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A simple and efficient PCR method was developed for generating dye- or radiolabeled single-stranded DNA targets or probes used for hybridization studies. The method involved the use of a pair of long primers with high annealing temperatures and a short, labeled primer with a low annealing temperature in a PCR consisting of two cycles at different temperatures. We used this method to generate dye Cy 5-labeled and [32P]-radiolabeled single-stranded DNA targets and probes. These labeled probes were used successfully for the microarray identification of point mutations in Mycobacterium tuberculosis genes and for the Northern blot detection of expression changes of the GATA-2 gene in Pneumocystis carinii-infected rat lungs.

Global public health is threatened by the emergence of potentially dangerous antibiotic drug-resistant strains of Mycobacterium tuberculosis. Point mutations in certain M. tuberculosis genes are associated with the resistance of M. tuberculosis strains to antibiotic drugs. The purpose of this study was to develop a suitable microarray-based protocol for the detection of point mutations in M. tuberculosis genes associated with drug resistance. We initially developed a conventional, oligonucleotide microarray protocol and used it to detect and identify on a single microarray slide a number of point mutation-containing rpoB and katG gene target sequences. However, the occurrence of some non-specific hybridization led us to the development of an improved protocol based on allele specific PCR combined with tags/anti-tags and microarrays. This protocol was evaluated by detecting point mutations in M. tuberculosis katG and rpoB gene templates produced by recombinant PCR. The methodology allowed sequences containing single point mutations to be readily distinguished from wild type sequences. The data obtained with the improved protocol had strong and specific signals and relatively low amounts of non-specific hybridization. We successfully used this protocol to detect and identify (<8 h) a number of clinically relevant point mutations in the rpoB, katG and rpsL genes of M. tuberculosis clinical isolates. Our allele specific PCR/tags and anti-tags/microarray protocol has several advantages over our conventional oligonucleotide microarray protocol, and it may have broad applications for point mutation detection.

A PCR method was developed to compare HIV-1 DNA loads in brain tissue samples. The method determines the ratio of the amplified product of an HIV DNA sequence to that of a host cellular DNA sequence using standard DNAs as reference. The standards include DNA from a line of human cells that harbor one HIV-1 provirus per cellular genome, and DNA from non-infected human cells. The standard DNAs were mixed in varying proportions and used to establish conditions of amplification under which the ratios of their PCR-amplified products corresponded with the ratios of the amounts of the DNAs themselves. The method was evaluated using known mixtures of the standard DNAs. Using the conditions thus obtained, ratios of HIV proviral DNA to cellular genomic DNA were obtained for tissue DNA samples taken from several different locations within the brain of two deceased HIV-infected patients. Results showed that HIV DNA was non-uniformly distributed within each brain (10-250 per 10(3) cellular genomes); the highest ratios were found in the hippocampus for each patient, independent of postmortem neuropathological findings. The criteria for quantitative PCR have general applicability to comparative studies of any proviral DNA loads in different tissue samples.

Background: Data concerning the distribution of HIV in the brains of AIDS patients at different stages of viral infection might contribute towards: (1) understanding the route(s) of HIV entry into the brain and virus dissemination within the brain and (2) establishing a possible correlation between the extent of CNS damage and the distribution of virus in AIDS brains., Objective: To determine the distribution of HIV-1 genomic DNA within the brains of three deceased AIDS patients by polymerase chain reaction (PCR)., Study Design: The brains of three deceased AIDS patients were examined. Two brains had limited neuropathologic findings (brains I and II), and one brain (brain III) showed primary HIV-specific neuropathologic damage. Tissues were taken from different locations within each brain, and high molecular weight DNA isolated from the tissues was assessed for HIV-1 genomic DNA by PCR., Results: HIV-1 genomic DNA was found in all three brains, but the amount was low: order of magnitude of 1 viral genome per 1,000 cells. Multiple PCR analyses of DNA from brain I showed that the viral genomic DNA in this brain was non-uniformly distributed; only samples taken from the brainstem were clearly positive for HIV-1. HIV-1 genomic DNA in brain II was found in portions of the lower and upper hemispheres. All but one of the brain III samples were clearly positive for HIV-1, and they had been taken from locations spread throughout this brain., Conclusions: Our results suggest that in early or latent stages of HIV-infection of the brain, viral genomic DNA is localized at restricted regions. At later stages this DNA is distributed more uniformly throughout the brain. Our data are compatible with the concept of rare infection events followed by viral spreading within brain tissues.

