Mutations designed to alter the stability of a putative RNA duplex in the HIV-1 pol gene.

In the HIV-1 integrase coding region there is a polypurine tract (PPT) involved in the initiation of provirus plus-strand synthesis. Upstream of this PPT there is a 15-nucleotide inverted repeat (IR) complementary to most of the PPT. We have constructed one mutant with five amino acid-neutral U to C and A to G changes in the IR and one mutant with corresponding amino acid-neutral changes in the PPT. Each set of changes abolished the complementarity and suppressed the replication of HIV-1 slightly. The combination of these ten changes restored the complementarity, and doubled the calculated free energy of the putative duplex between the IR and the PPT. This double mutant did not replicate under normal conditions, possibly because the reverse transcriptase was unable to penetrate the duplex. However, when high loads of the double mutant were added to permissive cells, replicative HIV did occasionally appear. The resurrected virus harvested from these cells replicated consistently, even though the ten nucleotide changes were left unchanged. There were no compensatory mutations in the vicinity of the IR/PPT or in the reverse transcriptase gene.

CAG repeats in various organisms studied by Southern blot analysis.

A 90-nucleotide (CAG)30, single-stranded DNA was used to probe Southern blots in order to indicate the quantity and distribution of long CAG repeats in selected genomes. Bovine and rat genomes were found to contain a particularly high content of CAG repeats, while the repeats were comparatively rare in the human genome. A particularly strong signal in the bovine genome was due to a CAG repeat associated with the 1.709 satellite. A similar element was found in goat and musk, but not in the other artiodactyls tested, suggesting that this particular CAG repeat developed some 10-20 million years ago within a 3.8-kb unit presently belonging to the satellite element and that this unit has later multiplied in the genome. Single-copy repeats could be discerned in yeast, but not in mammals. Thus the probe did not detect specific repeats in patients with CAG repeat diseases.

Sequence comparison and mutational analysis of elements that may be involved in the regulation of DNA synthesis in HIV-1.

The large number of sequenced clones of HIV-1 and related viruses made it possible to indicate conserved elements with potential regulatory or structural functions. Such analysis was combined with directed mutagenesis in order to investigate the importance of elements that may influence the initiation of plus-strand DNA synthesis. The main site for plus-strand initiation is a polypurine tract near the 3' end of the viral RNA (the 3' PPT). An exact copy of this PPT is located in the middle of the genome (the internal PPT). Upstream from the internal PPT there is an inverted repeat. Mutants designed to upset the internal PPT (i.e., purine to pyrimidine changes), as well as mutants designed to abolish the potential stem-loop formation (changes around the internal PPT or in the upstream inverted repeat) both resulted in viruses with a reduced ability to replicate. Upsetting the stem-loop formation was, however, less harmful than changing the polypurine nature of the PPT. Changing a conserved T on the 3' side of the PPT to a C did not affect the phenotype.

Mutations in the central polypurine tract of HIV-1 result in delayed replication.

The reverse transcription of HIV-1 generates a linear genomic DNA with a single-stranded gap. The gap is believed to be the result of plus-strand priming from a second, centrally located, polypurine tract (PPT). A mutant containing four amino-acid-neutral purine-to-pyrimidine changes within the central PPT did not replicate as fast as wild-type virus. Another mutant with the entire 15-bp PPT deleted was replication-deficient. The results indicate that plus-strand priming at the central PPT is important for viral replication, possibly by ensuring efficient DNA synthesis.

Survival of HIV-1 activity after disinfection, temperature and pH changes, or drying.

A recently developed assay for measuring infectious HIV-1 particles was used to determine the stability of the virus under various storage conditions as well as the effect of commonly used disinfectants. At the optimum pH of 7.1 the half life of the virus ranged from approx. twenty-four hours at 37 degrees C to no significant loss over 6 months at -75 degrees C. Drying the virus on a glass surface or freezing caused a 5-12 fold and 4-5 fold decrease of activity, respectively. The dried preparations, however, were about as stable as when stored in a buffered solution. A solution of iodine and detergent (2% Jodopax) was the only disinfectant examined which removed all detectable HIV-1 activity. Isopropanol and ethanol were more potent than acetone; however, all three solvents left some viable particles after a 30 min treatment with 70% solutions.

