Amplification of reiterated sequences of herpes simplex virus type 1 (HSV-1) genome to discriminate between clinical HSV-1 isolates.

Herpes simplex virus type 1 (HSV-1)-related disease ranges from a localized, self-limiting illness to fatal disease in immunocompromised individuals. The corneal disease herpetic keratitis may develop after reactivation of a latent virus or reinfection with an exogenous herpesvirus. Molecular analysis of the virus involved may allow distinction between these two options. The HSV-1 genome contains several hypervariable regions that vary in numbers of reiterating regions (reiterations I to VIII [ReI to ReVIII]) between individual strains. Twenty-four HSV-1 clones, derived by subcloning of HSV-1 (strain F) twice in limiting dilutions, were tested in a PCR-based assay to analyze the stabilities of ReI, ReIII, ReIV, and ReVII. ReI and ReIII proved to vary in size upon subcloning, whereas ReIV and ReVII were stable. Subsequently, 37 unrelated isolates and 10 sequential isolates from five patients, all with HSV-1-induced keratitis, were genotyped for ReIV and ReVII. Of the 37 unrelated samples, 34 (92%) could be discriminated, while the genotypes of the viruses in sequential samples were identical for each individual. Conclusively, the data show that the approach presented allows the rapid and accurate discrimination of HSV-1 strains in studies that address the transmission and pathogenesis of HSV-1 infections.

Helicobacter pylori and the birth cohort effect: evidence of a continuous decrease of infection rates in childhood.

OBJECTIVE: To investigate whether the decrease in rate of Helicobacter pylori infection in subsequent birth cohorts has continued during the last decades. METHODS: Determination by ELISA of IgG H. pylori antibodies in 314 serum samples from Dutch children (age 6-8 yr, n = 154) and young adolescents (age 12-15 yr, n = 160), collected in 1978 and 1993. RESULTS: The prevalence of H. pylori declined from 19% to 9% at age 6-8 yr and from 23% to 11% at age 12-15 yr. For the whole study population, a decline from 21% to 10% (p = 0.01) was observed between 1978 and 1993. On the basis of these data and an incidence of infection with H. pylori of 0.3% per year during the same period, a model for both past and future prevalence rates of H. pylori in the Dutch population was calculated. The outcome demonstrates a decrease from more than 50% around World War II to less than 20% for the whole population around year 2040. CONCLUSIONS: H. pylori infection rates in childhood have continued to decline until recent decades, demonstrating a persistent birth cohort effect. This decline will result in a very low prevalence of H. pylori infection in the Dutch population during the next decades, becoming even lower as the observed decline in children and young adolescents continues.

Virucidal effect of glycerol as used in donor skin preservation.

Glycerol has been used for a long time as a viral preservation medium in tissue samples at a 50 per cent concentration, however after a limited time span viruses could no longer be detected. This fact combined with the dehydrating action of glycerol, raised the suspicion that glycerol in a higher concentration could be virucidal. To test this hypothesis, experiments were done at various concentrations of glycerol at three different temperatures (4, 20 and 37 degrees C), using the following viruses: herpes simplex virus, a virus with an envelope, comparable to human immunodeficiency virus; and poliovirus as an example of small, hard to inactivate viruses without an envelope. Glycerol will dehydrate the skin, the extracted water being replaced by glycerol, preserving the original structure. The remaining water is optimally distributed throughout the tissue. However, the possibility exists that glycerol influences the enzymatic processes of nucleic acid breakdown. Plasmid DNA pBR322 was added to HeLa-cells in the presence and absence of glycerol. The outcome of the experiments showed that glycerol has a strong virucidal action. Preservation in 85 per cent glycerol was preferred, because using this concentration the glycerolized allograft skin retained its suppleness and was easy to manipulate during operations.

[HIV-2 in The Netherlands]. / HIV-2 in Nederland.

By comparing data from the HIV confirmation laboratories it could be established that in the period prior to February 1991 a HIV-2 infection was demonstrated in at least 18 persons in the Netherlands. Consequently, use of combined HIV-1/2 tests is urgently recommended for screening in diagnostic and blood bank laboratories in the Netherlands. A suspicion of presence of HIV-2 antibodies may arise in the interpretation of a combined HIV-1/2-ELISA, a specific HIV-1-ELISA and the pattern of HIV-1 western immunoblot. Confirmation of the presence of HIV-2 antibodies should be obtained by means of western blots of viral lysates of both HIV-1 and HIV-2 and peptide tests from the transmembranous region. Most persons with a HIV-2 infection originated from Africa south of the Sahara or the Cape Verdean Islands or had had a sexual relationship with a person from these areas. The vast majority was heterosexual and 13 of the 18 infected persons were women. This differs greatly from the epidemiological pattern of HIV-1 infections in the Netherlands. The possibility of further spread of HIV-2 in the Netherlands via heterosexual contacts should be seriously taken into account.

[Aids caused by HIV-2 in The Netherlands]. / AIDS door HIV-2 in Nederland.

