Herpes Simplex Virus DNA Sequences Research Articles

Excimer Laser Photokeratectomy Reactivates Latent Herpes Simplex Virus

It has been reported that excimer laser irradiation might elicit herpes simplex virus (HSV) genome activation. We describe a clinical case in which HSV DNA sequences were detected quantitatively after phototherapeutic keratectomy (PTK). A 90-year-old woman underwent excimer laser photokeratectomy for bilateral band-shaped keratopathy. Tear film was collected from both eyes using a Schirmer's strip before and 3 and 7 days after phototherapeutic keratectomy. HSV-DNA was quantified by a real-time polymerase chain reaction assay. HSV-DNA was detected only on the third day postoperatively in both eyes. The amount of viral DNA was 2.0 x 10(5) (OD) and 1.3 x 10(5) (OS) copies/sample, respectively. Excimer laser photokeratectomy stimulated viral shedding in the tear film. Ophthalmologists should be aware that laser irradiation can reactivate latent HSV.

Attempts to identify herpes simplex virus DNA in normal and diseased human peripheral nerve.

PCR analysis of DNA extracted from 31 sections of formalin-fixed sural nerve biopsies did not reveal herpes simplex virus (HSV) DNA. Unlike the presence of HSV DNA sequences in normal human brain, spinal cord, and ganglia, HSV DNA is not present in normal or diseased human distal peripheral nerve.

Evaluation of a non-isotopic polymerase chain reaction assay for detection in clinical specimens of herpes simplex virus type 2 DNA

Background: PCR assays for detection of herpes simplex virus DNA sequences in clinical specimens are more sensitive than cell culture. Objective: A non-isotopic PCR assay (glycoprotein B HSV-2 assay, Roche Molecular Systems) for detection of HSV-2 DNA sequences was evaluated on 234 clinical specimens. Study design: A total of 125 patients (73 women, 40 men, 12 unknown) provided 162 samples contained in viral transport medium. Samples were first inoculated in cell culture, centrifuged at 12 000 × g, lyzed and kept frozen. A total of 77 women provided 42 cervicovaginal lavages and 30 vaginal tampons that were lyzed, tested with two isotopic HSV-2 PCR assays and kept frozen. All these samples were subsequently thawed and amplified with the glycoprotein B HSV-2 assay using generic primers for HSV glycoprotein B gene. Amplicons were captured on microplates with a HSV-2-specific probe and were detected with avidin-peroxidase and substrate. Results: Of the 162 samples submitted to viral culture, HSV-2 was isolated from 73 while 89 did not contain HSV-2. All the 73 specimens with culture-proven HSV-2 infections tested positive with glycoprotein B HSV-2 assay (sensitivity of 100%). Herpesviruses other than HSV-2 were isolated from 34 samples that were negative with glycoprotein B HSV-2 assay. Two culture-negative samples tested positive in the glycoprotein B HSV-2 assay (specificity of 98.7%). The latter samples could not be retested in confirmatory isotopic HSV-2 PCR tests. HSV-2 DNA sequences could also be detected directly in cervical lavages or vaginal tampons from 13 women with the glycoprotein B HSV-2 assay. Conclusion: Detection and typing of HSV-2 in clinical samples, including those collected in viral transport medium, can be accomplished with PCR assays using the AMPLICOR format.

Retention of herpes simplex virus DNA sequences in the nuclei of mouse footpad keratinocytes after recovery from primary infection.

Persistence of herpes simplex virus (HSV) DNA in mouse footpad keratinocytes was studied by non-isotopic in situ hybridization. HSV DNA was retained in keratinocyte nuclei for more than 2 weeks after disappearance of infectious virus and viral antigens. The anatomical location of viral DNA became more superficial with increasing time post-infection, reflecting the migration of cells from the basal layer of the epidermis towards the stratum granulosum. Latency-associated transcripts (LATs) were not detected in footpad cells at any of the times studied. In contrast, LATs were detected readily in the nuclei of lumbar ganglionic neurons innervating HSV DNA positive footpads. It was concluded that, after termination of productive infection in the skin, HSV DNA persists transiently in keratinocyte nuclei, in the absence of abundant latency-associated transcription. An implication of these data is that detection of HSV DNA in the skin may reflect recent, but not necessarily current, cutaneous virus replication.

