Concatemeric forms of intracellular Tupaia herpesvirus DNA

Kinetics of Tupaia herpesvirus strain 2 (THV-2) DNA synthesis was measured by labeling with radioactive thymidine and determining the amount of newly synthesized DNA. Viral DNA synthesis reached a maximum between 24 to 36 hr postinfection, preceded by a transient stimulation of host cell DNA synthesis. Blot hybridizations of labeled recombinant plasmids harboring inserts of both genomic termini of THV-2 and of individual viral terminal DNA fragments to DNA of virus-infected cells and combined restriction enzyme analysis of labeled intracellular viral DNA revealed the presence of linear concatemeric and/or circular viral DNA molecules.     

Fate of parental herpesvirus DNA.

The fate of the DNA of infecting pseudorabies virions in rabbit kidney cells was followed. The rate of adsorption of infectious and noninfectious virions is the same. Most of the DNA in the adsorbed virions (infectious and noninfectious) in preparations of freshly purified virions is rapidly transferred to the cell nucleus and replicates. Storage of the virions at ?70° does not significantly affect infectivity but does affect the ability of the DNA in some of the adsorbed virions to be transferred to the cell nucleus and to replicate. Although the DNA in the noninfectious particles present in preparations of fresh virions replicates, this process is dependent upon coinfection of the cells with infectious virus, indicating that the replication of the DNA of noninfectious particles requires the expression of some functions of the DNA in infectious particles.Most of the single-stranded interruptions in mature pseudorabies viral DNA are ligated prior to the replication of the DNA. Few double-stranded fragments of viral DNA accumulate in the infected cell. Breakage and reunion of parental viral DNA is detectable and seems to occur preferentially around the middle of the molecules.     

Replication of herpesvirus DNA. II. Sedimentation characteristics of newly synthesized DNA.

The sedimentation behavior of viral DNA synthesized during short labeling periods at various times after infection was investigated. These experiments indicated that viral DNA synthesis may be divided into the following two phases: (1) During the early phase, newly synthesized DNA is associated with structures which sediment with S values up to approximately twice that of mature viral DNA; (2) during the later phase, newly synthesized DNA is associated with structures that sediment much more rapidly. Both at early and later times after infection, approximately 20% of the newly synthesized DNA sediments in sucrose gradients more slowly than does mature viral DNA. Furthermore, after isopycnic centrifugation in CsCl, most of the newly synthesized viral DNA sediments in sucrose gradients more slowly than does mature viral DNA. The smaller than unit-size, newly synthesized viral DNA molecules are breakage products resulting from the fragility of newly synthesized DNA. These molecules fragment, not only because of their fragility in the regions of the replicative forks but also because of the presence of fragile sites at other positions along the newly synthesized DNA molecule. Experiments dealing with the transfer of parental DNA to progeny virions show that most parental viral DNA strands that are transferred to progeny virions retain their integrity. Breakage and reunion of parental viral DNA strands with progeny DNA is a relatively rare event.     

A rapid method for purification of herpesvirus DNA.

A rapid and reliable method for purification of herpesvirus DNA from cell cultures is described. The method is based on the isolation of virus particles and/or nucleocapsids by differential centrifugation and exploits the solubilizing and denaturing capabilities of cesium trifluoroacetate during isopycnic centrifugation, so that phenol/chloroform extractions can be omitted. The method was used for the purification of DNA from several members of the Alfaherpesvirinae subfamily.     

Replication of herpesvirus DNA. IV: analysis of concatemers.

At late stages of the infective process, pseudorabies virus DNA replicates in the form of large, tangled masses. These tangles have been analyzed by cleavage with restriction endonucleases. The results of these experiments show that the tangled masses contain concatemeric forms of linear arrays of unit size molecules in head-to-tail alignment.     

Analysis of herpesvirus DNA substructure by means of restriction endonucleases.

The mol. wt. and molar ratios of the Hind III and Hpa I fragments of HSV-1 DNA and the Eco RI fragments of HSV-2 DNA have been determined. Results obtained suggest that DNA isolated from both HSV-1 and HSV-2 consists of molecules with four different sequence arrangements which are present in similar amounts. Our explanation of the cleavage patterns of these four genome arrangements with the different restriction enzymes is presented. Some of the possible implications of these four genome arrangements for genetic recombination are discussed.     

