The applicability of formalin-fixed and formalin fixed paraffin embedded tissues in forensic DNA analysis.

Historically, formalin fixed (FF) tissues could not be used as a source of DNA in forensic science due to the fact that the DNA was too degraded for DNA analysis. With the introduction of the polymerase chain reaction (PCR) technique to forensic science, the usefulness of DNA from this biological material has been re-evaluated. This study evaluates the potential use of DNA from FF and formalin fixed paraffin embedded (FFPE) tissues in 13 PCR systems; HLA DQ alpha, LDLR, GYPA, HBGG, D7S8, GC, D1S80, vWA31, THO1, F13A1, FES/FPS, TPOX, and CSF1PO. The first six, HLA DQ alpha, LDLR, GYPA, HBGG, D7S8, and GC are reverse dot blot systems, D1S80 is an amplified fragment length polymorphism (AmpFlp) system and the others are short tandem repeats (STRs). This study shows that FFPE tissue which has not been fixed in formalin for more than three days is a useful source of DNA for 12 of the 13 PCR systems. In contrast, FF tissue did not prove to be a reliable source of DNA for the PCR techniques examined here.

Chronic fatigue syndrome in northern Nevada.

The clinical and laboratory findings from studies of patients with chronic fatigue syndrome (CFS) from northern Nevada are summarized. Physicians caring for these patients have estimated that greater than 400 patients with CFS from northern Nevada and nearby communities in California were identified between 1984 and 1988. As a result of these studies, a cluster of clinical and laboratory features associated with the illness in moderately to severely affected patients has been identified: profound fatigue of prolonged duration; cervical lymphadenopathy; recurrent sore throat and/or symptoms of influenza; loss of cognitive function manifested by loss of memory and loss of ability to concentrate; myalgia; impairment of fine motor skills; abnormal findings on magnetic resonance imaging brain scan; depressed level of antibody to Epstein-Barr virus (EBV) nuclear antigen; elevated level of antibody to EBV early antigen restricted component; elevated ratio of CD4 helper to CD8 suppressor cells; and strong evidence of association of this syndrome with infection with human herpesvirus 6. More-serious and longer-lasting neurologic impairments, including seizures, psychosis, and dementia, have also been observed in some of these patients.

Genetic relatedness of disease-associated field isolates of bovid herpesvirus type 4.

Eight field isolates of bovid herpesvirus type 4 (BHV-4) were examined by restriction analysis and Southern blot hybridization with respect to their relatedness to one another and to the BHV-4 prototype strain DN-599. Isolates were obtained from cattle exhibiting a range of disease states including abortion, pneumonia, enteritis, metritis, and vaginal blisters. Initial growth studies of all 9 viruses were performed and revealed that the overall rate of virus growth was slow when compared with that of other herpesviruses. Infection with each virus also resulted in the formation of large fused cells, which in addition to the slow growth rate, indicated that the isolates were of the cytomegalovirus type. Further studies to characterize and compare the various BHV-4 isolates were undertaken by obtaining cell-free virus from infected cell populations. Viral isolates were purified and used as a source of BHV-4 DNA. Purified DNA, representing each of the 8 field isolates and the prototype strain DN-599, were each cleaved with 3 restriction enzymes and were separated by agarose-gel electrophoresis, and the resultant fragment patterns were compared. In general, genomic fragments of the field isolates corresponded to those generated by cleavage of DN-599 DNA, with the exception of the abortion-associated isolate 83-3572. Additional minor differences were also seen between DN-599 DNA and DNA from the other field isolates, but the overall restriction patterns were similar. To confirm that all isolates were members of the BHV-4 type, hybridization studies were performed using DN-599.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of autonomous replicating sequences (ars) in the genome of Epstein-Barr virus.

Epstein-Barr virus (EBV) DNA was analyzed for the presence of autonomous replicating sequences (designated ars) in a eukaryotic system consisting of a uracil auxotroph of Saccharomyces cerevisiae, YNN27, and a pBR322 hybrid plasmid, YIp5, containing the yeast uracil gene but apparently lacking a eukaryotic origin of replication. Cloned EBV DNA EcoRI restriction fragments, A, B, and DIJhet, were judged to function in this capacity by their ability to convert YNN27 cells to the uracil phenotype after transformation with each EBV-specific fragment ligated into YIp5. Additional analyses to confirm and to specify further the location of the ars were performed by cleavage of EcoRI fragments A and B into smaller BamHI fragments, which were subsequently cloned in YIp5 and tested for their ability to function as ars. BamHI fragment X, obtained from EcoRI fragment A, and BamHI fragment R, obtained from EcoRI fragment B, showed ars behavior. The successful recovery of the appropriate virus DNA segments in plasmid form from transformed yeast cells and the ability of these yeast cells to be propagated further substantiated the ars capability of the three EBV fragments.