The plus strand is discontinuous in a subpopulation of unintegrated HIV-1 DNA.

During reverse transcription the synthesis of plus strand viral DNA is initiated from an RNA polypurine primer immediately upstream of the U 3 region. The polypurine tract (PPT) sequence at this site is in HIV-1 also present in the middle of the genome. Here we demonstrate that a subpopulation of linear unintegrated HIV-1 DNA has a discontinuity in the plus strand within less than 50 bp from this central PPT, consistent with its utilization as a plus strand initiation site.

An assay for quantifying infectious HIV particles.

A method for assessing the number of infectious particles in preparations of HIV has been developed. Virus was mixed with cells to allow binding of virus. The cells were then cast in an agar gel to block any further transfer of virus between the cells. After 4 days of incubation the cells initially infected with HIV expressed viral antigens. The percentage of infected cells was then determined by indirect immunofluorescence. The method was developed for HIV, but is presumably suitable for any virus that can replicate in cells not attached to a surface.

Modified oligopeptides designed to interact with the HIV-1 proteinase inhibit viral replication.

The human immunodeficiency virus 1 (HIV-1) codes for a proteinase that cuts viral proteins at specific sites. We have tested 13 modified oligopeptides related to these cleavage sites to see if they inhibit viral replication. To indicate whether a decrease in replication could be due to a general inhibition of cell metabolism, we also measured the effect of the peptides on cellular protein synthesis. Three of the peptides tested (Ac-Gln-Asn-Sta-Val-NH2, Ac-Gln-Asn-Sta-Val-Val-NH2, and Ac-Glu-Asn-Sta-Ile-NH2) inhibited HIV-1 replication at concentrations that did not inhibit protein synthesis. Ac-Gln-Asn-Sta-Val-NH2 was the most potent, causing an approximately 40% decrease in viral replication, measured as the synthesis of HIV-1 antigens and the formation of infectious particles.

Virus quantification by immunofluorescence of cells grown in agar.

We have developed a method for assessing the number of infectious viral particles by measuring what we call fluorescence initiating units (FIV). The present work has been done with HIV, but the methods should be applicable to other viruses as well. Briefly described, cells are mixed with virus and then cast in an agar gel to block further transfer of virus. After a period of incubation sufficient to allow infected cells to express virus antigens, the percentage of infected cells is determined by indirect immunofluorescence.

The proteinase inhibitor pepstatin A inhibits formation of reverse transcriptase in H9 cells infected with human immunodeficiency virus 1.

Retroviruses depend on a virus-encoded proteinase. As this enzyme is an interesting target for antiviral therapy, we examined the effect of various low-molecular-weight proteinase inhibitors, as well as a few oligopeptides related to the proteolytic cleavage sites, on the replication of HIV-1 in H9 cells. The increase in reverse transcriptase activity during incubation was assumed to reflect viral replication. Cellular DNA synthesis was measured to quantitate the adverse effects of the inhibitors on the cells. Only one of the substances tested, pepstatin A, had an appreciable selective effect on viral replication. Substances that decreased DNA synthesis generally caused an equally large decrease in reverse transcriptase activity.

Detecting inhibition of coxsackievirus replication by measuring DNA synthesis--effect of proteinase inhibitors.

The replication of picornaviruses can be monitored by microscopic examination of the cytopathogenic effect. We found that measuring cellular DNA synthesis gave a more objective and reliable estimate of the viral effect on the cells. Furthermore, by simultaneously measuring DNA synthesis in uninfected cells, we obtained a sensitive indication of whether the agents tested influenced cellular activity. The method was employed to investigate the effect of various proteinase inhibitors on the replication of coxsackie-B3 virus in HEp2 cells. Certain inhibitors of metallo-proteinases had a limited, but consistent, protective effect against viral activity.