Replication of herpesvirus DNA. VI. Virions containing either isomer of pseudorabies virus DNA are infectious

Pseudorabies virus DNA exists in two isomeric forms in which the short unique sequence is present in two orientations with respect to the long unique sequence. The viral DNA present in the virions of 21 individual plaques was analyzed. In all cases, equimolar amounts of the two isomeric forms of the DNA were present, indicating that isomerization of the DNA is a rapid process which is complete by the time a small plaque (<2 mm) has developed. Virions containing either isomeric form of the DNA adsorb equally well to cells and either isomeric form of the DNA has the same likelihood of becoming associated with the cell nucleus and to form circles (or concatemers) before initiation of DNA synthesis. The two isomeric forms of viral DNA are also equally represented in the genomes that mature first from concatemeric replicating DNA in cells infected at low multiplicities (0.01 PFU/cell). Furthermore, during the first round of DNA replication in cells infected at low multiplicity, equimolar amounts of the two isomeric forms of the DNA replicate. Since, in this experiment, each cell was infected with a maximum of one viral particle per cell, we conclude that virions containing either isomeric form of the DNA can initiate infection. Previous data (J. M. DeMarchi, T. Ben-Porat, and A. S. Kaplan, 1979, Virology97,457–463) have indicated that each cell, in which infection is initiated, is able to produce a plaque. We therefore conclude that virions containing either isomeric form of the DNA are infectious.     

Replication of herpesvirus DNA. I. Electron microscopic analysis of replicative structures.

The replicative structures of pseudorabies virus DNA were examined by electron microscopy during the first round of parental viral DNA replication. Molecules with replicative “eyes” and branches were observed. Eyes, centering mainly between 18 and 24 μm from one of the ends of linear unit-size molecules, were observed indicating that internal initiation of replication occurred preferentially at that site. Y-shaped molecules with branches of varying sizes were also seen. Some of the molecules had relatively short branches, indicating that initiation of replication of linear molecules occurs near or at the end of the molecules as well. Circular and longer than unit-size molecules with eyes or branches were seen, indicating that parental viral DNA molecules that acquire single-stranded ends within the host cells and, consequently, can circularize and form concatemers proceed to replicate. Viral DNA molecules with terminal loops were also observed; the function of these structures in replication remains uncertain.     

Prevalence of human herpesvirus 8 DNA sequences in several patient populations.

We have undertaken a large-scale study of various tissues from normal controls and patients with Kaposi's sarcoma (KS) or other malignancies, both with and without human immunodeficiency virus infection, to determine the prevalence of human herpesvirus 8 (HHV-8) DNA. A total of 566 specimens were analyzed by PCR for the presence of HHV-8 DNA. Of the samples tested, 251 were obtained from patients with KS and 315 were obtained from patients without KS. HHV-8 DNA was detected in 103 (41%) of the 251 samples from patients with KS. In particular, 92% of KS tumor specimens were positive. None of the tissues from patients without KS showed evidence of HHV-8 DNA. Sequencing and phylogenetic analyses indicate a high degree of conservation (97.5 to 100%) among the HHV-8 strains tested.     

Detection of bovine herpesvirus 1 DNA immobilized on nitrocellulose by hybridization with biotinylated DNA probes.

A molecular hybridization technique using biotinylated DNA probes was used to detect bovine herpesvirus 1 (BHV-1) nucleic acid species immobilized on nitrocellulose. Seventeen recombinant plasmids containing HindIII restriction fragments of the BHV-1 genome were compared for their ability to detect immobilized BHV-1 DNA from purified virus and infected cells. One probe, pCB2, labeled by nick translation with either 3H or biotin, detected as little as 10 pg of viral DNA. In time course experiments, BHV-1 DNA could be detected by 2 h postinfection in 10(6) infected cells. BHV-1 DNA was detected in nasal swabs and exudate from experimentally infected cattle, even when specimens had been stored for over a year. In a retrospective study of a respiratory disease outbreak in a feedlot, hybridization was compared with virus isolation for diagnosis of BHV-1 infections. The sensitivity rate was 0.68 with virus isolation as the referent standard. Blot hybridization provides a novel approach with unique applications for the diagnosis of bovine herpesvirus infections.     

Biologic activity of oligomeric forms of SV40 DNA.

We have analyzed the biologic activity of supercoiled oligomeric forms of SV40 DNA during productive and transforming infection. These DNA molecules had equal infectivity as assayed by plaque formation; however, the ability of such DNA molecules to transform nonpermissive cells increased linearly with their size. Oligomeric linear and circular forms of SV40 DNA produced by in vitro ligation of linear SV40 monomeric DNA had similarly enhanced transforming activity. Nucleic acid hybridization studies suggest that the amount of viral DNA in rat cells transformed by the variously sized oligomeric DNAs is similar.     

Antigenic difference between intracellular and membrane antigens induced by herpesvirus of turkeys

Summary The difference between intracellular antigen (IA) and membrane antigen (MA) induced by herpesvirus of turkeys (HVT) was examined by immunofluorescence analysis. Convalescent sera from chickens infected with HVT (HVT convalescent serum) and hyperimmune sera from chickens immunized with partially purified virus (anti-HVT particle serum) or with a major HVT soluble precipitin antigen (anti-common Ag serum) were used as the source of antisera to specify both antigens in these experiments.Absorption of a convalescent serum from HVT-inoculated chicken with IA positive cells, resulted in reduction of their reactivity to IA but not to MA. Differential absorption of the hyperimmune sera by IA positive cells and MA positive cells resulted in reduction of their reactivity to homologous cells but not to heterologous cells which were used in the absorptions. When the relationship between the levels of anti-IA and anti-MA antibodies was examined by a blocking immunofluorescence test using 38 sera from chickens inoculated with HVT, no correlation was observed (p>0.25). These results provide evidence that MA and IA are antigenically different from each other.With 3 Figures     

Restriction maps of the DNA of cervid herpesvirus 1 and cervid herpesvirus 2, two viruses related to bovine herpesvirus 1

Summary Restriction maps of cervid herpesviruses 1 and 2 which are antigenically related to bovine herpesvirus 1, were deduced from Southern blot hybridization withHin dIII restriction fragments of BHV-1 DNA as probes.     