Argentine hemorrhagic fever is an often fatal human disease caused by Junin virus, an RNA-containing virus and member of the Arenavirus family. This virus was detected in vitro by the polymerase chain reaction (PCR) procedure. A pair of Junin virus-specific PCR DNA oligonucleotide primers and an oligonucleotide probe were designed from a known portion of the viral RNA sequence. RNA was isolated from Junin virus-infected monkey kidney cells and used to produce complementary DNA (cDNA) by reverse transcription. A DNA segment, 151 +/- 24 bp long, was amplified from this cDNA and characterized by agarose gel electrophoresis and Southern blot hybridization with the Junin virus-specific DNA probe. Sensitivity experiments showed that Junin virus could be detected with nanogram quantities of RNA isolated from virus-infected cells. The rapid and sensitive assay described here may contribute towards the development of a procedure for the early diagnosis of Argentine hemorrhagic fever.

The capacity of human fibroblasts to support plaque formation by Herpes simplex virus following treatment of the cells with gilvocarcin V, a polyaromatic C-glycoside, plus near ultraviolet radiation (UVA, 320-400 nm) was examined. Gilvocarcin V, plus UVA radiation, effectively inhibited host cell capacity at concentrations five orders of magnitude lower than that of 8-methyoxypsoralen required for capacity inhibition at similar levels of UVA radiation. This result extends the observation of unusual biological potency of UVA-activated gilvocarcins from bacterial cells to human cells.

The authors determined virus leakage from condoms made from processed sheep caecum using two viral probes simultaneously. They poured a mixture of two viruses, the bacteriophage, phi X174 (4 X 10(7) pfu/ml), and the human pathogen, herpes simplex virus (about 1 X 10(6) pfu/ml), in a buffered solution into condoms, which were suspended into beakers also containing buffered solution. The authors then assayed aliquots from the beakers to measure the extent of virus leakage from the condoms. With one brand of condom, 10 out of 24 samples leaked small amounts of phi X174; with the other brand of condom, 13 out of 24 samples gave similar leakage. The extent of leakage varied over two orders of magnitude from condom to condom within each brand. Of the 23 condoms that leaked the smaller virus, phi X174 (27 nm in diameter), only two also leaked the larger herpesvirus (120-150 nm in diameter). These data demonstrate that (1) large and small viruses can leak from natural membrane condoms; (2) there is considerable variation from condom to condom in allowing leakage of the viruses; and (3) leakage of a small virus does not necessarily indicate that a larger virus will leak from that particular condom. The authors explain some inconsistencies in the published literature.

The ability of UV-irradiated herpes simplex virus to form plaques was examined in monolayers of CV-1 monkey kidney cells preexposed to UV radiation at different intervals before virus assay. From analysis of UV reactivation (Weigle reactivation) curves it was found that as the interval between cell UV irradiation (0-20 J/m2) and initiation of the virus assay was increased over a period of five days, (1) the capacity of the cells to support unirradiated virus plaque formation, which was decreased immediately following UV exposure to the monolayers, increased and returned to approximately normal levels within five days, and (2) at five days an exponential increase was observed in the relative plaque formation of irradiated virus as a function of UV fluence to the monolayers. For high UV fluence (20 J/m2) to the cells, the relative plaque formation by the UV-irradiated virus at five days was about 10-fold higher than that obtained from assay on unirradiated cells. This enhancement in plaque formation is interpreted as a delayed expression of Weigle reactivation. The amount of enhancement resulting from this delayed reactivation was several fold greater than that produced by the Weigle reactivation which occurred when irradiated herpes virus was assayed immediately following cell irradiation.

Herpes simplex virus was grown in different lines of human tumor and normal cells. The progeny virus was assayed for resistance to iododeoxycytidine, an indicator of a forward mutation in the virus genome. Virus grown in cells from 4 of 5 tumor lines demonstrated greater fractions mutated to iododeoxycytidine resistance than did virus grown in 7 normal human skin cell lines. The data indicate that some lines of human tumor cells modify the herpesvirus replication process, making it more mutagenic. In 2 cases of osteosarcoma patients, normal skin fibroblasts of the patients yielded normal levels of mutagenesis, while their tumor cells gave enhanced mutagenesis.

Can simian virus 40 (SV40) be used to detect mutagenic DNA repair in cultured mammalian cells? The published evidence from different laboratories are in direct conflict. In order to decide between the conflicting evidence, we conducted experiments in two separate laboratories using experimental protocols similar to those previously used to investigate mutagenic repair with viral probes. Mutagenesis in SV40 virus stocks obtained by infecting ultraviolet (UV)-irradiated or unirradiated CV-1 monkey kidney cells with UV-irradiated or unirradiated temperature-sensitive SV40 mutant tsB201 was investigated. The frequency of reversion of the ts mutant to phenotypically wild-type virus was determined by assaying the virus stocks at permissive (33 degrees) and non-permissive (39 degrees) temperatures. These data show that (a) the reversion frequency for unirradiated virus propagated in irradiated cells was more than that in unirradiated cells; (b) irradiated virus gave more reversion than unirradiated virus in unirradiated and irradiated cells; and (c) irradiated virus had a lower reversion frequency in irradiated cells than in unirradiated cells. Reactivation experiments carried out in parallel; with the mutagenesis showed enhanced reactivation in UV-irradiated SV40 in UV-irradiated CV-1 cells. We conclude that enhanced reactivation of UV-irradiated SV40 was not mutagenic in monkey kidney cells.

