the amplified segment and the second step occurring via homologous recombination through the duplicated sequence. These results demonstrate that oriLyt has been amplified in some virus stocks and indicate that (i) origin amplification confers a growth advantage on the virus in cell culture and (ii) laboratory-passaged HHV-6B genomes can accommodate additional nucleotide sequences and thus may be useful gene transfer vectors. The structures of the amplified segment and its adjacent sequences together suggest that HHV-6B or a progenitor virus acquired oriLyt by transposition from an unknown source.

Alphaherpesvirus origin-binding protein homolog encoded by human herpesvirus 6B, a betaherpesvirus, binds to nucleotide sequences that are similar to ori regions of alphaherpesviruses.

We previously identified a human herpesvirus 6B (HHV-6B) homolog of the alphaherpesvirus origin-binding protein (OBP), exemplified by the herpes simplex virus type 1 UL9 gene product. This finding is of particular interest because HHV-6B is otherwise more closely related to members of the betaherpesvirus subfamily. The prototypic betaherpesvirus, human cytomegalovirus, does not encode an obvious OBP homolog and contains a more complex origin of replication than do alphaherpesviruses. Thus, analysis of the function of the HHV-6B OBP homolog is essential for understanding the mechanism of HHV-6B DNA replication initiation. The HHV-6B OBP homolog, OBPH6B, was expressed in vitro by coupled transcription and translation and in insect cells by infection with recombinant baculoviruses. The expressed protein bound to two DNA sequences located upstream of the HHV-6B major DNA-binding protein gene homolog, within a region that was predicted to serve as an origin of replication on the basis of its sequence properties. The binding sites lie within 23-bp segments and are similar to OBP-binding sites of herpes simplex virus type 1. The two OBPH6B-binding sequences are separated by an AT-rich region and have an imperfect dyad symmetry as do the alphaherpesvirus origin regions. We identified OBPH6B transcripts by reverse transcription PCR in HHV-6B-infected Molt-3 cells. These results suggest that OBPH6B functions in a manner analogous to the alphaherpesvirus OBP and that initiation of HHV-6B DNA replication may resemble that of alphaherpesviruses.

Identification of a lytic-phase origin of DNA replication in human herpesvirus 6B strain Z29.

DNA sequences which have structural features suggestive of their functioning as an origin of lytic-phase DNA replication were previously identified in both human herpesvirus 6B strain Z29 [HHV-6B (Z29)] and in HHV-6A (U1102). Plasmid constructs containing the putative HHV-6B (Z29) oriLyt element were replicated after transfection into permissive T cells, when trans-acting factors were provided by HHV-6B (R-1) infection. By using this assay, the HHV-6B (Z29) oriLyt was mapped to a minimal region of approximately 400 bp which lies upstream of the gene that is homologous to herpes simplex virus UL29, a region that carries an origin in other betaherpesviruses and in some alphaherpesviruses.

Molecular biology of human herpesviruses 6A and 6B.

Human herpesvirus 6 variant A (HHV-6A) and human herpesvirus 6 variant B (HHV-6B) are closely related herpesviruses. No disease has been specifically associated with HHV-6A, whereas HHV-6B is the major etiologic agent of exanthem subitum. Both viruses may be opportunistic pathogens in the immunocompromised patient. HHV-6 genomes have low G+C contents for herpesviruses (43%); they consist of a 141-kb unique segment that is flanked by single copies of a directly repeated sequence that can vary from 10 to 13 kb. HHV-6A and HHV-6B encode homologs of many conserved herpesvirus proteins and are classified as beta-herpesviruses based on their close genetic relationship with human cytomegalovirus. HHV-6A and HHV-6B are even more closely related to the recently discovered human herpesvirus 7. HHV-6 encodes homologs of the seven genes that are essential for origin-dependent herpes simplex virus type 1 DNA replication, including the origin-binding protein, which has no clear homolog in human cytomegalovirus. The HHV-6B origin-binding protein binds to sequences with similarities to alpha-herpesvirus replication origins that lie within a genomic segment that can serve as a replication origin in transient replication assays. Both HHV-6 variants encode homologs of the adeno-associated virus type 2 Rep protein; the role of this protein during infection is unknown. HHV-6 induces synthesis of a broad range of host cell proteins, including interferon alpha, CD4, interleukin-1 beta, and tumor necrosis factor alpha, and also induces expression of the human immunodeficiency virus type 1 LTR promoter. Little is known about the process by which HHV-6 regulates gene expression.

A strongly immunoreactive virion protein of human herpesvirus 6 variant B strain Z29: identification and characterization of the gene and mapping of a variant-specific monoclonal antibody reactive epitope.