Polymerase chain reaction reveals herpes simplex virus DNA in saliva of patients with Behçet's disease.

The etiology of Behçet's disease is unclear, but viral infection is thought to be one etiologic factor. The aims of this study were to detect herpes simplex virus (HSV) DNA in the saliva of patients with Behçet's disease and of healthy persons, to determine whether the presence of HSV in saliva is associated with the presence of intraoral ulcer, and to investigate the relationship between HSV and Behçet's disease. The polymerase chain reaction (PCR) was used to detect HSV DNA sequences in DNA extracted from the saliva of patients with Behçet's disease and of healthy control subjects. Of 66 patients with Behçet's disease diagnosed clinically, 19 were diagnosed as complete type, 29 as incomplete type and 18 as suspected type. Of 66 DNA preparations from the saliva of the patients, 26 (39.4%) showed the 289-bp band. This contrasts with 12 of 87 preparations (13.8%) from healthy controls (P < 0.01). There were no significant differences among the three patient groups. All the 289-bp bands analyzed by restriction endonuclease digestion yielded the expected 158-bp and 131-bp fragments when digested with PstI.HSV DNA was detected in 12 of 33 Behçet's disease patients (36.4%) with oral ulceration and 14 of 33 patients (42.4%) without oral ulceration at the time of testing. There was no statistically significant correlation in the PCR results between the two groups.

Detection of herpes simplex and varicella-zoster infection from cutaneous lesions in different clinical stages with the polymerase chain reaction

Background: The polymerase chain reaction (PCR) can be used to diagnose a variety of infectious processes. Objective: We sought to determine whether Tzanck smear debris, vesicle fluid swabs, crusts, or fixed tissue specimens are the best source for template herpes simplex virus (HSV) or varicella-zoster virus (VZV) DNA for the PCR. Methods: Patients with both clinical and histologic evidence of HSV ( n = 6) or VZV ( n = 16) infection were examined. Stained Tzanck smears, vesicle fluid swabs, dried crusts, and skin biopsy specimens were obtained at the same time from each patient. DNA was extracted from the different clinical specimens and then examined for HSV or VZV DNA with PCR. Fifteen control subjects did not have clinical or histologic evidence of herpesvirus infection. Results: In cases of suspected VZV infection, PCR detected VZV DNA sequences from all 15 Tzanck smears, all 15 vesicle swabs, one of one crust, and 14 of 16 fixed tissue specimens. HSV DNA sequences were detected from all six Tzanck smears, all four vesicle fluid swabs, two of two crusts, and five of six fixed tissue specimens. Conclusion: PCR can detect VZV and HSV DNA sequences from a variety of sources including formalin-fixed tissue specimens. Although viral DNA was detected slightly more frequently from Tzanck smear debris, crusts, and vesicle fluid swabs compared with fixed tissue specimens, each was an excellent source of target DNA for the PCR to confirm the diagnosis of herpesvirus infection.

In situ PCR localization of herpes simplex virus DNA sequences in disseminated neonatal herpes encephalitis.

To more precisely define the role of herpes simplex virus (HSV) in development of nervous system disease in neonates with disseminated infection, an in situ polymerase chain reaction (ISPCR) method was used to detect and localize HSV DNA sequences in paraffin sections of neural and non-neural autopsy tissues. In subregions of adjacent sections corresponding to ISPCR-labeled and unlabeled areas, HSV specificity was verified using solution PCR and Southern blots. In serial sections, ISPCR results were compared to lesions, HSV antigen and, in selected samples, to viral sequence detection by in situ hybridization. By ISPCR, HSV-specific labeling was limited to HSV-infected neonates and experimentally infected mouse controls. ISPCR-labeled cells corresponded to regions that were histologically abnormal and contained HSV antigen or in situ hybridization signal in some foci; in others, labeled cells were in areas with no evident lesions or antigen. Results suggest two routes of HSV spread to the CNS: (i) blood-borne infection, with HSV DNA in splenic lymphocytes, circulating cells, meningeal vessel walls and cells in intraventricular hemorrhage, and (ii) neural spread, with HSV detected in brain stem sensory neurons. In the brain of one neonate surviving acute infection, detection of HSV nucleic acid sequences suggests a latent or persistent viral genome. With other methods, this highly sensitive ISPCR technique permits a more complete definition of HSV infection in these infants and provides new insights into disease mechanisms. Fuller understanding of HSV persistence and recurrent neurological disease in survivors will require further studies using these and other techniques in human tissues and in animal models.