Infectious simian virus 40 (SV40) was induced from SV40-transformed hamster kidney cells by treatment with proflavine and visible fluorescent light. The optimum levels of SV40 induced were about three orders of magnitude above spontaneous background levels observed with untreated cells. No virus induction above background levels was found by treatment of cells with either proflavine or light alone.

A line of simian virus 40-transformed hamster kidney cells was exposed to ultraviolet radiation at eleven different wavelengths in the region 238-302 nm. An action spectrum derived from the resulting exposure-response curves for the induction of simian virus 40 from these cells exhibits a broad peak in the region 260-270 nm suggesting DNA as the major chromophore for this response. This conclusion is consistent with results obtained by other investigators who have noted viral induction by a number of DNA-damaging agents.

The responses of two in vitro mammalian virus-host cell systems to the photosensitizing chemicals proflavine sulfate and 8-methoxypsoralen (8-MOP) in the presence of light are described. Infectious simian virus 40 (SV40) could be induced from SV40-transformed hamster cells by treatment with proflavine plus visible light or 8-MOP plus near UV radiation. The same photosensitizing treatments inactivated the capacity of monkey cells to support the growth of herpes simplex virus. SV40 induction and inactivation of host cell capacity for herpesvirus growth might be useful as screening systems for testing the photosensitizing potential of chemicals. Advantages and disadvantages associated with each system are discussed.

Ultaviolet enhanced (Weigle) reactivation of UV-irradiated herpes simplex virus in UV-irradiated CV-1 monkey kidney cell monolayers was decreased by caffeine. X-ray enhanced reactivation of UV-irradiated virus in X-irradiated monolayers (X-ray reactivation) and UV- or X-ray-inactivated capacity of the cells to support unirradiated virus plaque formation were unaffected by caffeine. The results suggest that a caffeine-sensitive process is necessary for the expression of Weigle reactivation for herpes virus. Since cafeine did not significantly affect X-ray reactivation, different mechanisms may be responsible for the expression of Weigle reactivation and X-ray reactivation.

The capacity of monolayers of both normal human and xeroderma pigmentosum (XP) filbroblasts to support plaque formation by herpes simplex virus was decreased when the monolayers were ultraviolet (UV) irradiated and infected with virus. Fibroblasts of XP complementation groups A, B, and D were sensitive to UV, being 4-6 fold more sensitive than either fibroblasts of XP complementation group C or fibroblasts from a normal individual. When the monolayers were irradiated 4 days prior to infection, the capacity of normal fibroblasts to support herpes virus growth recovered, whereas the capacity of the XP strains decreased further compared to that measured when infection immediately followed irradiation. Concurrent experiments with UV-irradiated herpes virus showed that the survival of this virus did not increase when infection by irradiated virus immediately followed irradiation of the monolayers. However, if the monolayers were irradiated 4 days prior to infection, the survival of this virus increased by a factor of nearly 2. Such Weigle reactivation (WR) occurred at lower fluences to the XP fibroblasts than to normal fibroblasts, suggesting that WR results from residual cellular DNA damage left after excision repair.

Data are presented which show that bromegrass mosaic virus has a particularly low molecular weight and nucleic acid content. A molecular weight of 4.6 x 10(6) was calculated from the sedimentation coefficient, S degrees (20,w) = 86.2S, the diffusion coefficient, D(20,w) = 1.55 x 10(-7) cm(2)/sec., and an assumed partial specific volume, [UNK] = 0.708 ml/gm. The virus has a ribonucleic acid content of 1.0 x 10(6) atomic mass units. Electrophoresis experiments showed that the virus is stable in 0.10 ionic strength buffers in the pH range 3-6. Breakdown of the virus was observed outside this pH range. Some characteristics of the breakdown products are described.

When CV-1 mammalian cells were X-irradiated before infection with ultraviolet (UV)-irradiated herpes simplex virus, an increase in survival of this virus was observed. X-ray reactivation is proposed as the name of this phenomenon by analogy with UV reactivation. The amount of survival enhancement was about the same as that found for UV reactivation in the same virus-host system. The enhancement reached a plateau at approximately 1000 rads and appeared to be radiation-insensitive at high doses.