Oral acyclovir suppression of recurrent genital herpes: a double-blind, placebo-controlled, crossover study.

A randomised, double-blind, placebo-controlled, crossover study was conducted in 31 male patients with a history of frequently recurrent genital herpes who received consecutively 200 mg acyclovir and matching placebo by mouth four times a day for 12 weeks each. During acyclovir therapy recurrences were completely prevented in 24 (77%) and were reduced in both frequency and duration in the remainder compared with those occurring during treatment with placebo. The incidence and nature of adverse events reported during each treatment period was virtually identical. No long-term effects on recurrence rates were discernible but chronic suppressive therapy can be considered to offer the means of controlling the severe forms of disease experienced by some patients.

Reproducibility of an indirect immunofluorescence test for varicella-zoster antibodies.

The need has arisen for a rapid test for detection of antibody production in the central nervous system (CNS) in patients with varicella-zoster (VZV) encephalitis. We have developed an immunofluorescence test based on semiquantitative determination of antibody concentration. In this work we have systematically examined some of the most important errors that may influence the quantification of the system. In the reported experiments six samples with known antibody dilutions and one control sample were tested for fluorescence "in the blind" on 716 slides. On each slide two samples were tested in six two-fold dilutions. The reproducibility of the differences between two titres was best when the fluorescence of each test sample in each pair had been directly compared under the microscope. Pairs of samples which had been tested in random order with very small chance of being directly compared, showed a higher number of errors. Of 224 pairs having four-fold or greater concentration difference, only one was not detected by the improved test. However, with only two-fold difference, the failure increased to 35%. Only three out of 62 pairs with the same concentration of antibodies falsely showed two-fold or greater difference of titres. No paired titres showed a difference opposite to what was expected. Based on the results reported in this paper we have started to use the test in routine diagnosis of encephalitis.

Cytomegalovirus (CMV) and rubella virus infection during pregnancy. A study of CMV and rubella virus antibodies in 2014 pregnant women and follow-up studies of infants at risk for intrauterine CMV infections.

Two blood samples, one in the first and one in the third trimester, were collected from 2014 pregnant women. Serological tests for CMV and rubella antibodies were performed in the paired samples. Seroconversion by the CF test for CMV antibodies was demonstrated in 15 women. However, seroconversion also by the IF test was found in only one of these. A rise in titer during pregnancy by the CF test was found in 16 woman. None of these specimens contained specific IgM. High CMV-CF antibody titer (greater than 128) in the first serum sample was found in 28 women, but none of the sera contained specific IgM. It is concluded that no single serological test can serve at present as a screening test for the diagnosis of CMV infection during pregnancy. In children thought to be at risk contracting congenital CMV infection, no case with CNS malfunction that could be attributed to a congenital CMV infection could be demonstrated at the age of 7-8 years. One case of seroconversion in the examination for rubella antibodies was found. The infant of this mother showed no clinical signs of rubella infection.

Efficacy of oral acyclovir in the treatment of initial and recurrent genital herpes.

A double-blind, randomised trial of acyclovir versus placebo was conducted in 31 patients with initial and 85 patients with recurrent genital herpes. 17 patients with initial and 42 with recurrent disease were treated with 200 mg acyclovir by mouth five times a day for 5 days, and the remaining patients received matching placebo. In patients with initial genital herpes shedding virus acyclovir significantly reduced the duration of viral shedding, itching, and pain, the time to crusting and complete healing, and new lesion formation compared with controls. In patients with recurrence disease acyclovir significantly reduced the duration of viral shedding, time to complete healing, and new lesion formation. The reported incidence of adverse events was similar in both acyclovir and placebo groups. Oral acyclovir is effective and well tolerated in patients with initial and recurrent genital herpes and can be used in outpatient therapy.