Detection of herpes simplex virus DNA from cerebrospinal fluid by PCR and a rapid, nonradioactive hybridization technique

A molecular assay for the detection of herpes simplex virus (HSV), including a novel, nonradioactive hybridization technique, was evaluated with a total of 123 cerebrospinal fluid specimens. After DNA extraction, specific HSV DNA sequences were amplified with digoxigenin-labeled primers derived from the DNA polymerase gene-coding region from HSV. Amplified products were detected by the Enzymun-Test DNA detection assay (Boehringer, Mannheim, Federal Republic of Germany), which uses biotinylated probes. Amplification with nonlabeled primers and then Southern blotting and nonradioactive detection of hybrids by the digoxigenin technique was the reference system. The sensitivities of the molecular assays were determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene obtained from the HSV type 1 strain Angelotti. The Enzymun assay was able to detect all of the 16 positive samples, giving 100% agreement with the Southern blot hybridization results. Optical density values were widely separated for the positive and negative groups of specimens. Ten copies of plasmid pS4 per microliter could be distinctly detected by the Enzymun assay. The cutoff was determined for the hybridization assay, and an equivocal zone was defined. The whole molecular assay including the Enzymun-Test DNA detection proved to be sensitive and easy to use. It may contribute to the rapid and safe detection of HSV DNA in cerebrospinal fluid.

Detection of herpes simplex virus DNA sequences in human blood and bone marrow cells

Herpes simplex virus type 1 (HSV1) establishes latent infections in neural tissues of humans and experimental animals. Utilizing a sensitive polymerase chain reaction (PCR) assay we detected HSV DNA sequences in blood cells of healthy prospective bone marrow transplant (BMT) donors and patients. In three healthy individuals studied, HSV DNA sequences were found in all blood cell types and also in bone marrow cells as well as in stem cell progenitor colonies isolated from in vitro cultures. Studies of BMT donor-recipient pairs suggested that HSV reactivation may occur in hematopoietic cells after transplantation, as the PCR signal intensity increased over time simultaneous with an increased antibody titer to HSV. In a mouse model for HSV infection, HSV DNA sequences were found in blood and bone marrow cells at the latent stage of infection, after intravenous (IV) inoculation, but not after ocular inoculation. These studies suggest that bone marrow cells may be an additional site of HSV latency capable of reactivation after BMT. These studies have broad implications for understanding pathogenesis of HSV disease and are of particular significance in situations where allogeneic bone marrow cells are given therapeutically.

Comparison of Tzanck smear, viral culture, and DNA diagnostic methods in detection of herpes simplex and varicella-zoster infection