Epstein-Barr virus polypeptides: identification of early proteins and their synthesis and glycosylation.

We have identified at least six early polypeptides induced by Epstein-Barr virus in cells or under conditions which are nonpermissive for Epstein-Barr virus DNA replication ranging in molecular weight from 140,000 to 26,000.

Epstein-Barr virus polypeptides: effect of inhibition of viral DNA replication on their synthesis.

After Epstein-Barr virus superinfection of the human lymphoblastoid cell line Raji, a Burkitt lymphoma-derived line that contains Epstein-Barr virus genomes in an episomal form, at least 40 polypeptides could be resolved by polyacrylamide gel electrophoresis. Eleven of the 40 polypeptides were immunoprecipitable by early antigen+/viral capsid antigen+ antiserum. The polypeptides could be divided into six classes, immediate-early, early, intermediate, late, very late, and persistent, depending upon the time of synthesis. Ten of the 40 polypeptides appeared to preexist before superinfection and persisted despite general cessation of host protein synthesis; none of the persistent proteins was immunoprecipitated by the Epstein-Barr virus antibody-containing serum. When viral DNA replication was blocked by a variety of inhibitors of DNA synthesis, a number of different patterns of polypeptide synthesis could be detected. The synthesis of six polypeptides was blocked by the most virus-specific inhibitors, acyclovir and phosphonoacetic acid. Additionally, in the presence of 1-beta-D-arabinofuranosylcytosine, 1-beta-D-arabinofuranosyladenine, and methotrexate, seven polypeptides showed oversynthesis.

Alterations in virus protein synthesis and capsid production in infection with DI particles of herpesvirus.

High multiplicity, undiluted passage of equine herpesvirus type 1 (EHV-1) in L-M cells resulted in the rapid production of virus particles whose genome was genetically less complex, contained more reiterated DNA sequences and exhibited a greater buoyant density (rho = 1.724 g/ml) than the DNA (rho = 1.716 g/ml) of standard virus. These data and the finding that these particles inhibited the replication of standard virus in interference assays confirmed that these were defective interfering (DI) particles (Henry et al. 1979). Additional evidence for this has been obtained from the pattern of cyclic fluctuation in infectious virus titre through 17 serial passages as well as from the pronounced variation in the particle to plaque ratio for each passage. Total particle production was markedly reduced in cells infected with virus preparations containing DI particles and quantification of major cell-associated EHV-1 capsid species by electron microscopy and analysis in Renografin density gradients indicated that this reduction occurred at the level of capsid assembly. Although total capsid production was reduced in cells infected with DI particle preparations, the synthesis of I (immature) capsids increased relative to that of L (empty) capsids and these alterations in the assembly of capsid species could be related to changes in the synthesis of capsid proteins. In cells infected with EHV-1 preparations rich in DI particles, the synthesis of major capsid protein 150000 was greatly reduced, whereas core protein 46000, a major component of I capsids, was overproduced as compared to standard virus infection. Capsids produced in cells infected with virus preparations rich in DI particles were identical in polypeptide composition to those made in standard virus infection.

Biochemical and morphological characterization of mycobacteriophage R1.

Large-scale propagation of mycobacteriophage R1 in broth culture has allowed the isolation of quantities of virus sufficient for characterization of its nucleic acid and lipid components as well as investigation of its ultrastructural attributes. Analysis of R1 DNA indicates that it is double stranded and possesses a molecular weight of 2.5 X 10(7) and a guanine-plus-cytosine content of 65.7 +/- 0.5%. The lipid fraction of R1 accounts for 14% of the total dry weight of the virus, 20% of which was identified as free or esterified sterols. A rapid loss of viral titer occurred after seconds of exposure to organic solvents. This result suggests that the lipid fractions of R1 is essential for its infectivity. Electron microscopic investigation of solvent-extracted R1 showed extensive deterioration of its normal morphology, including nucleocapsid disintegration and base plate separation. Routine phosphotungstate preparations demonstrated a particle with an oval head and a noncontractile tail. Altering the pH of the phosphotungstate negative stain from neutrality damage the viral particles. Uranyl formate-contrasted specimens displayed an elongated hexagonal nucleocapsid with a neck region; the cross-striated tail possessed a starlike base plate.