We previously identified a 101-kDa apparent molecular mass polypeptide (101K) as the major immunoreactive virion protein of human herpesvirus 6 variant B strain Z29 [HHV-6B(Z29)] and found that the human immune response to this protein is HHV-6-specific (Yamamoto, M., Black, J. B., Stewart, J. A., Lopez, C., and Pellett, P. E., 1990, J. Clin. Microbiol. 28, 1957-1962). We report here the identification and characterization of the gene encoding 101K. We found 81% amino acid identity between an HHV-6B(Z29) open reading frame (ORF) and its homolog in HHV-6A strain U1102 [HHV-6A(U1102)]. The product of this gene was identified as 101K on the basis of both the reactivity of a 101K-specific monoclonal antibody (MAb C3108-103) with a bacterially expressed portion of the gene and the reactivity of polyclonal rabbit antibodies raised against the bacterially expressed protein with 101K expressed by HHV-6B(Z29)-infected cells. MAb C3108-103 reacted with eight of eight variant B isolates and none of six variant A isolates, indicating that it is a variant-specific MAb. The MAb reactivity was mapped to an eight-amino-acid segment of 101K. HHV-6A(U1102) differs from HHV-6B(Z29) by two amino acids in this region; substitution mapping with synthetic oligopeptides mapped the variant B specificity to Asp723, this explaining the failure of the MAb to react with variant A proteins. A set of transcripts appropriately sized for expression of 101K was identified and precisely mapped. The transcripts originated down-stream from either of two TATA boxes located 139 bp apart in the region 5' to the 101K ORF, with one 5'-species being much more abundant. Two independent polyadenylation sites were identified; the canonical polyadenylation signal located 3' to the 101K ORF was used much more frequently than was the atypical polyadenylation signal located within the 101K ORF. These results suggest a complex regulatory mechanism for this gene.

Primary human herpesvirus 6 infection in young children.

BACKGROUND: Human herpesvirus 6 (HHV-6) is a recently discovered virus that, on the basis of serologic evidence, appears to infect most children by the age of three years. However, the clinical manifestations of primary HHV-6 infection have not been well defined. METHODS: We studied consecutive children two years old or younger who presented to an emergency ward with febrile illnesses. Our evaluation included the isolation of HHV-6 from peripheral-blood mononuclear cells, an immunofluorescent-antibody assay, the detection of HHV-6 by the polymerase chain reaction (PCR), and restriction-endonuclease-fragment profiles of HHV-6 isolates. RESULTS: HHV-6 was isolated from 34 of 243 acutely ill children (14 percent). The children with viremia had irritability, high temperatures (mean, 39.7 degrees C), and inflammation of tympanic membranes (in 21), but few other localizing signs. Two children were hospitalized, but all 34 recovered after an average of four days of fever. The rash characteristic of roseola, which has been associated with HHV-6 infection, was noted in only three children. In 29 children (85 percent), serum samples obtained during convalescence had at least a fourfold increase in IgG antibody titers; 4 infants less than three months old who presumably had maternal antibody did not have this increase. HHV-6 was isolated from blood obtained during convalescence in only one child, but in two thirds of the children the virus could be detected by PCR. The isolates had genomic heterogeneity, indicating the presence of multiple strains. CONCLUSIONS: Primary infection with HHV-6 is a major cause of acute febrile illness in young children. Such infection is associated with varied clinical manifestations, viremia, and the frequent persistence of the viral genome in mononuclear cells.

Identification and nucleotide sequences of two similar tandem direct repeats in Epstein-Barr virus DNA.

Epstein-Barr virus DNA is known to have partially homologous segments, designated DL and DR, near the left and right ends of the long unique region (Raab-Traub et al., Cell 22:257-267, 1980). DL and DR are each partially composed of tandem direct repeat sequences. DL contains 11 to 14 repeats of a 124-base-pair sequence designated IR2. DR contains approximately 30 direct repeats of a 103-base-pair sequence designated IR4. The DL and DR sequences have colinear partial homology for approximately 2.4 and 1.5 kilobase pairs to the right of IR2 and IR4, respectively. IR2 and IR4 are similar sequences and evolved in part from a common ancestor. Both sequences are 84% guanine and cytosine and have limited homology to Epstein-Barr virus IR1 and to the herpes simplex virus type 1 inverted terminal repeat "a" sequence. IR2 encodes part of an abundant 2.5-kilobase persistent early EBV RNA expressed in productively infected cells, but does not encode part of the 3-kilobase Epstein-Barr virus RNA which is transcribed from the adjacent IR1-U2 region of the Epstein-Barr virus genome in latently infected cells.