<h3>Objective.</h3> —To compare Tzanck smears, viral cultures, and DNA diagnostic methods using the polymerase chain reaction (PCR) in detection of herpes simplex virus (HSV) or varicella-zoster virus (VZV) infection in clinically suspected cases. <h3>Design.</h3> —A 12-month trial comparing PCR with viral cultures and Tzanck smears in patients with clinically suspected HSV or VZV infection. <h3>Setting.</h3> —Both ambulatory and hospitalized patients were recruited from a tertiary referral center and the Miami (Fla) Veterans Affairs Medical Center. <h3>Patients.</h3> —Convenience samples of patients clinically suspected to have HSV (n=48) or VZV (n=35). To be included in the final analysis patients needed to have a positive Tzanck smear, viral culture, or PCR result. Patients who were clinically suspected to have HSV but had VZV by viral culture or PCR were analyzed in the VZV group. Similarly, patients who were clinically suspected to have VZV, but had HSV by viral culture or PCR were analyzed in the HSV group. Seventy-seven patients were available for final analysis: HSV (n=30), VZV (n=32), and 15 control cases who did not have evidence of viral infection. <h3>Results.</h3> —For HSV, PCR detected HSV DNA sequences in 73% of stained smears and 83% of unstained smears. For VZV infection, VZV DNA sequences were detected in 88% of stained smears and 97% of unstained smears. Viral DNA sequences were not detected in the 15 control cases. Viral cultures were positive in 83% and 44% of HSV and VZV cases, respectively. The Tzanck smear was positive in 60% and 75% of HSV and VZV cases, respectively. <h3>Conclusions.</h3> —PCR is a reliable method for detecting HSV and VZV DNA sequences from single stained and unstained Tzanck smears. It is clearly superior to viral culture in identifying VZV infection and is equivalent to conventional culture techniques in identifying cases of HSV. (<i>JAMA</i>. 1992;268:2541-2544)

Comparison of Tzanck Smear, Viral Culture, and DNA Diagnostic Methods in Detection of Herpes Simplex and Varicella-Zoster Infection

To compare Tzanck smears, viral cultures, and DNA diagnostic methods using the polymerase chain reaction (PCR) in detection of herpes simplex virus (HSV) or varicella-zoster virus (VZV) infection in clinically suspected cases. A 12-month trial comparing PCR with viral cultures and Tzanck smears in patients with clinically suspected HSV or VZV infection. Both ambulatory and hospitalized patients were recruited from a tertiary referral center and the Miami (Fla) Veterans Affairs Medical Center. Convenience samples of patients clinically suspected to have HSV (n = 48) or VZV (n = 35). To be included in the final analysis patients needed to have a positive Tzanck smear, viral culture, or PCR result. Patients who were clinically suspected to have HSV but had VZV by viral culture or PCR were analyzed in the VZV group. Similarly, patients who were clinically suspected to have VZV, but had HSV by viral culture or PCR were analyzed in the HSV group. Seventy-seven patients were available for final analysis: HSV (n = 30), VZV (n = 32), and 15 control cases who did not have evidence of viral infection. For HSV, PCR detected HSV DNA sequences in 73% of stained smears and 83% of unstained smears. For VZV infection, VZV DNA sequences were detected in 88% of stained smears and 97% of unstained smears. Viral DNA sequences were not detected in the 15 control cases. Viral cultures were positive in 83% and 44% of HSV and VZV cases, respectively. The Tzanck smear was positive in 60% and 75% of HSV and VZV cases, respectively. PCR is a reliable method for detecting HSV and VZV DNA sequences from single stained and unstained Tzanck smears. It is clearly superior to viral culture in identifying VZV infection and is equivalent to conventional culture techniques in identifying cases of HSV.

A prospective study of the polymerase chain reaction for detection of herpes simplex virus in cerebrospinal fluid submitted to the Clinical Virology Laboratory

Polymerase chain reaction (PCR) was prospectively performed with cerebrospinal fluid (CSF) from 51 patients whose CSF was available for analysis and was submitted for viral culture and/or herpes simplex virus (HSV) serology and 20 patients whose CSF was submitted exclusively to the Clinical Biochemistry Laboratory. Primers were used that flanked a 92 bp segment of the HSV DNA polymerase gene (35 cycles). Amplified products were electrophoresed on agarose gel, blotted onto nylon membrane, and probed with a 32P-labelled sequence internal to the primers. For nested PCR, 1 μl of PCR product was amplified for an additional 35 cycles before electrophoresis and Southern blot analysis. Review of the clinical records revealed that 15 patients had central nervous system (CNS) infections. Specific HSV DNA sequences were detected in CSF specimens of three of the individuals [PCR(2), nested PCR(1)]. Two of these patients had disseminated HSV infection including encephalitis and one patient had aseptic meningitis. The diagnoses of the 12 patients with CNS infection who did not have HSV DNA detected in CSF included encephalitis [varicella-zoster virus (1), cytomegalovirus (1), Mycoplasma pneumoniae (1)], meningitis [ Neisseria meningitidis (1), Coccidioides immitis (1), Enterovirus (1), aseptic meningitis (1)], varicella-zoster radiculitis (2), human immuno-deficiency virus dementia (2), and transverse myelitis due to Epstein-Barr virus (1). Importantly, HSV DNA was also not detected in the CSF of the 36 patients who did not have CNS infection and 20 samples submitted exclusively to the Clinical Biochemistry Laboratory. Our findings demonstrate the utility of PCR as a rapid, non-invasive method for the routine laboratory diagnosis of CNS infection due to HSV.