Real-time quantitative PCR for human herpesvirus 6 DNA

The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection. We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids. The test uses a 5' nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system. The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 10(6) viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule. The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions. Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys.     

Development of a polymerase chain reaction for the detection of Anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene

Anguillid herpesvirus (AnHV, also known as Herpesvirus anguillae or HVA) is found in both Japanese and European eels. Based on restriction enzyme analysis a small number of differences were found between AnHV isolated from Japanese eels and from European eels. The total genome size of both is about 245 kb, which is confirmed by alternating-field electrophoresis. Using a set of degenerate primers based on conserved regions within DNA-directed DNA polymerase coding regions, a 463 base pair fragment was isolated from both Japanese and European AnHV. Nucleotide sequence analysis showed that the cloned regions of both viruses have identical sequences. Based on this part of the DNA-polymerase sequence, primers were selected and used to develop a sensitive PCR to detect AnHV DNA in eel tissue samples. To avoid false negative results and to estimate the number of AnHV genome copies found in tissues, 100 copies of an internal control plasmid were added to the tissue samples. This semi-quantitative AnHV PCR can be used for both the European and Japanese isolates of AnHV, detects as few as 10 genome copies and is 100 times more sensitive than standard virus isolation.     

Replication of vaccinia DNA in mouse L cells III. Intracellular forms of viral DNA

Parental and replicating vaccinia DNA molecules, labeled with [³H]thymidine or [¹⁴C]thymidine, present in infected L cells were analyzed by sedimentation in neutral and alkaline sucrose gradients. Alkaline sucrose gradient sedimentation analysis of labeled parental genomes present in infected cells showed that: (a) Such genomes were not degraded to acid-soluble products during the infection cycle; (b) cell-associated, cross-linked parental molecules (102 and 90–92 S) were “nicked”; parental DNA molecules sedimenting at 70–72 S were detected, but further nicking or degradation did not occur; and (c) molecules sedimenting at 90–92 S appeared to accumulate in the cytoplasm of infected cells and could serve as the templates for semiconservative DNA replication. When analyzed in neutral sucrose gradients, about 90% of viral DNA molecules labeled from 1 to 2 hr postinfection were associated with large aggregates or complexes which pelleted under the conditions of analysis used in these studies. With time (2–3 hr) after infection, viral DNA molecules could be dissociated from such aggregates and resolved by sedimentation in neutral sucrose gradients. The dissociation of labeled viral DNA molecules from complexes required continuous protein synthesis. Viral DNA could be released from complexes by treatment with alkali or digestion with S-1 nuclease, but not with RNase or Pronase. The results suggest that single-stranded (ss) DNA regions per se or proteins having affinity for ssDNA may bind the replicating vaccinia DNA molecules together in complexes.     

No detection of parvovirus B19 or herpesvirus DNA in giant cell arteritis.

BACKGROUND: Compelling arguments exist for a role of infectious agent in giant cell arteritis (GCA). Parvovirus B19 and several herpesviruses have focussed the attention in recent years, but the few studies to date have yielded inconsistent results. OBJECTIVES: To study the relationship between the presence of parvovirus B19 DNA or major known herpesviruses and the histopathological features of GCA. STUDY DESIGN: Between January 1997 and March 2002, 147 consecutive temporal artery biopsies were performed in our center because of a clinical suspicion of GCA. Using polymerase chain reaction (PCR) procedures validated by the World Health Organization and employed routinely by our laboratory, we examined the paraffin-embedded specimens for DNA from parvovirus B19, herpes simplex viruses (HSV) 1 and 2, Epstein-Barr virus (EBV), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and human herpesvirus 6 (HHV-6). We investigated positive results further with immunohistochemistry studies. RESULTS: Fifty of the 147 temporal artery biopsies (34%) showed histological features of GCA. Three biopsies (2.5%) were initially PCR positive for parvovirus B19. None of the herpesvirus PCR assays were positive. Upon repeat testing by both PCR and immunohistochemistry, none of the three initially positive parvovirus B19 assays were confirmed. The results of both positive and negative control assays in these studies validated these findings. We confirmed the presence of amplifiable DNA in the temporal artery biopsy specimens using PCR primers for beta-globin and indoleamine 2,3-dioxygenase (IDO). CONCLUSIONS: The results of our study do not support a role in the etiopathogenesis of GCA for either parvovirus B19 or any of these six herpesviruses.