In four patients in Rotterdam AIDS was diagnosed, and it was subsequently demonstrated that they were infected with HIV type 2. One of the patients was an immigrant from the Cape Verde islands, two of the others had heterosexual contacts with persons of this area, and the fourth patient was a male homosexual without known contacts with Africans. The Cape Verde islands are one of the areas in which HIV-2 infection occurs, and because of immigration into The Netherlands and in particular to Rotterdam, the possibility of HIV-2 infection should be considered in persons of this population group and their sexual contacts. Furthermore, problems with serologic investigations should be avoided by the application of HIV-2 specific confirmatory assays.

[HIV-2 infection in a Portuguese woman with an AIDS-dementia complex living in The Netherlands]. / HIV-2-infectie bij een in Nederland wonende Portugese vrouw met een AIDS-dementiecomplex.

The case history is presented of the first patient with a fatal HIV-2 infection in The Netherlands, a Portuguese woman aged 51 yr. The infection resulted in AIDS, the AIDS-dementia complex and death. Her partner, a retired Cape Verde sailor, also proved to be infected with HIV-2. Epidemiology, virology and clinical manifestations of HIV-2 infection are discussed.

Diagnosis and prevalence of HIV-2 antibodies in different population subsets in The Netherlands.

A serum panel obtained from male homosexuals (n = 278); i.v. drug abusers (n = 99), patients attending a VD clinic (n = 390), blood donors who visited Central or West Africa (n = 573), blood donors who had sexual contact with natives from Central or West Africa (n = 38), blood donors from Surinam, South America (n = 481), individuals positive for anti-HIV-1 (n = 94), individuals with indeterminate HIV-1 western blot (WB) reactions (n = 73), African patients with AIDS or AIDS-related symptoms (n = 30), and random Dutch blood donors (n = 555), was tested with HIV-2 ELISA (ELAVIA-2). Of these 2,611 samples, 32 (1.2%) were repeatedly reactive. Antibodies to gp140/105env were found in 4/32 by HIV-2 WB, and in 1/4 by radioimmunoprecipitation (RIPA). These 4 HIV-2 WB-positive samples were also reactive with gp160/120env in HIV-1 WB, suggesting cross-reactivity. In a spot test with synthetic peptides of the transmembrane glycoprotein of both HIV types, 3/4 were only HIV-1 positive and 1/4 was strongly HIV-2 positive and weakly HIV-1 positive. In inhibition assays with soluble HIV-1 or HIV-2 synthetic peptides in HIV-1 and HIV-2 peptide ELISA, cross-reactivity was excluded, which indicates an HIV variant or HIV-1/HIV-2 double infection. It is concluded that for the moment HIV-2 infection is at low prevalence in risk groups in The Netherlands, and that in addition to WB and RIPA, synthetic peptide assays are useful for differentiation between HIV-1 and HIV-2 antibodies.

A simple and rapid method for typing adenoviruses 40 and 41 without cultivation.

Fastidious adenovirus DNA in faecal samples obtained from children with acute gastroenteritis was extracted by a simple and rapid method. The extracted viral DNA was characterized by restriction endonuclease SmaI treatment. Fastidious adenovirus DNA was detected in 58 of 65 cases. If faecal samples were too small it was not always possible to identify virus DNA.

Characterization of rotaviral RNA isolated from children with gastroenteritis in two hospitals in Rotterdam.

Rotavirus RNA was extracted from stools collected from children with gastroenteritis in two hospitals in Rotterdam. Electrophoresis of the RNAs showed that at least 19 different electropherotypes were isolated from stools collected in the period from 1977 to 1982. It was possible to identify the index case in some hospital-acquired cases of rotaviral gastroenteritis.

Characterization of SV40-transformed human liver cells before and after passage through crisis.

SV40-transformed human liver cells could be passed through crisis after transfection with various DNAs (SV40, BKV, or salmon sperm). The pre- and post-crisis cells were compared with respect to growth behavior and the state of the viral genome. Comparison of the viral inserts in the pre- and post-crisis cells showed minor changes in the integration pattern of the SV40 DNA after recovery from crisis.

Transformation in Bacillus subtilis: biological and physical evidence for a novel DNA-intermediate in synchronously transforming cells.

Competent B. subtilis cells exposed to transforming DNA in the presence of EDTA bind, but do not take up DNA. Rapid and almost synchronous uptake of the bound DNA is achieved by the addition of Mg2+ ions in excess of the EDTA. At 30 degrees and at 17 degrees comparable numbers of transformants are produced from cells pre-loaded with DNA at 30 degrees (after termination of uptake by the addition of DNA ase the samples were incubated at 37 degrees). However, almost no transformants are produced when cells are exposed to DNA at 17 degrees, although binding does take place then. Because DNA is taken up at 17 degrees after having loaded the cells at 30 degrees, whereas no uptake occurs after binding at 17 degrees, it is suggested that binding of DNA to the cellular surface involves at least two steps. In DNA re-extracted from cells at 17 degrees, pre-loaded with DNA at 30 degrees, little recombinant type activity is present, indicating that integration is blocked at 17 degrees. However, physico-chemical analysis of the re-extracted DNA indicates that a complex between single-stranded donor DNA and the recipient chromosome is formed at 17 degrees. This complex has a higher buoyant density than donor-recipient complexes formed at 30 degrees.