Detection of herpes simplex virus in gingival tissue.

The presence of herpes simplex virus (HSV) antigens was shown by immunofluorescence staining in 26 of 66 (39.3%) specimens of clinically healthy gingiva, but only one sample contained infectious virus. HSV DNA sequences were clearly identified in intact gingival cells by dot blot hybridization in one specimen, and a weak pattern in a second one. Both specimens harbored viral antigens. These findings of viral genome and protein expression suggest that the virus is present in the latent form in the gingiva.

Two patterns of persistence of herpes simplex virus DNA sequences in the nervous systems of latently infected mice.

The number of herpes simplex virus (HSV) genome equivalents recovered from latently infected mouse spinal ganglia was compared with the proportion of neurons containing latency-associated transcripts (LATs). Two distinct patterns of HSV persistence were observed, depending on the anatomical location of ganglia with respect to the site of cutaneous inoculation. The location of the bulk of latent viral DNA did not correspond with the highest prevalence of LAT+ neurons. Viral DNA was most abundant in spinal ganglia directly innervating the inoculation site and the amount recovered, which was similar to that found previously in human trigeminal ganglia, suggested that LAT+ neurons each contain hundreds of copies of HSV DNA. In stark contrast, although LAT+ neurons were most abundant in neighbouring ganglia, viral DNA was scarce (approx. 20 copies/LAT+ cell). These data indicate that amplification of HSV DNA sequences is greatest in ganglia previously shown to be associated with viral antigen expression during the productive phase of primary infection.

Detection of herpes simplex virus DNA sequences in corneal transplant recipients by polymerase chain reaction assays.

Polymerase chain reaction (PCR) assays were used to amplify herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) sequences in DNA extracted from formalin-fixed, paraffin embedded corneas of patients undergoing corneal transplantation. PCR reamplification with an internal (nested) set of primers was required for detection in 10 of the 12 positive corneas indicating very low abundance of viral sequences. Three of the positive corneal samples were from failed corneal grafts. Overall, TK sequences were detected in 8 of 11 corneas from subjects with a past history of herpes keratitis and in 4 of 11 corneas from subjects with no past history of herpetic eye disease.

Monoclonal antibodies to HSV-infection-related antigens cross-react with tumor cell lines and tumor tissue sections

The purpose of our study was to evaluate a possible association between herpes simplex virus (HSV) and various tumors, including oral squamous cell carcinoma (SCC). To this end, we tested the binding of appropriate monoclonal antibodies to a panel of cell lines and tumor sections. The 25 monoclonal antibodies were reactive with HSV-infected cells but not with uninfected cells. Of these antibodies, three bound to several SCC cell lines and to one non-SCC cell line (K562). One of these three antibodies also reacted with sections of oral SCC tumors, the adjacent mucosa, and normal esophageal epithelium. In contrast, it did not bind to sections of kidney, spleen, esophageal smooth muscle, and skin. To evaluate whether the observed antibody binding could reflect an actual infection by HSV, hybridization experiments between K562 DNA and HSV DNA were performed. HSV DNA sequences were found in K562 DNA at a ratio of 0.1 genome-equivalents/cell. Although these data do not characterize the nature of the relationship, they clearly confirm the postulated association of HSV with some tumors (in particular oral SCC).

Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the alpha 47 gene.

The herpes simplex virus genome consists of two components, L and S, that invert relative to each other to yield four isomeric arrangements, prototype (P), inversion of the S component (Is), inversion of the L component (Il), and inversion of both components (Isl). Previous studies have shown that the 500-base-pair a sequences flanking the two components contain a cis-acting site for inversion. In an attempt to insert a third copy of the alpha 4 gene, the major regulatory gene mapping in the repeats flanking the S component, a fragment containing the alpha 4 gene and an origin of DNA synthesis, was recombined into the thymidine kinase gene mapping in the unique sequences of the L component. The resulting recombinants showed massive rearrangements and deletions mapping in the S component and in the junction between the L and S components. One recombinant (R7023) yielded two isomeric DNA arrangements, a major component consisting of Is and a minor component consisting of Isl. In these arrangements, the genome lacked the gene specifying glycoprotein E and all contiguous genes located between it and the alpha 0 gene in the inverted repeats of the L component. Among the deleted sequences were those encoding an origin of viral DNA synthesis, the alpha 47 gene, and the a sequences located at the junction between the L and S-components. The recombinant grew well in rabbit skin, 143TK-, and Vero cell lines. We conclude that the four unique genes deleted in R7023 are not essential for the growth of herpes simplex virus, at least in the cell lines tested, and that the b sequence of the inverted repeats of the L component also contains cis-acting sites for the inversion of herpes simplex virus DNA sequences.

Detection of viral DNA and RNA by in situ hybridization.

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.

Herpes simplex virus sequences involved in the initiation of oncogenic morphological transformation of rat cells are not required for maintenance of the transformed state.

We have determined the herpes simplex virus (HSV) type 2 DNA sequences responsible for the initiation of morphological transformation and have investigated the retention and expression of these sequences in morphologically transformed cells and in tumours derived from these cells. All the transformed cells analysed were selected by a focus formation assay and are oncogenic in the inbred host rat. Cloned HindIII and Bg/II fragments from the HSV-2 genome were assayed for the ability to initiate morphological transformation of rat embryo cells. Only the HindIII a (map units 0.52 to 0.72) and the Bg/II n (0.582 to 0.612) clones gave transformed foci. This shows that the Bg/II n region is responsible for initiation of transformation. Southern blot analysis of DNA extracted from these transformed cells and from tumours derived from these transformed cells revealed that neither the Bg/II n fragment nor fragments of 500 bp mapping within it are detected at the level of one copy per cell and therefore need not be retained in the cell to maintain the oncogenic phenotype. In addition there was no evidence of expression of the HSV-specified ribonucleotide reductase activity which is partially encoded within the Bg/II n fragment of HSV-2. We also analysed DNA from rat embryo cells transformed by ts mutants of HSV-2 (HG52) or HSV-1 (HFEM or 17) at non-permissive temperature or by virus at supraoptimal temperature or by sheared virus DNA and DNA from tumours derived from lines of these transformed cells. In addition, we cloned both transformed and tumour cell lines and analysed these similarly. In no case could we detect HSV DNA sequences at the level of one copy per cell.

Novel rearrangements of herpes simplex virus DNA sequences resulting from duplication of a sequence within the unique region of the L component.

We constructed insertion mutants of herpes simplex virus type 1 that contained a duplication of DNA sequences from the BamHI-L fragment (map units 0.706 to 0.744), which is located in the unique region of the L component (UL) of the herpes simplex virus type 1 genome. The second copy of the BamHI-L sequence was inserted in inverted orientation into the viral thymidine kinase gene (map units 0.30 to 0.32), also located within UL. A significant fraction of the progeny produced by these insertion mutants had genomes with rearranged DNA sequences, presumably resulting from intramolecular or intermolecular recombination between the BamHI-L sequences at the two different genomic locations. The rearranged genomes either had an inversion of the DNA sequence flanked by the duplication or were recombinant molecules in which different regions of the genome had been duplicated and deleted. Genomic rearrangements similar to those described here have been reported previously but only for herpes simplex virus insertion mutants containing an extra copy of the repetitive a sequence. Such rearrangements have not been reported for insertion mutants that contain duplications of herpes simplex virus DNA sequences from largely unique regions of the genome. The implications of these results are discussed.